alpha-synuclein and Protein-Aggregation--Pathological

Aberrant aggregation of the misfolded presynaptic protein, α-Synuclein (α-Syn) into Lewy body (LB) and Lewy neuritis (LN) is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Numerous studies have suggested that prefibrillar and fibrillar species of the misfolded α-Syn aggregates are responsible for cell death in PD pathogenesis. However, the precise molecular events during α-Syn aggregation, especially in the early stages, remain elusive. Emerging evidence has demonstrated that liquid-liquid phase separation (LLPS) of α-Syn occurs in the nucleation step of α-Syn aggregation, which offers an alternate non-canonical aggregation pathway in the crowded microenvironment. The liquid-like α-Syn droplets gradually undergo an irreversible liquid-to-solid phase transition into amyloid-like hydrogel entrapping oligomers and fibrils. This new mechanism of α-Syn LLPS and gel formation might represent the molecular basis of cellular toxicity associated with PD. This review aims to demonstrate the recent development of α-Syn LLPS, the underlying mechanism along with the microscopic events of aberrant phase transition. This review further discusses how several intrinsic and extrinsic factors regulate the thermodynamics and kinetics of α-Syn LLPS and co-LLPS with other proteins, which might explain the pathophysiology of α-Syn in various neurodegenerative diseases.

Topics: alpha-Synuclein; Humans; Lewy Bodies; Parkinson Disease; Protein Aggregation, Pathological

The formation of aggregates due to protein misfolding is encountered in various neurodegenerative diseases. α-Synuclein (α-Syn) aggregation is linked to Parkinson's disease (PD). It is one of the most prevalent neurodegenerative disorders after Alzheimer's disease. Aggregation of α-Syn is associated with Lewy body formation and degeneration of the dopaminergic neurons in the brain. These are the pathological hallmarks of PD progression. α-Syn aggregates in a multi-step process. The native unstructured α-Syn monomers combine to form oligomers, followed by amyloid fibrils, and finally Lewy bodies. Recent evidence suggests that α-Syn oligomerization and fibrils formation play major roles in PD development. α-Syn oligomeric species is the main contributor to neurotoxicity. Therefore, the detection of α-Syn oligomers and fibrils has drawn significant attention for potential diagnostic and therapeutic development. In this regard, the fluorescence strategy has become the most popular approach for following the protein aggregation process. Thioflavin T (ThT) is the most frequently used probe for monitoring amyloid kinetics. Unfortunately, it suffers from several significant drawbacks including the inability to detect neurotoxic oligomers. Researchers developed several small molecule-based advanced fluorescent probes compared to ThT for the detection/monitoring of α-Syn aggregates states. These are summarized here.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Fluorescent Dyes; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Lewy bodies that contain aggregated α-synuclein in dopamine neurons are the main culprit for neurodegeneration in Parkinson's disease. However, mitochondrial dysfunction has a well-established and prominent role in the pathogenesis of Parkinson's disease. The exact mechanism by which α-synuclein causes dopamine neuronal loss is unclear. Recent evidence suggests that aggregated α-synuclein localises in the mitochondria contributes to oxidative stress-mediated apoptosis in neurons. Therefore, the involvement of aggregated α-synuclein in mitochondrial dysfunction-mediated neuronal loss has made it an emerging drug target for the treatment of Parkinson's disease. However, the exact mechanism by which α-synuclein permeabilises through the mitochondrial membrane and affects the electron transport chain remains under investigation. In the present study, we describe mitochondria-α-synuclein interactions and how α-synuclein aggregation modulates mitochondrial homeostasis in Parkinson's disease pathogenesis. We also discuss recent therapeutic interventions targeting α-synuclein aggregation that may help researchers to design novel therapeutic treatments for Parkinson's disease.

Topics: alpha-Synuclein; Apoptosis; Dopaminergic Neurons; Humans; Lewy Bodies; Mitochondria; Mitochondrial Diseases; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological

Parkinson's disease is the second most prevalent neurodegenerative disease. The loss of dopaminergic neurons in the substantia nigra is one of the pathological hallmarks of PD. PD also belongs to the class of neurodegenerative disease known as 'Synucleinopathies' as α-synuclein is responsible for disease development. The presence of aggregated α-synuclein associated with other proteins found in the Lewy bodies and Lewy neurites in the substantia nigra and other regions of the brain including locus ceruleus, dorsal vagal nucleus, nucleus basalis of Meynert and cerebral cortex is one of the central events for PD development. The complete biological function of α-synuclein is still debated. Besides its ability to propagate, it undergoes various post-translational modifications which play a paramount role in PD development and progression. Also, the aggregation of α-synuclein is modulated by various post-translational modifications. Here, we present a summary of multiple PTMs involved in the modulation of α-synuclein directly or indirectly and to identify their neuroprotective or neurotoxic roles, which might act as potential therapeutic targets for Parkinson's disease.

Topics: alpha-Synuclein; Brain; Dopaminergic Neurons; Humans; Lewy Bodies; Neuroprotective Agents; Neurotoxins; Parkinson Disease; Protein Aggregation, Pathological; Protein Processing, Post-Translational

Specific protein misfolding and aggregation are mechanisms underlying various neurodegenerative diseases such as prion disease and Alzheimer's disease (AD). The misfolded proteins are involved in prions, amyloid-β (Aβ), tau, and α-synuclein disorders; they share common structural, biological, and biochemical characteristics, as well as similar mechanisms of aggregation and self-propagation. Pathological features of AD include the appearance of plaques consisting of deposition of protein Aβ and neurofibrillary tangles formed by the hyperphosphorylated tau protein. Although it is not clear how protein aggregation leads to AD, we are learning that the cellular prion protein (PrPC) plays an important role in the pathogenesis of AD. Herein, we first examined the pathogenesis of prion and AD with a focus on the contribution of PrPC to the development of AD. We analyzed the mechanisms that lead to the formation of a high affinity bond between Aβ oligomers (AβOs) and PrPC. Also, we studied the role of PrPC as an AβO receptor that initiates an AβO-induced signal cascade involving mGluR5, Fyn, Pyk2, and eEF2K linking Aβ and tau pathologies, resulting in the death of neurons in the central nervous system. Finally, we have described how the PrPC-AβOs interaction can be used as a new potential therapeutic target for the treatment of PrPC-dependent AD.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Humans; Neurofibrillary Tangles; Neurons; Prion Proteins; Protein Aggregation, Pathological; Randomized Controlled Trials as Topic; Receptor, Metabotropic Glutamate 5; tau Proteins

Aggregation of specific proteins are histopathological hallmarks of several neurodegenerative diseases, such as, Amyloid β (Aβ) plaques and tau neurofibrillary tangles in Alzheimer's disease (AD); morphologically different inclusions of ratiometric 3 repeat (3 R) and 4 repeat (4 R) tau isoforms in progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD); α-Synuclein (α-Syn) containing Lewy bodies (LBs) and dystrophic Lewy neurites (LNs) in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, mixed brain protein pathologies have been frequently observed in many of these diseases and in normal aging brains, among which Aβ/tau and tau/α-Syn crosstalks have received increased attention. Interestingly, studies have also shown synergistic interplay among Aβ, tau, and α-Syn in several neurodegenerative diseases, suggesting a protein triumvirate. In this review, we summarize the emerging evidence of Aβ, tau, and α-Syn aggregation in pathophysiology, and their overlap in a spectrum of neurodegenerative diseases including AD, PSP, PiD, CBD, PD and DLB. We discuss the prognostic advancements made in biomarker and imaging techniques in the triumvirate proteinopathies. Finally, we discuss the combined therapeutic modality involving biomarkers and imaging techniques for future combinatorial immunotherapeutic targeting more than one protein aggregates. We hope that such a multitarget therapeutic approach will have synergistic or additive effects to manage neurodegenerative diseases with two or more protein pathologies that might uncover a promising strategy for personalized combination therapies. Managing neurodegenerative diseases by optimizing the diagnostic criteria and the correct combination of immunotherapies will be a key factor in the success of future treatment.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Humans; Lewy Bodies; Neurodegenerative Diseases; Parkinson Disease; Plaque, Amyloid; Protein Aggregation, Pathological; tau Proteins

The pathological features of PDD are represented by dopaminergic neuronal death and intracellular

Topics: alpha-Synuclein; Alzheimer Disease; Dementia; Ferroptosis; Humans; Iron; Iron Regulatory Protein 1; Iron Regulatory Protein 2; Neurodegenerative Diseases; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; tau Proteins

According to emerging studies, the excessive activation of microglia and the subsequent release of pro-inflammatory cytokines play important roles in the pathogenesis and progression of Parkinson's disease (PD). However, the exact mechanisms governing chronic neuroinflammation remain elusive. Findings demonstrate an elevated level of NLRP3 inflammasome in activated microglia in the substantia nigra of PD patients. Activated NLRP3 inflammasome aggravates the pathology and accelerates the progression of neurodegenerative diseases. Abnormal protein aggregation of α-synuclein (α-syn), a pathologically relevant protein of PD, were reported to activate the NLRP3 inflammasome of microglia through interaction with toll-like receptors (TLRs). This eventually releases pro-inflammatory cytokines through the translocation of nuclear factor kappa-B (NF-κB) and causes an impairment of mitochondria, thus damaging the dopaminergic neurons. Currently, therapeutic drugs for PD are primarily aimed at providing relief from its clinical symptoms, and there are no well-established strategies to halt or reverse this disease. In this review, we aimed to update existing knowledge on the role of the α-syn/TLRs/NF-κB/NLRP3 inflammasome axis and microglial activation in PD. In addition, this review summarizes recent progress on the α-syn/TLRs/NF-κB/NLRP3 inflammasome axis of microglia as a potential target for PD treatment by inhibiting microglial activation.

Topics: alpha-Synuclein; Animals; Biomarkers; Disease Management; Disease Susceptibility; Humans; Inflammasomes; Microglia; Molecular Targeted Therapy; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Protein Binding; Signal Transduction; Toll-Like Receptors

Parkinson's disease (PD) is characterized by four pathognomonic hallmarks: (1) motor and non-motor deficits; (2) neuroinflammation and oxidative stress; (3) pathological aggregates of the α-synuclein (α-syn) protein; (4) neurodegeneration of the nigrostriatal system. Recent evidence sustains that the aggregation of pathological α-syn occurs in the early stages of the disease, becoming the first trigger of neuroinflammation and subsequent neurodegeneration. Thus, a therapeutic line aims at striking back α-synucleinopathy and neuroinflammation to impede neurodegeneration. Another therapeutic line is restoring the compromised dopaminergic system using neurotrophic factors, particularly the glial cell-derived neurotrophic factor (GDNF). Preclinical studies with GDNF have provided encouraging results but often lack evaluation of anti-α-syn and anti-inflammatory effects. In contrast, clinical trials have yielded imprecise results and have reported the emergence of severe side effects. Here, we analyze the discrepancy between preclinical and clinical outcomes, review the mechanisms of the aggregation of pathological α-syn, including neuroinflammation, and evaluate the neurorestorative properties of GDNF, emphasizing its anti-α-syn and anti-inflammatory effects in preclinical and clinical trials.

Topics: alpha-Synuclein; Animals; Clinical Trials as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Glial Cell Line-Derived Neurotrophic Factor; Humans; Neuroinflammatory Diseases; Parkinson Disease; Protein Aggregation, Pathological

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson's disease (PD), multiple system atrophy, Alzheimer's disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.

Topics: alpha-Synuclein; Amyloidogenic Proteins; Amyloidosis; Animals; Disease Susceptibility; Humans; Hydrophobic and Hydrophilic Interactions; Liquid-Liquid Extraction; Models, Molecular; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Protein Multimerization; Structure-Activity Relationship

The neuronal protein α-synuclein (αS) is central to the pathogenesis of Parkinson's disease and other progressive brain diseases such as Lewy body dementia and multiple system atrophy. These diseases, collectively referred to as 'synucleinopathies', have long been considered purely proteinopathies: diseases characterized by the misfolding of a protein into small and large aggregates mainly consisting of that protein (in this case: α-synuclein). However, recent morphological insights into Lewy bodies, the hallmark neuropathology of human synucleinopathies, suggests these lesions are also rich in vesicles and other membranous organelles. Moreover, αS physiology and pathology are both strongly associated with various aspects of intracellular vesicle trafficking and lipid biology. αS physiologically binds to synaptic and other small vesicles, and several functions of αS in regulating vesicle biology have been proposed. Familial PD-linked αS excess and missense mutations have been shown to impair vesicle trafficking and alter lipid homeostasis. On the other hand, vesicle trafficking and lipid-related genes have emerged as Parkinson's risk factors, suggesting a bidirectional relationship. The answer to the question "Does abnormal αS accumulation cause impaired vesicle trafficking and lipid dyshomeostasis or is αS aggregation the consequence of such impairments?" may be "Both". Here, we review current knowledge of the αS-lipid and αS-vesicle trafficking interplay, with a special focus on Parkinson's disease and Lewy body dementia.

Topics: alpha-Synuclein; Animals; Cytoplasmic Vesicles; Humans; Lipid Metabolism; Protein Aggregation, Pathological; Protein Transport; Synucleinopathies

The ubiquitin-proteasome system is the major pathway for the maintenance of protein homeostasis. Its inhibition causes accumulation of ubiquitinated proteins; this accumulation has been associated with several of the most common neurodegenerative diseases. Several genetic factors have been identified for most neurodegenerative diseases, however, most cases are considered idiopathic, thus making the study of the mechanisms of protein accumulation a relevant field of research. It is often mentioned that the biggest risk factor for neurodegenerative diseases is aging, and several groups have reported an age-related alteration of the expression of some of the 26S proteasome subunits and a reduction of its activity. Proteasome subunits interact with proteins that are known to accumulate in neurodegenerative diseases such as α-synuclein in Parkinson's, tau in Alzheimer's, and huntingtin in Huntington's diseases. These interactions have been explored for several years, but only until recently, we are beginning to understand them. In this review, we discuss the known interactions, the underlying patterns, and the phenotypes associated with the 26S proteasome subunits in the etiology and progression of neurodegenerative diseases where there is evidence of proteasome involvement. Special emphasis is made in reviewing proteasome subunits that interact with proteins known to have an age-related altered expression or to be involved in neurodegenerative diseases to explore key effectors that may trigger or augment their progression. Interestingly, while the causes of age-related reduction of some of the proteasome subunits are not known, there are specific relationships between the observed neurodegenerative disease and the affected proteasome subunits.

Topics: alpha-Synuclein; Animals; Humans; Huntingtin Protein; Neurodegenerative Diseases; Proteasome Endopeptidase Complex; Protein Aggregation, Pathological; Protein Binding; Protein Subunits; tau Proteins; Ubiquitin

The cause of most Parkinson's disease cases is unknown. However, it is well documented that mitochondrial dysfunction and misfolded α synuclein aggregation are important cellular abnormalities associated with the disease. In this paper, we use the microcompetition model to show how latent viruses, which infect the central and peripheral nervous systems, can cause the observed mitochondrial dysfunction and excess α synuclein aggregation, and eventually, Parkinson's disease.

Topics: alpha-Synuclein; Animals; Humans; Latent Infection; Mitochondria; Parkinson Disease; Protein Aggregation, Pathological; Virus Latency; Viruses

In protein aggregation disorders, we assume that, during the process of protein aggregation, different types of aggregated species (oligomers, protofibrils, fibrils, etc.) are formed, some of which can be toxic to cells/tissues/organs. Recent evidence from numerous studies in cell and animal models of disease suggest that oligomeric species of different proteins might be more toxic that the larger, fibrillar forms. However, we still lack definitive data on the nature of the toxic species, mostly due to our inability to detect and define the various protein species that form as protein aggregate. The terms used are often broad and do not capture inter-laboratory variation in protocols and methods used for the characterization of aggregates. Even antibody-based methods can be ambiguous, as antibodies are delicate tools. Therefore, systematic and interdisciplinary studies are essential in order to guide future developments in the field.

Topics: alpha-Synuclein; Antibodies; Humans; Protein Aggregation, Pathological

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer,

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Amyotrophic Lateral Sclerosis; Animals; Diabetes Mellitus, Type 2; Humans; Islet Amyloid Polypeptide; Models, Molecular; Neurodegenerative Diseases; Parkinson Disease; Protein Aggregation, Pathological; Proteostasis Deficiencies; Superoxide Dismutase-1; tau Proteins

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of human COVID-19, not only causes flu-like symptoms and gut microbiome complications but a large number of infected individuals also experience a host of neurological symptoms including loss of smell and taste, seizures, difficulty concentrating, decreased alertness, and brain inflammation. Although SARS-CoV-2 infections are not more prevalent in Parkinson's disease patients, a higher mortality rate has been reported not only associated with older age and longer disease duration, but also through several mechanisms, such as interactions with the brain dopaminergic system and through systemic inflammatory responses. Indeed, a number of the neurological symptoms seen in COVID-19 patients, as well as the alterations in the gut microbiome, are also prevalent in patients with Parkinson's disease. Furthermore, biochemical pathways such as oxidative stress, inflammation, and protein aggregation have shared commonalities between Parkinson's disease and COVID-19 disease progression. In this review, we describe and compare the numerous similarities and intersections between neurodegeneration in Parkinson's disease and RNA viral infections, emphasizing the current SARS-CoV-2 global health crisis.

Topics: Aged; alpha-Synuclein; Cognition Disorders; COVID-19; Cytokines; Diet; Disease Progression; Dysbiosis; Gastrointestinal Microbiome; Humans; Inflammation; Metals, Heavy; Models, Neurological; Nerve Degeneration; Olfactory Bulb; Oxidative Stress; Parkinson Disease; Practice Guidelines as Topic; Protein Aggregation, Pathological; Reactive Oxygen Species; RNA Virus Infections; SARS-CoV-2; Sensation Disorders

Lewy bodies (LBs), one of the neuropathological defining hallmarks of Parkinson's disease (PD), are composed of a complex mixture of alpha-synuclein (aSyn) filaments and hundreds of proteins, lipids, and membranous organelles. However, these proteins' role in aSyn aggregation and the biogenesis of LBs remains poorly understood. Previous studies have focused on investigating the role of these proteins as modifiers of aSyn aggregation, inclusion formation, and toxicity; very often, one protein at a time. In a recent study, Ham et al. suggest that one of these proteins, aminoacyl tRNA synthase complex-interacting multifunctional protein 2 (AIMP2), plays a primary role in the initiation of aSyn aggregation and is essential for aSyn inclusion formation and toxicity in cells and several models of synucleinopathies (Ham et al., 2020). Based on in vitro aggregation studies, they proposed a model in which AIMP2 self-associates to form amyloid-like aggregates that interact with monomeric aSyn and catalyze/seed the formation of aSyn fibrils and, eventually, LB-like inclusions. Herein, we present a critical analysis of their results and conclusions, review previous studies on AIMP2 aggregation, and reexamine the role of AIMP2 in regulating aSyn inclusion formation and clearance and aSyn-induced neurodegeneration in Parkinson's disease. We conclude by presenting lesson learned and recommendations on experimental factors and approaches that should be considered in future studies aimed at investigating the potential of targeting LBs-associated proteins, including AIMP2, for developing therapies to treat PD and other synucleinopathies.

Topics: alpha-Synuclein; Animals; Humans; Lewy Bodies; Nuclear Proteins; Protein Aggregation, Pathological; Substantia Nigra

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable β-sheet enriched intermediates, which are stabilized by intermolecular interactions with other β-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.

Topics: alpha-Synuclein; Amyloid; Humans; Intrinsically Disordered Proteins; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation, beta-Strand; tau Proteins

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disease caused by degeneration of dopaminergic neurons in the substantia nigra. Existing pharmaceutical treatments offer alleviation of symptoms but cannot delay disease progression and are often associated with significant side effects. Clinical studies have demonstrated that acupuncture may be beneficial for PD treatment, particularly in terms of ameliorating PD symptoms when combined with anti-PD medication, reducing the required dose of medication and associated side effects. During early stages of PD, acupuncture may even be used to replace medication. It has also been found that acupuncture can protect dopaminergic neurons from degeneration via antioxidative stress, anti-inflammatory, and antiapoptotic pathways as well as modulating the neurotransmitter balance in the basal ganglia circuit. Here, we review current studies and reflect on the potential of acupuncture as a novel and effective treatment strategy for PD. We found that particularly during the early stages, acupuncture may reduce neurodegeneration of dopaminergic neurons and regulate the balance of the dopaminergic circuit, thus delaying the progression of the disease. The benefits of acupuncture will need to be further verified through basic and clinical studies.

Topics: Acupuncture Therapy; alpha-Synuclein; Antiparkinson Agents; Apoptosis; Basal Ganglia; Bibliometrics; Clinical Trials as Topic; Combined Modality Therapy; Dopamine; Dopaminergic Neurons; Humans; Nerve Net; Neuroinflammatory Diseases; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Treatment Outcome

The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson's disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn accumulation in the CNS are not fully understood, growing evidence suggests that de novo α-syn misfolding and/or neuronal internalization of aggregated α-syn facilitates conformational templating of endogenous α-syn monomers in a mechanism reminiscent of prions. A refined understanding of the biochemical and cellular factors mediating the pathological neuron-to-neuron propagation of misfolded α-syn will potentially elucidate the etiology of PD and unravel novel targets for therapeutic intervention. Here, we discuss recent developments on the hypothesis regarding trans-synaptic propagation of α-syn pathology in the context of neuronal vulnerability and highlight the potential utility of novel experimental models of synucleinopathies.

Topics: alpha-Synuclein; Animals; Humans; Parkinson Disease; Prions; Protein Aggregation, Pathological

A neuropathological hallmark of Parkinson's disease (PD) is the cerebral deposition of abnormally aggregated α-synuclein (αSyn). PD-associated αSyn (αSyn

Topics: alpha-Synuclein; Animals; Brain; Enteric Nervous System; Gastrointestinal Tract; Humans; Mice; Parkinson Disease; Prion Proteins; Prions; Protein Aggregation, Pathological

Parkinson's disease (PD) is a movement disorder associated with severe loss of mainly dopaminergic neurons in the substantia nigra. Pathological hallmarks include Lewy bodies, and loss of neuromelanin, due to degeneration of neuromelanin-containing dopaminergic neurons. Despite being described over 200 years ago, the etiology of PD remains unknown. Here, we highlight the roles of reactive oxygen species (ROS), iron, alpha synuclein (α-syn) and neuromelanin in a toxic feedback loop culminating in neuronal death and spread of the disease. Dopaminergic neurons are particularly vulnerable due to decreased antioxidant concentration with aging, constant exposure to ROS and presence of neurotoxic compounds (e.g. ortho-quinones). ROS and iron increase each other's levels, creating a state of oxidative stress. α-Syn aggregation is influenced by ROS and iron but also increases ROS and iron via its induced mitochondrial dysfunction and ferric-reductase activity. Neuromelanin's binding affinity is affected by increased ROS and iron. Furthermore, during neuronal death, neuromelanin is degraded in the extracellular space, releasing its bound toxins. This cycle of events continues to neighboring neurons in the form of a toxic loop, causing PD pathology. The increase in ROS and iron may be an important target for therapies to disrupt this toxic loop, and therefore diets rich in certain 'nutraceuticals' may be beneficial. Turmeric is an attractive candidate, as it is known to have anti-oxidant and iron chelating properties. More studies are needed to test this theory and if validated, this would be a step towards development of lifestyle-based therapeutic modalities to complement existing PD treatments.

Topics: alpha-Synuclein; Animals; Autophagy; Brain Chemistry; Curcuma; Dopamine; Dopaminergic Neurons; Feedback, Physiological; Ferroptosis; Homeostasis; Humans; Iron; Melanins; Mice; Oxidative Stress; Parkinson Disease; Parkinsonian Disorders; Phytotherapy; Protein Aggregation, Pathological; Reactive Oxygen Species; Substantia Nigra

Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are characterized by the aggregation of misfolded proteins, including Aβ, tau and α-synuclein. It is well recognized that these misfolded proteins are able to self-propagate and spread throughout the nervous system and cause neuronal injury in a way that resembles prion disease. These disease-specific misfolded proteins demonstrate unique features, including the seeding barrier, the conformational memory effect, strain selection and strain evolution, based on the presence of various strains. However, the accurate definition of the term strain remains to be clarified. Here, a clear interpretation is proposed by a retrospective of its history in prion research and the recent progress in neurodegeneration research. Furthermore, the causes contributing to the genesis of various strains are also summarized. Deeper insight into strains helps us to understand the phenomena we observe in this field and it also enlightens us on the elusive mechanisms and management of neurodegeneration.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Humans; Neurodegenerative Diseases; Prions; Protein Aggregation, Pathological; Protein Folding; tau Proteins

The pathophysiology of Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and many others converge at alpha-synuclein (α-Syn) aggregation. Although it is still not entirely clear what precise biophysical processes act as triggers, cumulative evidence points towards a crucial role for protein quality control (PQC) systems in modulating α-Syn aggregation and toxicity. These encompass distinct cellular strategies that tightly balance protein production, stability, and degradation, ultimately regulating α-Syn levels. Here, we review the main aspects of α-Syn biology, focusing on the cellular PQC components that are at the heart of recognizing and disposing toxic, aggregate-prone α-Syn assemblies: molecular chaperones and the ubiquitin-proteasome system and autophagy-lysosome pathway, respectively. A deeper understanding of these basic protein homeostasis mechanisms might contribute to the development of new therapeutic strategies envisioning the prevention and/or enhanced degradation of α-Syn aggregates.

Topics: alpha-Synuclein; Animals; Humans; Metabolic Networks and Pathways; Protein Aggregation, Pathological; Synucleinopathies

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins. The majority of PD cases are, however, idiopathic, i.e., having no clear etiology. PD is characterized by progressive accumulation of insoluble inclusions, known as Lewy bodies, mostly composed of α-synuclein and membrane components. The cause of PD is currently attributed to cellular proteostasis deregulation and mitochondrial dysfunction, which are likely interdependent. In addition, neuroinflammation is present in brains of PD patients, but whether it is the cause or consequence of neurodegeneration remains to be studied. Rodents do not develop PD or PD-like motor symptoms spontaneously; however, neurotoxins, genetic mutations, viral vector-mediated transgene expression and, recently, injections of misfolded α-synuclein have been successfully utilized to model certain aspects of the disease. Here, we critically review the advantages and drawbacks of rodent PD models and discuss approaches to advance pre-clinical PD research towards successful disease-modifying therapy. © 2020 The Authors.

Topics: alpha-Synuclein; Animals; Corpus Striatum; Dopaminergic Neurons; Drug Evaluation, Preclinical; Forecasting; Genome-Wide Association Study; Histological Techniques; Humans; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neuroprotective Agents; Neurotoxins; Parkinson Disease; Parkinsonian Disorders; Pesticides; Protein Aggregation, Pathological; Rats; Substantia Nigra; Synucleinopathies

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the accumulation of α-synuclein (α-syn) into insoluble aggregates known as Lewy bodies and Lewy neurites in the brain. However, prior to the formation of these large aggregates, α-syn forms oligomers and small fibrils, which are believed to be the pathogenic species leading to the death of neurons in the substantia nigra in disease. The majority of aggregated α-syn is phosphorylated, and it is thought that this post-translational modification may be critical in disease pathogenesis. Thus, early detection of the toxic forms of α-syn may provide a window of opportunity for an intervention to halt or slow the progression of neurodegeneration in PD. Expression of α-syn is not restricted to the central nervous system and the protein can be found elsewhere, including bodily fluids and peripheral tissues. This review will examine current methods for detecting toxic forms of α-syn in accessible biospecimens and outline emerging techniques that may provide reliable identification of biomarkers for PD.

Topics: alpha-Synuclein; Animals; Biomarkers; Brain; Humans; Lewy Bodies; Parkinson Disease; Phosphorylation; Protein Aggregation, Pathological

The two main pathological hallmarks of Parkinson's disease are loss of dopamine neurons in the substantia nigra pars compacta and proteinaceous amyloid fibrils composed mostly of α-synuclein, called Lewy pathology. Levodopa to enhance dopaminergic transmission remains one of the most effective treatment for alleviating the motor symptoms of Parkinson's disease (Olanow, Mov Disord 34:812-815, 2019). In addition, deep brain stimulation (Bronstein et al., Arch Neurol 68:165, 2011) to modulate basal ganglia circuit activity successfully alleviates some motor symptoms. MRI guided focused ultrasound in the subthalamic nucleus is a promising therapeutic strategy as well (Martinez-Fernandez et al., Lancet Neurol 17:54-63, 2018). However, to date, there exists no treatment that stops the progression of this disease. The findings that α-synuclein can be released from neurons and inherited through interconnected neural networks opened the door for discovering novel treatment strategies to prevent the formation and spread of Lewy pathology with the goal of halting PD in its tracks. This hypothesis is based on discoveries that pathologic aggregates of α-synuclein induce the endogenous α-synuclein protein to adopt a similar pathologic conformation, and is thus self-propagating. Phase I clinical trials are currently ongoing to test treatments such as immunotherapy to prevent the neuron to neuron spread of extracellular aggregates. Although tremendous progress has been made in understanding how Lewy pathology forms and spreads throughout the brain, cell intrinsic factors also play a critical role in the formation of pathologic α-synuclein, such as mechanisms that increase endogenous α-synuclein levels, selective expression profiles in distinct neuron subtypes, mutations and altered function of proteins involved in α-synuclein synthesis and degradation, and oxidative stress. Strategies that prevent the formation of pathologic α-synuclein should consider extracellular release and propagation, as well as neuron intrinsic mechanisms.

Topics: alpha-Synuclein; Animals; Brain; Disease Progression; Dopaminergic Neurons; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Most neurodegenerative diseases are characterized by the intracellular or extracellular aggregation of misfolded proteins such as amyloid-β and tau in Alzheimer disease, α-synuclein in Parkinson disease, and TAR DNA-binding protein 43 in amyotrophic lateral sclerosis. Accumulating evidence from both human studies and disease models indicates that intercellular transmission and the subsequent templated amplification of these misfolded proteins are involved in the onset and progression of various neurodegenerative diseases. The misfolded proteins that are transferred between cells are referred to as 'pathological seeds'. Recent studies have made exciting progress in identifying the characteristics of different pathological seeds, particularly those isolated from diseased brains. Advances have also been made in our understanding of the molecular mechanisms that regulate the transmission process, and the influence of the host cell on the conformation and properties of pathological seeds. The aim of this Review is to summarize our current knowledge of the cell-to-cell transmission of pathological proteins and to identify key questions for future investigation.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Amyotrophic Lateral Sclerosis; Axonal Transport; Brain; Cell Communication; DNA-Binding Proteins; Endocytosis; Exosomes; Genetic Predisposition to Disease; Humans; Huntingtin Protein; Huntington Disease; Membrane Fusion; Nanotubes; Neurodegenerative Diseases; Neuroglia; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Protein Transport; tau Proteins

Parkinson's Disease (PD) is a progressive neurodegenerative disorder with no cure. Clinical presentation is characterized by postural instability, resting tremors, and gait problems that result from progressive loss of A9 dopaminergic neurons in the substantia nigra pars compacta. Traumatic brain injury (TBI) has been implicated as a risk factor for several neurodegenerative diseases, but the strongest evidence is linked to development of PD. Mild TBI (mTBI), is the most common and is defined by minimal, if any, loss of consciousness and the absence of significant observable damage to the brain tissue. mTBI is responsible for a 56% higher risk of developing PD in U.S. Veterans and the risk increases with severity of injury. While the mounting evidence from human studies suggests a link between TBI and PD, fundamental questions as to whether TBI nucleates PD pathology or accelerates PD pathology in vulnerable populations remains unanswered. Several promising lines of research point to inflammation, metabolic dysregulation, and protein accumulation as potential mechanisms through which TBI can initiate or accelerate PD. Amyloid precursor protein (APP), alpha synuclein (α-syn), hyper-phosphorylated Tau, and TAR DNA-binding protein 43 (TDP-43), are some of the most frequently reported proteins upregulated following a TBI and are also closely linked to PD. Recently, upregulation of Leucine Rich Repeat Kinase 2 (LRRK2), has been found in the brain of mice following a TBI. Subset of Rab proteins were identified as biological substrates of LRRK2, a protein also extensively linked to late onset PD. Inhibition of LRRK2 was found to be neuroprotective in PD and TBI models. The goal of this review is to survey current literature concerning the mechanistic overlap between TBI and PD with a particular focus on inflammation, metabolic dysregulation, and aforementioned proteins. This review will also cover the application of rodent TBI models to further our understanding of the relationship between TBI and PD.

Topics: alpha-Synuclein; Amyloid beta-Protein Precursor; Animals; Blood-Brain Barrier; Brain Injuries, Traumatic; DNA-Binding Proteins; Energy Metabolism; Humans; Inflammation; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Parkinson Disease; Phosphorylation; Protein Aggregation, Pathological; rab GTP-Binding Proteins; Risk; tau Proteins; Up-Regulation

Several lines of evidence from neuropathological studies, human genetics, in vitro aggregation studies and cellular and animal models support the hypothesis that aSyn plays a central role in the formation of Lewy pathologies. These are cytoplasmic proteinaceous and lipid-rich inclusions that represent key pathological hallmarks of Parkinson's disease (PD) and other neurodegenerative diseases, collectively referred to as synucleinopathies. For decades, light microscopy and electron microscopy studies of these inclusions have consistently shown that they are rich in filamentous structures that exhibit distinct distribution and organizational patterns depending on where they occur in the brain (e.g., classical brain-stem Lewy bodies (LBs) and cortical LBs) and the type of synucleinopathies. Although the identity of the protein that form these filaments was a subject of debate for decades, the discovery of PD-linked aSyn mutations, the demonstration that LBs are enriched in insoluble forms of aSyn, and the ability of aSyn to form fibrils of similar dimensions have led to convergence on the hypothesis that aSyn fibrils are key components of LBs. In a recent study, Shahmoradian et al used a combination of advanced electron microscopy and immunofluorescence based imaging techniques to investigate the structure, composition, and architecture of LBs from postmortem brain tissues of individuals with PD or other synucleinopathies (Shahmoradian et al., 2019). The paper's main conclusions suggest that "lipid membrane fragments and distorted organelles together with a non-fibrillar form of αSyn are the main structural building blocks for the formation of Lewy pathology". Their proposal that LBs are devoid of aSyn fibrils or that LB formation occurs independently of aSyn fibril formation casts doubts on a substantial body of work that forms the foundation of many of the current basic and translational research programs in academia and industry. In this article, I present a critical analysis of their data and claims in the context of the existing literature In addition, I examine the extent to which their findings and proposed models of the mechanisms of LB formation are consistent with existing data and are supported by other experimental evidence. The results from this analysis caution against overinterpretation of observations from a single report, especially given the limitations of the techniques and experimental approaches used by Shahmoradian et al and for more co

Topics: alpha-Synuclein; Animals; Brain; Humans; Lewy Bodies; Neurons; Protein Aggregation, Pathological; Synucleinopathies

α-Synuclein (αsyn) is an abundant brain neuronal protein that can misfold and polymerize to form toxic fibrils coalescing into pathologic inclusions in neurodegenerative diseases, including Parkinson's disease, Lewy body dementia, and multiple system atrophy. These fibrils may induce further αsyn misfolding and propagation of pathologic fibrils in a prion-like process. It is unclear why αsyn initially misfolds, but a growing body of literature suggests a critical role of partial proteolytic processing resulting in various truncations of the highly charged and flexible carboxyl-terminal region. This review aims to 1) summarize recent evidence that disease-specific proteolytic truncations of αsyn occur in Parkinson's disease, Lewy body dementia, and multiple system atrophy and animal disease models; 2) provide mechanistic insights on how truncation of the amino and carboxyl regions of αsyn may modulate the propensity of αsyn to pathologically misfold; 3) compare experiments evaluating the prion-like properties of truncated forms of αsyn in various models with implications for disease progression; 4) assess uniquely toxic properties imparted to αsyn upon truncation; and 5) discuss pathways through which truncated αsyn forms and therapies targeted to interrupt them. Cumulatively, it is evident that truncation of αsyn, particularly carboxyl truncation that can be augmented by dysfunctional proteostasis, dramatically potentiates the propensity of αsyn to pathologically misfold into uniquely toxic fibrils with modulated prion-like seeding activity. Therapeutic strategies and experimental paradigms should operate under the assumption that truncation of αsyn is likely occurring in both initial and progressive disease stages, and preventing truncation may be an effective preventative strategy against pathologic inclusion formation.

Topics: alpha-Synuclein; Animals; Humans; Neurodegenerative Diseases; Protein Aggregation, Pathological

The aggregation and deposition of α-synuclein (αS) are major pathologic features of Parkinson's disease, dementia with Lewy bodies, and other α-synucleinopathies. The propagation of αS pathology in the brain plays a key role in the onset and progression of clinical phenotypes. Thus, there is increasing interest in developing strategies that attenuate αS aggregation and propagation. Based on cumulative evidence that αS oligomers are neurotoxic and critical species in the pathogenesis of α-synucleinopathies, we and other groups reported that phenolic compounds inhibit αS aggregation including oligomerization, thereby ameliorating αS oligomer-induced cellular and synaptic toxicities. Heterogeneity in gut microbiota may influence the efficacy of dietary polyphenol metabolism. Our recent studies on the brain-penetrating polyphenolic acids 3-hydroxybenzoic acid (3-HBA), 3,4-dihydroxybenzoic acid (3,4-diHBA), and 3-hydroxyphenylacetic acid (3-HPPA), which are derived from gut microbiota-based metabolism of dietary polyphenols, demonstrated an in vitro ability to inhibit αS oligomerization and mediate aggregated αS-induced neurotoxicity. Additionally, 3-HPPA, 3,4-diHBA, 3-HBA, and 4-hydroxybenzoic acid significantly attenuated intracellular αS seeding aggregation in a cell-based system. This review focuses on recent research developments regarding neuroprotective properties, especially anti-αS aggregation effects, of phenolic compounds and their metabolites by the gut microbiome, including our findings in the pathogenesis of α-synucleinopathies.

Topics: alpha-Synuclein; Brain; Humans; Lewy Body Disease; Parkinson Disease; Phenols; Protein Aggregation, Pathological; Synucleinopathies

Prion-like propagation has been proposed to underlie the pathogenesis and progression of many progressive neurodegenerative diseases, and considerable experimental evidence has been accumulated to support this idea. However, only limited evidence is available from the brains of patients, and it is not clear how well various experimental models reflect the clinical situation. In this review, I discuss experimental models of prion-like propagation, focusing on three major disease-associated intracellular proteins, α-synuclein, tau and transactivation response DNA-binding protein 43 kDa, which provide a molecular basis for evaluating the spread of pathologies in diseased brains, known as Braak staging. Although some issues remain, and further biochemical and structural analyses are needed, it seems clear that the concept of prion-like propagation is the key to understanding disease progression, as well as for the development of disease-modifying therapies.

Topics: alpha-Synuclein; Animals; Brain; Disease Models, Animal; Disease Progression; DNA-Binding Proteins; Humans; Models, Neurological; Neurodegenerative Diseases; Prion Diseases; Protein Aggregation, Pathological; tau Proteins

The presence of intraneuronal Lewy bodies (LBs) and Lewy neurites (LNs) in the substantia nigra (SN) composed of aggregated α-synuclein (α-syn) has been recognized as a hallmark of pathological changes in Parkinson's disease (PD). Numerous studies have shown that aggregated α-syn is necessary for neurotoxicity. Meanwhile, the mitochondrial and lysosomal dysfunctions are associated with α-syn pathogenicity The hypothesis that α-syn transmission in the human brain contributes to the instigation and progression of PD has provided insights into PD pathology. This review will provide a brief overview of increasing researches that shed light on the relationship of α-syn aggregation with mitochondrial and lysosomal dysfunctions, and highlight recent understanding of α-syn transmission in PD pathology.

Topics: alpha-Synuclein; Animals; Cell Communication; Humans; Lewy Bodies; Lysosomes; Mitochondria; Neurons; Parkinson Disease; Protein Aggregation, Pathological

This review presents the main properties of hydroxycinnamic acid (HCA) derivatives and their potential application as agents for the prevention and treatment of neurodegenerative diseases. It is partially focused on the successful use of these compounds as inhibitors of amyloidogenic transformation of proteins. Firstly, the prerequisites for the emergence of interest in HCA derivatives, including natural compounds, are described. A separate section is devoted to synthesis and properties of HCA derivatives. Then, the results of molecular modeling of HCA derivatives with prion protein as well as with α-synuclein fibrils are summarized, followed by detailed analysis of the experiments on the effect of natural and synthetic HCA derivatives, as well as structurally similar phenylacetic and benzoic acid derivatives, on the pathological transformation of prion protein and α-synuclein. The ability of HCA derivatives to prevent amyloid transformation of some amyloidogenic proteins, and their presence not only in food products but also as natural metabolites in human blood and tissues, makes them promising for the prevention and treatment of neurodegenerative diseases of amyloid nature.

Topics: alpha-Synuclein; Amyloidogenic Proteins; Animals; Coumaric Acids; Humans; Neurodegenerative Diseases; Protein Aggregation, Pathological

α-Synuclein amyloid aggregation is a defining molecular feature of Parkinson's disease, Lewy body dementia, and multiple system atrophy, but can also be found in other neurodegenerative disorders such as Alzheimer's disease. The process of α-synuclein aggregation can be initiated through alternative nucleation mechanisms and dominated by different secondary processes giving rise to multiple amyloid polymorphs and intermediate species. Some aggregated species have more inherent abilities to induce cellular stress and toxicity, while others seem to be more potent in propagating neurodegeneration. The preference for particular types of polymorphs depends on the solution conditions and the cellular microenvironment that the protein encounters, which is likely related to the distinct cellular locations of α-synuclein inclusions in different synucleinopathies, and the existence of disease-specific amyloid polymorphs. In this review, we discuss our current understanding on the nature and structure of the various types of α-synuclein aggregated species and their possible roles in pathology. Precisely defining these distinct α-synuclein species will contribute to understanding the molecular origins of these disorders, developing accurate diagnoses, and designing effective therapeutic interventions for these highly debilitating neurodegenerative diseases.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Animals; Humans; Multiple System Atrophy; Parkinson Disease; Protein Aggregation, Pathological

Neurodegenerative disorders are associated with intra- or extra-cellular deposition of aggregates of misfolded insoluble proteins. These deposits composed of tau, amyloid-β or α-synuclein spread from cell to cell, in a prion-like manner. Novel evidence suggests that the circulating soluble oligomeric species of these misfolded proteins could play a major role in pathology, while insoluble aggregates would represent their protective less toxic counterparts. Recent convincing data support the proposition that the cellular prion protein, PrP

Topics: alpha-Synuclein; Animals; Humans; Neurodegenerative Diseases; Prion Proteins; Protein Aggregation, Pathological; Signal Transduction; tau Proteins

The aberrant aggregation of normally soluble proteins into amyloid fibrils is the pathological hallmark of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Understanding this process will be key to developing both diagnostic and therapeutic approaches for neurodegenerative diseases. Recent advances in biophysical techniques, coupled with kinetic analyses have enabled a thorough description of the key molecular steps involved in protein aggregation. In this review, we discuss these advances and how they have been applied to study the ability of one such protein, α-Synuclein, to form neurotoxic oligomers.

Topics: alpha-Synuclein; Humans; Parkinson Disease; Protein Aggregation, Pathological; Protein Binding

Neuropathological diagnostic criteria of neurodegenerative disorders are based on the presence of specific inclusions in a specific area of brain tissue that correlate with clinical manifestations. Concomitant neurodegenerative disorders correspond to a combination of two (or more) different fully developed diseases in the same patient. Concomitant neurodegenerative pathology represents the presence of definite neurodegeneration and deposits of pathological proteins specific for another disease, which is not, however, fully developed. Very frequent overlaps include Alzheimer's disease and alpha-synuclein inclusions. Nevertheless, careful neuropathological investigations reveal an increasing frequency of different co-pathologies in examined brains. In Alzheimer's disease, protein TDP-43 may co-aggregate, but it is not clear whether this is atypical isolated Alzheimer's disease or overlap of Alzheimer's disease with early frontotemporal lobar degeneration. Comorbidities of Alzheimer's disease and tauopathies are relatively rare. A combination of vascular pathology with primary neurodegeneration (mostly Alzheimer's disease or dementia with Lewy bodies) is historically called mixed dementia. Overlap of different neuropathologically confirmed neurodegenerations could lead to atypical and unusual clinical presentations and may be responsible for faster disease progression. Several CSF biomarkers have been evaluated for their utility in diagnostic processes in different neurodegenerative dementias; however, evidence regarding their role in neurodegenerative overlaps is still limited.

Topics: alpha-Synuclein; Alzheimer Disease; Biomarkers; DNA-Binding Proteins; Humans; Lewy Body Disease; Protein Aggregation, Pathological; tau Proteins

Amyloid formation is a pathological hallmark of many neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's. While it is unknown how these disorders are initiated, in vitro and cellular experiments confirm the importance of membranes. Ubiquitous in vivo, membranes induce conformational changes in amyloidogenic proteins and in some cases, facilitate aggregation. Reciprocally, perturbations in the bilayer structure can be induced by amyloid formation. Here, we review studies in the last 10 years describing α-synuclein (α-syn) and its interactions with membranes, detailing the roles of anionic and zwitterionic lipids in aggregation, and their contribution to Parkinson's disease. We summarize the impact of α-syn - comparing monomeric, oligomeric, and fibrillar forms - on membrane structure, and the effect of membrane remodeling on amyloid formation. Finally, perspective on future studies investigating the interplay between α-syn aggregation and membranes is discussed. This article is part of a Special Issue entitled: Amyloids.

Topics: alpha-Synuclein; Amyloid; Animals; Cell Membrane; Humans; Lipid Metabolism; Parkinson Disease; Protein Aggregation, Pathological

Alpha synuclein (αS) is a ~14 kDa intrinsically disordered protein. Decades of research have increased our knowledge on αS yet its physiological function remains largely elusive. The conversion of monomeric αS into oligomers and amyloid fibrils is believed to play a central role of the pathology of Parkinson's disease (PD). It is becoming increasingly clear that the interactions of αS with cellular membranes are important for both αS's functional and pathogenic actions. Therefore, understanding interactions of αS with membranes seems critical to uncover functional or pathological mechanisms. This review summarizes our current knowledge of how physicochemical properties of phospholipid membranes affect the binding and aggregation of αS species and gives an overview of how post-translational modifications and point mutations in αS affect phospholipid membrane binding and protein aggregation. We discuss the disruptive effects resulting from the interaction of αS aggregate species with membranes and highlight current approaches and hypotheses that seek to understand the pathogenic and/or protective role of αS in PD.

Topics: alpha-Synuclein; Amyloid; Animals; Cell Membrane; Humans; Lipid Bilayers; Parkinson Disease; Phospholipids; Protein Aggregation, Pathological

A host of human diseases, including Parkinson's disease and Dementia with Lewy bodies, are suspected to be directly linked to protein aggregation. Amyloid protein aggregates and oligomeric intermediates of α-synuclein are observed in synucleinopathies and considered to be mediators of cellular toxicity. Hence, α-synuclein has seen as one of the leading and most compelling targets and is receiving a great deal of attention from researchers. Nevertheless, there is no neuroprotective approach directed toward Parkinson's disease or other synucleinopathies so far. In this review, we summarize the available data concerning inhibitors of α-synuclein aggregation and their advancing towards clinical use. The compounds are grouped according to their chemical structures, providing respective insights into their mechanism of action, pharmacology, and pharmacokinetics. Overall, shared structure-activity elements are emerging, as well as specific binding modes related to the ability of the modulators to establish hydrophobic and hydrogen bonds interactions with the protein. Some molecules with encouraging in vivo data support the possibility of translation to the clinic.

Topics: alpha-Synuclein; Amyloidogenic Proteins; Drug Discovery; Humans; Protein Aggregation, Pathological; Structure-Activity Relationship

The gold standard for a definitive diagnosis of Parkinson disease (PD) is the pathologic finding of aggregated α-synuclein into Lewy bodies and for Alzheimer disease (AD) aggregated amyloid into plaques and hyperphosphorylated tau into tangles. Implicit in this clinicopathologic-based nosology is the assumption that pathologic protein aggregation at autopsy reflects pathogenesis at disease onset. While these aggregates may in exceptional cases be on a causal pathway in humans (e.g., aggregated α-synuclein in

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Brain; Causality; Humans; Parkinson Disease; Protein Aggregation, Pathological

Alpha-synuclein (α-Syn) is a central player in Parkinson's disease (PD) and in a spectrum of neurodegenerative diseases collectively known as synucleinopathies. The protein was first associated with PD just over 20 years ago, when it was found to (i) be a major component of Lewy bodies and (ii) to be also associated with familial forms of PD. The characterization of α-Syn pathology has been achieved through postmortem studies of human brains. However, the identification of toxic mechanisms associated with α-Syn was only achieved through the use of experimental models. In vitro models are highly accessible, enable relatively rapid studies, and have been extensively employed to address α-Syn-associated neurodegeneration. Given the diversity of models used and the outcomes of the studies, a cumulative and comprehensive perspective emerges as indispensable to pave the way for further investigations. Here, we subdivided in vitro models of α-Syn pathology into three major types: (i) models simulating α-Syn fibrillization and the formation of different aggregated structures in vitro, (ii) models based on the intracellular expression of α-Syn, reporting on pathogenic conditions and cellular dysfunctions induced, and (iii) models using extracellular treatment with α-Syn aggregated species, reporting on sites of interaction and their downstream consequences. In summary, we review the underlying molecular mechanisms discovered and categorize protective strategies, in order to pave the way for future studies and the identification of effective therapeutic strategies. This article is part of the Special Issue "Synuclein".

Topics: alpha-Synuclein; Amyloid; Animals; Autophagy; Cell Line; Cell Membrane; Endosomes; Humans; In Vitro Techniques; Lewy Bodies; Lysosomes; Mice; Mice, Knockout; Mitochondria; Neurons; Oxidative Stress; Protein Aggregation, Pathological; Proteolysis; Synaptic Transmission; Synucleinopathies

Synucleinopathies including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are characterized by the abnormal accumulation and propagation of α-synuclein (α-syn) pathology in the central and peripheral nervous system as Lewy bodies or glial cytoplasmic inclusions. Several antibodies against α-syn have been developed since it was first detected as the major component of Lewy bodies and glial cytoplasmic inclusions. Over the years, researchers have generated specific antibodies that alleviate the accumulation of intracellular aggregated α-syn and associated pathology in cellular and preclinical models of synucleinopathies. So far, antibodies have been the first choice as tools for research and diagnosis and currently, a wide variety of antibody fragments have been developed as an alternative to full-length antibodies for increasing its therapeutic usefulness. Recently, conformation specific antibody-based approaches have been found to be promising as therapeutic strategies, both to block α-syn aggregation and ameliorate the resultant cytotoxicity, and as diagnostic tools. In this review, we summarize different α-syn specific antibodies and provide their usefulness in tackling synucleinopathies. This article is part of the Special Issue "Synuclein".

Topics: alpha-Synuclein; Antibodies; Antibodies, Bispecific; Antibodies, Monoclonal; Antibody Specificity; Biomarkers; Delayed Diagnosis; Epitopes; Humans; Immunoglobulin Fragments; Immunologic Tests; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation; Protein Engineering; Recombinant Proteins; Single-Domain Antibodies; Synucleinopathies

The misfolding and aggregation of α-synuclein (αsyn) in the central nervous system is associated with a group of neurodegenerative disorders referred to as the synucleinopathies. In addition to being a pathological hallmark of disease, it is now well-established that upon misfolding, αsyn acquires pathogenic properties, such as neurotoxicity, that can contribute to disease development. The mechanisms that produce αsyn misfolding and the molecular events underlying the neuronal damage caused by these misfolded species are not well-defined. A consistent observation that may be relevant to αsyn's pathogenicity is its ability to associate with lipids. This appears important not only to how αsyn aggregates, but also to the mechanism by which the misfolded protein causes intracellular damage. This review discusses the current literature reporting a role of lipids in αsyn misfolding and neurotoxicity in various synucleinopathy disorders and provides an overview of current methods to assess protein misfolding and pathogenicity both

Topics: alpha-Synuclein; Central Nervous System; Fatty Acids, Unsaturated; Humans; Lipids; Protein Aggregation, Pathological; Proteostasis Deficiencies

The levels and conformers of alpha-synuclein are critical in the pathogenesis of Parkinson's Disease and related synucleinopathies. Homeostatic mechanisms in protein degradation and secretion have been identified as regulators of alpha-synuclein at different stages of its intracellular trafficking and transcellular propagation. Here we review pathways involved in the removal of various forms of alpha-synuclein from both the intracellular and extracellular environment. Proteasomes and lysosomes are likely to play complementary roles in the removal of intracellular alpha-synuclein species, in a manner that depends on alpha-synuclein post-translational modifications. Extracellular alpha-synuclein is cleared by extracellular proteolytic enzymes, or taken up by neighboring cells, especially microglia and astrocytes, and degraded within lysosomes. Exosomes, on the other hand, represent a vehicle for egress of excess burden of the intracellular protein, potentially contributing to the transfer of alpha-synuclein between cells. Dysfunction in any one of these clearance mechanisms, or a combination thereof, may be involved in the initiation or progression of Parkinson's disease, whereas targeting these pathways may offer an opportunity for therapeutic intervention. This article is part of the Special Issue "Synuclein".

Topics: alpha-Synuclein; Astrocytes; Disease Progression; Exosomes; Extracellular Fluid; Genetic Therapy; Humans; Immunotherapy; Intracellular Fluid; Lewy Bodies; Lysosomes; Microglia; Parkinson Disease; Phosphorylation; Proteasome Endopeptidase Complex; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Proteolysis; Ubiquitination

Parkinson's disease is the second most common neurodegenerative disorder worldwide. Neurodegeneration in this pathology is characterized by the loss of dopaminergic neurons in the substantia nigra, coupled with cytoplasmic inclusions known as Lewy bodies containing α-synuclein. The brain is an organ that concentrates metal ions, and there is emerging evidence that a break-down in metal homeostasis may be a critical factor in a variety of neurodegenerative diseases. α-synuclein has emerged as an important metal-binding protein in the brain, whereas these interactions play an important role in its aggregation and might represent a link between protein aggregation, oxidative damage, and neuronal cell loss. Additionally, α-synuclein undergoes several post-translational modifications that regulate its structure and physiological function, and may be linked to the aggregation and/or oligomer formation. This review is focused on the interaction of this protein with physiologically relevant metal ions, highlighting the cases where metal-AS interactions profile as key modulators for its structural, aggregation, and membrane-binding properties. The impact of α-synuclein phosphorylation and N-terminal acetylation in the metal-binding properties of the protein are also discussed, underscoring a potential interplay between PTMs and metal ion binding in regulating α-synuclein physiological functions and its role in pathology. This article is part of the Special Issue "Synuclein".

Topics: Acetylation; alpha-Synuclein; Binding Sites; Brain; Cations, Divalent; Humans; Metals; Oxidative Stress; Oxygen; Parkinson Disease; Phosphorylation; Protein Aggregation, Pathological; Protein Binding; Protein Domains; Protein Processing, Post-Translational; Structure-Activity Relationship; Sumoylation

Accumulation of alpha-synuclein protein aggregates is the hallmark neuropathologic feature of synucleinopathies such as Parkinson's disease. Rare point mutations and multiplications in SNCA, the gene encoding alpha-synuclein, as well as other genetic alterations are linked to familial Parkinson's disease cases with high penetrance and hence constitute major genetic risk factors for Parkinson's disease. However, the preponderance of cases seems sporadic, most likely based on a complex interplay between genetic predispositions, aging processes and environmental influences. Deciphering the impact of these environmental factors and their interactions with the individual genetic background in humans is challenging and often requires large cohorts, complicated study designs, and longitudinal set-ups. In contrast, rodent models offer an ideal system to study the influence of individual environmental aspects under controlled genetic background and standardized conditions. In this review, we highlight findings from studies examining effects of environmental enrichment mimicking stimulation of the brain by its physical and social surroundings as well as of environmental stressors on brain health in the context of Parkinson's disease. We discuss possible internal molecular transducers of such environmental cues in Parkinson's disease rodent models and emphasize their potential in developing novel avenues to much-needed therapies for this still incurable disease. This article is part of the Special Issue "Synuclein".

Topics: alpha-Synuclein; Animals; Brain; Diseases in Twins; Epigenesis, Genetic; Gene-Environment Interaction; Humans; Lewy Bodies; Mice; Mice, Knockout; Motor Activity; Parkinson Disease; Parkinsonian Disorders; Pesticides; Physical Stimulation; Protein Aggregation, Pathological; Risk Factors; Stress, Physiological; Stress, Psychological; Synucleinopathies

Parkinson's disease (PD) is a movement disorder, and its common characteristics include the loss of dopaminergic neurons and the accumulation of a special type of cytoplasmic inclusions called Lewy bodies in the substantia nigra pars compacta, which are more prevalent in the elderly. However, the pathophysiology of PD is still elusive. In this review, we summarized five common factors involved in PD, namely, (i) oxidative stress, (ii) mitochondrial dysfunction, (iii) inflammation, (iv) abnormal α-synuclein, and (v) endogenous neurotoxins, and proposed a hypothesis involving a damaging cycle. Oxidative stress-triggered aldehydes react with biogenic amines to produce endogenous neurotoxins. They cause mitochondrial dysfunction and the formation of inflammasomes, which induce the activation of neuroglial cells and the infiltration of T lymphocytes. The synergistic effect of these processes fosters chronic inflammation and α-synuclein aggregation and further exacerbates the impact of oxidative stress to establish a damaging cycle that eventually results in the degeneration of dopaminergic neurons. This damaging cycle provides an explanation of progressive neuronal death during the pathogenesis of PD and provides new potential targets beneficial for developing new drugs and approaches for clinical neuroprotection.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Aldehydes; alpha-Synuclein; Antiparkinson Agents; Biogenic Amines; Drug Design; Gene-Environment Interaction; Humans; Inflammasomes; MicroRNAs; Mitochondria; Models, Neurological; Mutation; Neuroglia; Oxidative Stress; Oxidopamine; Parkinson Disease; Protein Aggregation, Pathological; T-Lymphocyte Subsets

After the first research declaring the generation of human induced pluripotent stem cells (hiPSCs) in 2007, several attempts have been made to model neurodegenerative disease in vitro during the past decade. Parkinson's disease (PD) is the second most common neurodegenerative disorder, which is mainly characterized by motor dysfunction. The formation of unique and filamentous inclusion bodies called Lewy bodies (LBs) is the hallmark of both PD and dementia with LBs. The key pathology in PD is generally considered to be the alpha-synuclein (α-syn) accumulation, although it is still controversial whether this protein aggregation is a cause or consequence of neurodegeneration. In the present work, the recently published researches which recapitulated the α-syn aggregation phenomena in sporadic and familial PD hiPSC models were reviewed. Furthermore, the advantages and potentials of using patient-derived PD hiPSC with focus on α-syn aggregation have been discussed. [BMB Reports 2019; 52(6): 349-359].

Topics: alpha-Synuclein; Humans; Induced Pluripotent Stem Cells; Lewy Bodies; Neurodegenerative Diseases; Parkinson Disease; Protein Aggregation, Pathological

This review article provides an overview of the different species that α-synuclein aggregates can populate. It also attempts to reconcile conflicting views regarding the cytotoxic roles of oligomers versus fibrils. α-synuclein, while highly dynamic in the monomeric state, can access a large number of different assembly states. Depending on assembly conditions, these states can interconvert over different timescales. The fibrillar state is the most thermodynamically favored due to the many stabilizing interactions formed between each monomeric unit, but different fibrillar types form at different rates. The end distribution is likely to reflect kinetic partitioning as much as thermodynamic equilibra. In addition, metastable oligomeric species, some of which are on-pathway and others off-pathway, can be populated for remarkably long periods of time. Chemical modifications (phosphorylation, oxidation, covalent links to ligands, etc.) perturb these physical interconversions and invariably destabilize the fibrillar state, leading to small prefibrillar assemblies which can coalesce into amorphous states. Both oligomeric and fibrillar species have been shown to be cytotoxic although firm conclusions require very careful evaluation of particle concentrations and is complicated by the great variety and heterogeneity of different experimentally observed states. The mechanistic relationship between oligomers and fibrils remains to be clarified, both in terms of assembly of oligomers into fibrils and potential dissolution of fibrils into oligomers. While oligomers are possibly implicated in the collapse of neuronal homeostasis, the fibrillar state(s) appears to be the most efficient at propagating itself both in vitro and in vivo, pointing to critical roles for multiple different aggregate species in the progression of Parkinson's disease (https://onlinelibrary.wiley.com/page/journal/14714159/homepage/virtual_issues.htm). This article is part of the Special Issue "Synuclein".

Topics: alpha-Synuclein; Amyloid; Humans; Kinetics; Lewy Bodies; Lipid Peroxidation; Metals; Models, Molecular; Mutation, Missense; Point Mutation; Protein Aggregation, Pathological; Protein Conformation; Protein Folding; Protein Multimerization; Protein Processing, Post-Translational; Solubility; Structure-Activity Relationship; Synucleinopathies; Thermodynamics

α-Synuclein (αS) is the major component of the filamentous inclusions that constitute the defining characteristic of neurodegenerative synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. αS is deposited in a hyperphosphorylated and ubiquitinated form with a β-sheet-rich fibrillar structure in diseased brains. In 2008, some researchers reported that embryonic neurons transplanted into Parkinson's disease brains had Lewy body-like pathologies, suggesting that pathological αS propagates from diseased neurons to young neurons. Subsequently, a growing body of evidence supported the cell-to-cell spread of αS pathologies. Recent studies have revealed that intracerebral injection of insoluble αS into wild-type mice can induce prion-like propagation of phosphorylated αS pathology even 1 month after injection, while injection into αS-knockout mice failed to induce any pathology. We also showed that intracerebral injection of insoluble αS into adult common marmoset brains results in the spreading of abundant αS pathology. These in vivo experiments clearly indicate that insoluble αS has prion-like properties and that it propagates through neural networks. The underlying mechanisms of αS propagation are still poorly understood, but αS propagation model animals could be helpful in elucidating the pathogenetic mechanisms and developing drugs for synucleinopathies.

Topics: alpha-Synuclein; Animals; Brain; Callithrix; Humans; Lewy Body Disease; Mice; Neurodegenerative Diseases; Parkinson Disease; Phosphorylation; Prions; Protein Aggregation, Pathological

Topics: alpha-Synuclein; Brain; Humans; In Vitro Techniques; Lewy Body Disease; Prions; Protein Aggregation, Pathological; Protein Folding

Misfolding and aggregation of alpha-synuclein (α-synuclein) with concomitant cytotoxicity is a hallmark of Lewy body related disorders such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Although it plays a pivotal role in pathogenesis and disease progression, the function of α-synuclein and the molecular mechanisms underlying α-synuclein-induced neurotoxicity in these diseases are still elusive. Many in vitro and in vivo experimental models mimicking α-synuclein pathology such as oligomerization, toxicity and more recently neuronal propagation have been generated over the years. In particular, cellular models have been crucial for our comprehension of the pathogenic process of the disease and are beneficial for screening of molecules capable of modulating α-synuclein toxicity. Here, we review α-synuclein based cell culture models that reproduce some features of the neuronal populations affected in patients, from basic unicellular organisms to mammalian cell lines and primary neurons, to the cutting edge models of patient-specific cell lines. These reprogrammed cells known as induced pluripotent stem cells (iPSCs) have garnered attention because they closely reproduce the characteristics of neurons found in patients and provide a valuable tool for mechanistic studies. We also discuss how different cell models may constitute powerful tools for high-throughput screening of molecules capable of modulating α-synuclein toxicity and prevention of its propagation. This article is part of the Special Issue "Synuclein".

Topics: alpha-Synuclein; Cell Culture Techniques; Cells, Cultured; Cellular Reprogramming; Dopamine; Drug Evaluation, Preclinical; HEK293 Cells; Humans; In Vitro Techniques; Induced Pluripotent Stem Cells; Lewy Bodies; Models, Neurological; Neurodegenerative Diseases; Neurons; Protein Aggregation, Pathological; Protein Folding; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Synucleinopathies

α-Synuclein (αS) and tau have a lot in common. Dyshomeostasis and aggregation of both proteins are central in the pathogenesis of neurodegenerative diseases: Parkinson's disease, dementia with Lewy bodies, multi-system atrophy and other 'synucleinopathies' in the case of αS; Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy and other 'tauopathies' in the case of tau. The aggregated states of αS and tau are found to be (hyper)phosphorylated, but the relevance of the phosphorylation in health or disease is not well understood. Both tau and αS are typically characterized as 'intrinsically disordered' proteins, while both engage in transient interactions with cellular components, thereby undergoing structural changes and context-specific folding. αS transiently binds to (synaptic) vesicles forming a membrane-induced amphipathic helix; tau transiently interacts with microtubules forming an 'extended structure'. The regulation and exact nature of the interactions are not fully understood. Here we review recent and previous insights into the dynamic, transient nature of αS and tau with regard to the mode of interaction with their targets, the dwell-time while bound, and the cis and trans factors underlying the frequent switching between bound and unbound states. These aspects are intimately linked to hypotheses on how subtle changes in the transient behaviors may trigger the earliest steps in the pathogenesis of the respective brain diseases. Based on a deeper understanding of transient αS and tau conformations in the cellular context, new therapeutic strategies may emerge, and it may become clearer why existing approaches have failed or how they could be optimized.

Topics: alpha-Synuclein; Animals; Brain; Humans; Neurodegenerative Diseases; Protein Aggregation, Pathological; Protein Folding; tau Proteins

The deposition of misfolded β-sheet enriched amyloid protein is a shared feature of many neurodegenerative diseases. Recent studies demonstrated the existence of conformationally diverse strains as a common property for multiple amyloidogenic proteins including α-Synuclein (α-Syn). α-Syn is misfolded and aggregated in a group of neurodegenerative diseases collectively known as α-Synucleinopathies, which include Parkinson's disease (PD), dementia with Lewy body, multiple system atrophy and also a subset of Alzheimer's disease patients with concomitant PD-like Lewy bodies and neurites. While sharing the same pathological protein, different α-Synucleinopathies demonstrate distinct clinical and pathological phenotypes, which could result from the existence of diverse pathological α-Syn strains in patients. In this review, we summarized the characteristics of different α-Synucleinopathies and α-Syn strains generated with recombinant α-Syn monomers. We also make predictions of α-Syn strains that could potentially exist in patients based on the knowledge from other amyloid proteins and the clinical and pathological features of different α-Synucleinopathies.

Topics: alpha-Synuclein; Animals; Humans; Neurodegenerative Diseases; Protein Aggregation, Pathological; Protein Conformation

The formation of protein aggregates and inclusions in the brain and spinal cord is a common neuropathological feature of a number of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and many others. These are commonly referred as neurodegenerative proteinopathies or protein-misfolding diseases. The main characteristic of protein aggregates in these disorders is the fact that they are enriched in amyloid fibrils. Since protein aggregation is considered to play a central role for the onset of neurodegenerative proteinopathies, research is ongoing to develop strategies aimed at preventing or removing protein aggregation in the brain of affected patients. Numerous studies have shown that small molecule-based approaches may be potentially the most promising for halting protein aggregation in neurodegenerative diseases. Indeed, several of these compounds have been found to interact with intrinsically disordered proteins and promote their clearing in experimental models. This notwithstanding, at present small molecule inhibitors still awaits achievements for clinical translation. Hopefully, if we determine whether the formation of insoluble inclusions is effectively neurotoxic and find a valid biomarker to assess their protein aggregation-inhibitory activity in the human central nervous system, the use of small molecule inhibitors will be considered as a cure for neurodegenerative protein-misfolding diseases.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Humans; Neurodegenerative Diseases; Prion Proteins; Protein Aggregation, Pathological; Superoxide Dismutase-1

Amyloid-β and α-synuclein are intrinsically disordered proteins (IDPs), which are at the center of Alzheimer's and Parkinson's disease pathologies, respectively. These IDPs are extremely flexible and do not adopt stable structures. Furthermore, both amyloid-β and α-synuclein can form toxic oligomers, amyloid fibrils and other type of aggregates in Alzheimer's and Parkinson's diseases. Experimentalists face challenges in investigating the structures and thermodynamic properties of these IDPs in their monomeric and oligomeric forms due to the rapid conformational changes, fast aggregation processes and strong solvent effects. Classical molecular dynamics simulations complement experiments and provide structural information at the atomic level with dynamics without facing the same experimental limitations. Artificial missense mutations are employed experimentally and computationally for providing insights into the structure-function relationships of amyloid-β and α-synuclein in relation to the pathologies of Alzheimer's and Parkinson's diseases. Furthermore, there are several natural genetic variations that play a role in the pathogenesis of familial cases of Alzheimer's and Parkinson's diseases, which are related to specific genetic defects inherited in dominant or recessive patterns. The present review summarizes the current understanding of monomeric and oligomeric forms of amyloid-β and α-synuclein, as well as the impacts of artificial and pathological missense mutations on the structural ensembles of these IDPs using molecular dynamics simulations. We also emphasize the recent investigations on residual secondary structure formation in dynamic conformational ensembles of amyloid-β and α-synuclein, such as β-structure linked to the oligomerization and fibrillation mechanisms related to the pathologies of Alzheimer's and Parkinson's diseases. This information represents an important foundation for the successful and efficient drug design studies.

Topics: Alleles; alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Intrinsically Disordered Proteins; Molecular Dynamics Simulation; Mutation, Missense; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Protein Multimerization; Structure-Activity Relationship

Parkinson's disease (PD) is a chronic progressive neurodegenerative disease, which is characterized by severe loss of dopaminergic neurons and formation of Lewy bodies, which are rich in aggregated alpha-synuclein (α-syn). Two decades of intensive research have compiled a massive body of evidence that aggregation of α-syn is a critical process in PD and other synucleinopathies. The dissemination of Lewy body pathology throughout the central nervous system strongly suggests a cell-to-cell transmission of α-syn. Although in vitro and in vivo evidence has convincingly demonstrated that aggregation-prone α-syn can spread from cell to cell, the exact mechanisms and the role for the disease pathology remain elusive. Except for cases of direct contact, the transmission of α-syn from cell to cell requires that α-syn is released to the extracellular space and taken up by recipient cells. Furthermore, internalized α-syn needs to gain access to the cytoplasm and/or target organelles of the recipient cell. Here, we review the current state of knowledge about release and uptake of α-syn and discuss the key questions that remain unanswered.

Topics: alpha-Synuclein; Animals; Extracellular Space; Humans; Models, Biological; Protein Aggregation, Pathological

Parkinson's disease (PD) is an adult onset neurodegenerative disease that is characterized by selective degeneration of neurons primarily in the substantia nigra. At present, the pathogenesis of PD is incompletely understood and there are no neuroprotective treatments available. Accurate animal models of PD provide the opportunity to elucidate disease mechanisms and identify therapeutic targets. This review focuses on C. elegans models of PD, including both genetic and toxicant models. This microscopic worm offers several advantages for the study of PD including ease of genetic manipulation, ability to complete experiments rapidly, low cost, and ability to perform large scale screens for disease modifiers. A number of C. elegans models of PD have been generated including transgenic worms that express α-synuclein or LRRK2, and worms with deletions in PRKN/pdr-1, PINK1/pink-1, DJ-1/djr-1.1/djr-1.2 and ATP13A2/catp-6. These worms have been shown to exhibit multiple phenotypic deficits including the loss of dopamine neurons, disruption of dopamine-dependent behaviors, increased sensitivity to stress, age-dependent aggregation, and deficits in movement. As a result, these phenotypes can be used as outcome measures to gain insight into disease pathogenesis and to identify disease modifiers. In this way, C. elegans can be used as an experimental tool to elucidate mechanisms involved in PD and to find novel therapeutic targets that can subsequently be validated in other models.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Antiparkinson Agents; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; Dopaminergic Neurons; Drug Evaluation, Preclinical; Feeding Behavior; Gene-Environment Interaction; Genes, Reporter; Humans; Mitochondria; Movement Disorders; Nerve Degeneration; Neurotoxins; Parkinsonian Disorders; Phenotype; Protein Aggregation, Pathological; Recombinant Fusion Proteins; RNA Interference; Species Specificity

Over 100 years ago, Lewy bodies and Lewy neurites were defined as a pathologic hallmark of Parkinson disease. Eighty years later, α-synuclein was found to be the primary component of these inclusions. Emerging evidence suggests that α-synuclein pathology propagates across interconnected networks throughout the nervous system in a prion-like manner. Pathologic α-synuclein seeds aggregation of native α-synuclein, resulting in the formation of insoluble inclusions. These seeds can propagate within the neuron and to interconnected neurons, resulting in the spread of pathology throughout the brain. Here, we discuss how the findings that α-synuclein pathology spreads throughout the nervous system has revolutionized our understanding about Parkinson disease pathogenesis and resulted in the development of novel therapeutic strategies to halt disease progression.

Topics: alpha-Synuclein; Animals; Brain; Disease Progression; Humans; Parkinson Disease; Prions; Protein Aggregation, Pathological; Proteostasis Deficiencies

Synucleinopathies are a group of neurodegenerative diseases that share a common pathological lesion of intracellular protein inclusions largely composed of aggregates of alpha-synuclein protein. Accumulating evidence, including genome-wide association studies, has implicated the alpha-synuclein (SNCA) gene in the etiology of synucleinopathies and it has been suggested that SNCA expression levels are critical for the development of these diseases. This review focuses on genetic variants from the class of structural variants (SVs), including multiplication of large genomic segments and short (<50bp) genomic variants such as simple sequence repeats (SSRs), within the SNCA locus. We provide evidence that SNCA-SVs play a key role in the pathogenesis of synucleinopathies via their effects on gene expression and on regulatory mechanisms including transcription and splicing.

Topics: alpha-Synuclein; Genomic Structural Variation; Humans; Microsatellite Repeats; Neurodegenerative Diseases; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation

α-synuclein is a small protein abundantly expressed in the brain and mainly located in synaptic terminals. The conversion of α-synuclein into oligomers and fibrils is the hallmark of a range of neurodegenerative disorders including Parkinson's disease and dementia with Lewy bodies. α-synuclein is disordered in solution but can adopt an α-helical conformation upon binding to lipid membranes. This lipid-protein interaction plays an important role in its proposed biological function, i.e., synaptic plasticity, but can also entail the aggregation of the protein. Both the chemical properties of the lipids and the lipid-to-protein-ratio have been reported to modulate the aggregation propensity of α-synuclein. In this review, the influence of changes in the nature and levels of lipids on the aggregation propensity of α-synuclein in vivo and in vitro will be discussed within a common general framework. In particular, while biophysical measurements and kinetic analyses of the time courses of α-synuclein aggregation in the presence of different types of lipid vesicles allow a mechanistic dissection of the influence of the lipids on α-synuclein aggregation, biological studies of cellular and animal models of Parkinson's disease allow the determination of changes in lipid levels and properties associated with the disease.

Topics: alpha-Synuclein; Animals; Humans; Membrane Lipids; Parkinson Disease; Protein Aggregation, Pathological

PD is a common and a debilitating degenerative movement disorder. The number of patients is increasing worldwide and as yet there is no cure for the disease. The majority of existing treatments target motor symptom control. Over the last two decades the impact of the genetic contribution to PD has been appreciated. Significant discoveries have been made, which have advanced our understanding of the pathophysiological and molecular basis of PD. In this chapter we outline current knowledge of the clinical aspects of PD and the basic mechanistic understanding.

Topics: alpha-Synuclein; Autonomic Nervous System Diseases; Brain; Dementia; Glucosylceramidase; Humans; Hypokinesia; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Lewy Bodies; Mitochondria; Muscle Rigidity; Olfaction Disorders; Oxidative Stress; Parkinson Disease; Postural Balance; Protein Aggregation, Pathological; Protein Deglycase DJ-1; Sleep Wake Disorders; Tremor; Ubiquitin-Protein Ligases

Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Amyotrophic Lateral Sclerosis; Animals; Cell Membrane; Extracellular Space; Humans; Huntingtin Protein; Huntington Disease; Neurodegenerative Diseases; Parkinson Disease; Prions; Protein Aggregation, Pathological; Protein Folding; Protein Transport; Superoxide Dismutase-1; tau Proteins

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving the formation of cytoplasmic aggregates by proteins including TDP-43 and SOD1, in affected cells in the central nervous system (CNS). Pathology spreads from an initial site of onset to contiguous anatomical regions. There is evidence that for disease-associated proteins, including TDP-43 and SOD1, non-native protein conformers can promote misfolding of the natively folded counterparts, and cell-to-cell transfer of pathological aggregates may underlie the spread of the disease throughout the CNS. A variety of studies have demonstrated that SOD1 is released by neuron-like cells into the surrounding culture medium, either in their free state or encapsulated in extracellular vesicles such as exosomes. Extracellular SOD1 can then be internalised by naïve cells incubated in this conditioned medium, leading to the misfolding and aggregation of endogenous intracellular SOD1; an effect that propagates over serial passages. A similar phenomenon has also been observed with other proteins associated with protein misfolding and progressive neurological disorders, including tau, α-synuclein and both mammalian and yeast prions. Conditioned media experiments using TDP-43 have been less conclusive, with evidence for this protein undergoing intercellular transfer being less straightforward. In this review, we describe the properties of TDP-43 and SOD1 and look at the evidence for their respective abilities to participate in cell-to-cell transfer via conditioned medium, and discuss how variations in the nature of cell-to-cell transfer suggests that a number of different mechanisms are involved in the spreading of pathology in ALS.

Topics: alpha-Synuclein; Amyotrophic Lateral Sclerosis; Animals; Cell Communication; DNA-Binding Proteins; Humans; Protein Aggregation, Pathological; Proteostasis Deficiencies; Superoxide Dismutase-1

Alpha-synuclein is a protein implicated in Parkinson's disease and thought to be one of the main pathological drivers in the disease, although it remains unclear how this protein elicits its neurotoxic effects. Recent findings indicate that the assembly of toxic oligomeric species of alpha-synuclein may be one of the key processes for the pathology and spread of the disease. The absence of a sensitive in situ detection method has hindered the study of these oligomeric species and the role they play in the human brain until recently. In this review, we assess the evidence for the toxicity and prion-like activity of oligomeric forms of alpha-synuclein and discuss the advances in our understanding of the role of alpha-synuclein in Parkinson's disease that may be brought about by the specific and sensitive detection of distinct oligomeric species in post-mortem patient brain. Finally, we discuss current approaches being taken to therapeutically target alpha-synuclein oligomers and their implications.

Topics: alpha-Synuclein; Animals; Antiparkinson Agents; Biomarkers; Humans; Parkinson Disease; Protein Aggregation, Pathological

Among the diseases affecting the central nervous system (CNS), neurodegenerations attract the interest of both the clinician and the medicinal chemist. The increasing average age of population, the growing number of patients, and the lack of long-term effective remedies push ahead the quest for novel tools against this class of pathologies. We present a review on the state of the art of the molecules (or combination of molecules) of natural origin that are currently under study against two well-defined pathologies: Parkinson's disease (PD) and Huntington's disease (HD). Nowadays, very few tools are available for preventing or counteracting the progression of such diseases. Two major parameters were considered for the preparation of this review: particular attention was reserved to these research works presenting well-defined molecular mechanisms for the studied compounds, and where available, papers reporting in vivo data were preferred. A literature search for peer-reviewed articles using PubMed, Scopus, and Reaxys databases was performed, exploiting different keywords and logical operators: 91 papers were considered (preferentially published after 2015). The review presents a brief overview on the etiology of the studied neurodegenerations and the current treatments, followed by a detailed discussion of the natural and semisynthetic compounds dividing them in different paragraphs considering their several mechanisms of action.

Topics: alpha-Synuclein; Animals; Anti-Dyskinesia Agents; Antioxidants; Antiparkinson Agents; Autophagy; Biological Products; Dementia; Dopamine; Drug Discovery; Drug Evaluation, Preclinical; Humans; Huntington Disease; Microglia; Mitochondria; Molecular Targeted Therapy; Monoamine Oxidase Inhibitors; Oxidative Stress; Parkinson Disease; Plant Preparations; Protein Aggregation, Pathological; Signal Transduction

Alzheimer's and Parkinson's diseases are the most prevalent neurodegenerative diseases that generate important health-related direct and indirect socio-economic costs. They are characterized by severe neuronal losses in several disease-specific brain regions associated with deposits of aggregated proteins. In Alzheimer's disease, β-amyloid peptide-containing plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein tau are the two main neuropathological lesions, while Parkinson's disease is defined by the presence of Lewy Bodies that are intraneuronal proteinaceous cytoplasmic inclusions. α-Synuclein has been identified as a major protein component of Lewy Bodies and heavily implicated in the pathogenesis of Parkinson's disease. In the past few years, evidence has emerged to explain how these aggregate-prone proteins can undergo spontaneous self-aggregation, propagate from cell to cell, and mediate neurotoxicity. Current research now indicates that oligomeric forms are probably the toxic species. This article discusses recent progress in the understanding of the pathogenesis of these diseases, with a focus on the underlying mechanisms of protein aggregation, and emphasizes the pathophysiological molecular mechanisms leading to cellular toxicity. Finally, we present the putative direct link between β-amyloid peptide and tau in causing toxicity in Alzheimer's disease as well as α-synuclein in Parkinson's disease, along with some of the most promising therapeutic strategies currently in development for those incurable neurodegenerative disorders.

Topics: alpha-Synuclein; Alzheimer Disease; Animals; Humans; Nerve Degeneration; Neurodegenerative Diseases; Parkinson Disease; Protein Aggregation, Pathological; Tauopathies

α-Synuclein (α-Syn) aggregation is directly associated with Parkinson's disease (PD) pathogenesis. In vitro aggregation and in vivo animal model studies of α-Syn recapitulate many features of the disease pathogenesis. Six familial PD associated mutations of α-Syn have been discovered; many of which are associated with early onset PD. Three of PD associated mutations have been shown to accelerate the α-Syn aggregation, whereas other three are shown to delay the aggregation kinetics. The membrane binding studies also suggest that few of these PD mutants strongly bind to synthetic membrane vesicles, while others are shown to have attenuated membrane binding ability. Furthermore, the PD mutations do not drastically alter the toxicity of α-Syn oligomers/fibrils. Although according to recent suggestions that early formed oligomers are the most potent toxic species responsible for PD, only p.A30P mutant is shown to form faster oligomers and delayed conversion from oligomers to fibrils. Therefore, it is difficult to establish a unifying mechanism of how familial PD associated mutations affect the α-Syn structure, aggregation and function for their disease association. It is possible that each PD associated mutation alters α-Syn biology in a unique way, which might be responsible for disease pathogenesis. In this review, we discuss the structure function of α- Syn and how these are altered due to the PD associated mutations and their relationship to disease pathogenesis.

Topics: alpha-Synuclein; Amino Acid Sequence; Animals; Cell Membrane; Gene Expression; Humans; Mutation; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Structure-Activity Relationship

Protein misfolding under stressful environmental conditions cause several cellular problems owing to the disturbed cellular protein homeostasis, which may further lead to neurological disorders like Parkinson's disease (PD), Alzheimer's disease (AD), Amyloid lateral sclerosis and Huntington disease (HD). The presence of cellular defense mechanisms like molecular chaperones and proteasomal degradation systems prevent protein misfolding and aggregation. Molecular chaperones plays primary role in preventing protein misfolding by mediating proper native folding, unfolding and refolding of the polypeptides along with vast number of cellular functions. In past few years, the understanding of molecular chaperone mechanisms has been expanded enormously although implementation to prevent protein aggregation diseases is still deficient. We in this review evaluated major classes of molecular chaperones and their mechanisms relevant for preventing protein aggregation, specific case of α-synuclein aggregation. We also evaluate the molecular chaperone function as a novel therapeutic approach and the chaperone inhibitors or activators as small molecular drug targets.

Topics: alpha-Synuclein; Animals; Heat-Shock Proteins; Humans; Parkinson Disease; Protein Aggregation, Pathological; Protein Folding; Proteostasis Deficiencies

The abnormal aggregation of a small number of known proteins underlies the most common human neurodegenerative diseases. In tauopathies and synucleinopathies, the normally soluble intracellular proteins tau and α-synuclein become insoluble and filamentous. In recent years, non-cell autonomous mechanisms of aggregate formation have come to the fore, suggesting that nucleation-dependent aggregation may occur in a localized fashion in human tauopathies and synucleinopathies, followed by seed-dependent propagation. There is a long prodromal phase between the formation of protein aggregates and the appearance of the first clinical symptoms, which manifest only after extensive propagation, opening novel therapeutic avenues.

Topics: alpha-Synuclein; Animals; Humans; Neurodegenerative Diseases; Prion Proteins; Protein Aggregation, Pathological; tau Proteins

Parkinson's disease (PD), dementia with Lewy Bodies (DLB), and multiple system atrophy (MSA) are three major synucleinopathies characterized by α-synuclein-containing inclusions in the brains of patients. Because the cell types and brain structures that are affected vary markedly between the disorders, the patients have different clinical manifestations in addition to some overlapping symptoms, which are the basis for differential diagnosis. Cognitive impairment and depression associated with hippocampal dysfunction are frequently observed in these disorders. While various α-synuclein-containing inclusions are found in the hippocampal formation, increasing evidence supports that small α-synuclein aggregates or oligomers may be the real culprit, causing deficits in neurotransmission and neurogenesis in the hippocampus and related brain regions, which constitute the major mechanism for the hippocampal dysfunctions and associated neuropsychiatric manifestations in synucleinopathies.

Topics: alpha-Synuclein; Animals; Hippocampus; Humans; Inclusion Bodies; Lewy Body Disease; Multiple System Atrophy; Neurogenesis; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Synaptic Transmission

Alterations in α-synuclein dosage lead to familial Parkinson's disease (PD), and its accumulation results in synucleinopathies that include PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Furthermore, α-synuclein contributes to the fibrilization of amyloid-b and tau, two key proteins in Alzheimer's disease, which suggests a central role for α-synuclein toxicity in neurodegeneration. Recent studies of factors contributing to α-synuclein toxicity and its disruption of downstream cellular pathways have expanded our understanding of disease pathogenesis in synucleinopathies. In this Review, we discuss these emerging themes, including the contributions of aging, selective vulnerability and non-cell-autonomous factors such as α-synuclein cell-to-cell propagation and neuroinflammation. Finally, we summarize recent efforts toward the development of targeted therapies for PD and related synucleinopathies.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Autophagy; Axonal Transport; Endoplasmic Reticulum; Golgi Apparatus; Humans; Lewy Body Disease; Lysosomes; Mitochondria; Molecular Targeted Therapy; Multiple System Atrophy; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Synapses; tau Proteins

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Topics: alpha-Synuclein; GTP Phosphohydrolases; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Models, Genetic; Mutation; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological; tau Proteins

Intracellular aggregates of the α-synuclein protein result in cell loss and dysfunction in Parkinson's disease and atypical Parkinsonism, such as multiple system atrophy and dementia with Lewy bodies. Each of these neurodegenerative conditions, known collectively as α-synucleinopathies, may be characterized by a different suite of molecular triggers that initiate pathogenesis. The mechanisms whereby α-synuclein aggregates mediate cytotoxicity also remain to be fully elucidated. However, recent studies have implicated the cell-to-cell spread of α-synuclein as the major mode of disease propagation between brain regions during disease progression. Here, we review the current evidence for different modes of α-synuclein cellular release, movement and uptake, including exocytosis, exosomes, tunneling nanotubes, glymphatic flow and endocytosis. A more detailed understanding of the major modes by which α-synuclein pathology spreads throughout the brain may provide new targets for therapies that halt the progression of disease.

Topics: alpha-Synuclein; Animals; Astrocytes; Cell Communication; Exosomes; Extracellular Space; Humans; Intracellular Space; Lewy Bodies; Lewy Body Disease; Microglia; Models, Biological; Multiple System Atrophy; Parkinson Disease; Protein Aggregation, Pathological; Protein Binding; Protein Transport

Synucleinopathies are a group of neurodegenerative diseases that share a common pathological lesion of intracellular protein inclusions largely composed by aggregates of alpha-synuclein protein. Accumulating evidence, including genome wide association studies, has implicated alpha-synuclein (SNCA) gene in the etiology of synucleinopathies. However, the precise variants within SNCA gene that contribute to the sporadic forms of Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and other synucleinopathies and their molecular mechanisms of action remain elusive. It has been suggested that SNCA expression levels are critical for the development of these diseases. Here, we review several model systems that have been developed to advance the understanding of the role of SNCA expression levels in the etiology of synucleinopathies. We also describe different molecular mechanisms that regulate SNCA gene expression and discuss possible strategies for SNCA down-regulation as means for therapeutic approaches. Finally, we highlight some examples that underscore the relationships between the genetic association findings and the regulatory mechanisms of SNCA expression, which suggest that genetic variability in SNCA locus is directly responsible, at least in part, to the changes in gene expression and explain the reported associations of SNCA with synucleinopathies. Future studies utilizing induced pluripotent stem cells (iPSCs)-derived neuronal lines and genome editing by CRISPR/Cas9, will allow us to validate, characterize, and manipulate the effects of particular cis-genetic variants on SNCA expression. Moreover, this model system will enable us to compare different neuronal and glial lineages involved in synucleinopathies representing an attractive strategy to elucidate-common and specific-SNCA-genetic variants, regulatory mechanisms, and vulnerable expression levels underlying synucleinopathy spectrum disorders. This forthcoming knowledge will support the development of precision medicine for synucleinopathies.

Topics: alpha-Synuclein; Animals; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation; Humans; Lewy Body Disease; Multiple System Atrophy; Parkinson Disease; Protein Aggregation, Pathological; Up-Regulation

There has been an explosion in the number of papers discussing the hypothesis of 'pathogenic spread' in neurodegenerative disease - the idea that abnormal forms of disease-associated proteins, such as tau or α-synuclein, physically move from neuron to neuron to induce disease progression. However, whether inter-neuronal spread of protein aggregates actually occurs in humans and, if so, whether it causes symptom onset remain uncertain. Even if pathogenic spread is proven in humans, it is unclear how much this would alter the specific therapeutic approaches that are in development. A critical appraisal of this increasingly popular hypothesis thus seems both important and timely.

Topics: alpha-Synuclein; Animals; Humans; Neurodegenerative Diseases; Neurons; Protein Aggregation, Pathological; Protein Transport; tau Proteins

Misfolded protein aggregates are the hallmark of several neurodegenerative diseases in humans. The main protein constituent of these aggregates and the regions within the brain that are affected differ from one neurodegenerative disorder to another. A plethora of reports suggest that distinct diseases have in common the ability of protein aggregates to spread and amplify within the central nervous system. This review summarizes briefly what is known about the nature of the protein aggregates that are infectious and the reason they are toxic to cells. The chameleon property of polypeptides which aggregation into distinct high-molecular weight assemblies is associated to different diseases, in particular, that of alpha-synuclein which aggregation is the hallmark of distinct synucleinopathies, is discussed. Finally, strategies targeting the formation and propagation of structurally distinct alpha-synuclein assemblies associated to different synucleinopathies are presented and their therapeutic and diagnostic potential is discussed.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Brain; Humans; Neurodegenerative Diseases; Protein Aggregation, Pathological; Proteostasis Deficiencies; tau Proteins

In a number of neurological diseases including Parkinson's disease (PD), α-synuclein is aberrantly folded, forming abnormal oligomers, and amyloid fibrils within nerve cells. Strong evidence exists for the toxicity of increased production and aggregation of α-synuclein in vivo. The toxicity of α-synuclein is popularly attributed to the formation of "toxic oligomers": a heterogenous and poorly characterized group of conformers that may share common molecular features. This review presents the available evidence on the properties of α-synuclein oligomers and the potential molecular mechanisms of their cellular disruption. Toxic α-synuclein oligomers may impact cells in a number of ways, including the disruption of membranes, mitochondrial depolarization, cytoskeleton changes, impairment of protein clearance pathways, and enhanced oxidative stress. We also examine the relationship between α-synuclein toxic oligomers and amyloid fibrils, in the light of recent studies that paint a more complex picture of α-synuclein toxicity. Finally, methods of studying and manipulating oligomers within cells are described.

Topics: alpha-Synuclein; Amyloid; Animals; Humans; Mutation; Neurodegenerative Diseases; Protein Aggregation, Pathological; Protein Multimerization

Parkinson's disease (PD) is a very common neurodegenerative disorder characterized by the accumulation of α-synuclein (α-syn) into Lewy body (LB) inclusions and the loss of neuronmelanin (NM) containing dopamine (DA) neurons in the substantia nigra (SN). Pathological α-syn and NM are two prominent hallmarks in this selective and progressive neurodegenerative disease. Pathological α-syn can induce dopaminergic neuron death by various mechanisms, such as inducing oxidative stress and inhibiting protein degradation systems. Therefore, to explore the factors that trigger α-syn to convert from a non-toxic protein to toxic one is a pivotal question to clarify the mechanisms of PD pathogenesis. Many triggers for pathological α-syn aggregation have been identified, including missense mutations in the α-syn gene, higher concentration, and posttranslational modifications of α-Syn. Recently, the role of NM in inducing α-syn expression and aggregation has been suggested as a mechanism for this pigment to modulate neuronal vulnerability in PD. NM may be responsible for PD and age-associated increase and aggregation in α-syn. Here, we reviewed our previous study and other recent findings in the area of interaction between NM and α-syn.

Topics: alpha-Synuclein; Animals; Humans; Melanins; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease associated with selective loss of dopaminergic neurons. The characteristic hallmark of the disease is intracytoplasmic proteinacious inclusion bodies called Lewy bodies, primarily consisting of a presynaptic protein α-synuclein. Oxidative stress-mediated damage to macromolecules have been shown to occur frequently in PD. Oxidative damage to DNA in the form of oxidized guanine (8-oxodG) accumulates in both the mitochondrial and nuclear DNA of dopaminergic neurons of the substantia nigra in PD. 8-oxodG-mediated transcriptional mutagenesis has been shown to have the potential to alter phenotype of cells through production of mutant pool of proteins. This review comprehensively summarizes the role of oxidative stress-mediated damage incurred during neurodegeneration, and highlights the scope of transcriptional mutagenesis event in leading to α-synuclein aggregation as seen in PD.

Topics: 8-Hydroxy-2'-Deoxyguanosine; alpha-Synuclein; Amino Acid Sequence; Animals; Deoxyguanosine; Humans; Molecular Sequence Data; Mutagenesis; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Substantia Nigra; Transcription, Genetic

Currently, several α-synuclein immunotherapies are being tested in experimental Parkinson's disease models and in clinical trials. Recent research has revealed that α-synuclein is not just an intracellular synaptic protein but also exists extracellularly. Moreover, the transfer of misfolded α-synuclein between cells might be a crucial step in the process leading to a progressive increase in deposition of α-synuclein aggregates throughout the Parkinson's disease brain. The revelation that α-synuclein is present outside cells has increased the interest in antibody-based therapies and opens up for the notion that microglia might play a key role in retarding Parkinson's disease progression. The objectives of this review are to describe and contrast the use of active and passive immunotherapy in treating α-synucleinopathies and highlight the likely important role of microglia in clearing misfolded α-synuclein from the extracellular space.

Topics: alpha-Synuclein; Animals; Antibodies; Blood-Brain Barrier; Brain; Clinical Trials as Topic; Encephalitis; Humans; Immunotherapy; Microglia; Neurons; Parkinson Disease; Protein Aggregation, Pathological

Despite decades of research, therapy for diseases caused by abnormal protein folding and aggregation (amyloidoses) is limited to treatment of symptoms and provides only temporary and moderate relief to sufferers. The failure in developing successful disease-modifying drugs for amyloidoses stems from the nature of the targets for such drugs - primarily oligomers of amyloidogenic proteins, which are distinct from traditional targets, such as enzymes or receptors. The oligomers are metastable, do not have well-defined structures, and exist in dynamically changing mixtures. Therefore, inhibiting the formation and toxicity of these oligomers likely will require out-of-the-box thinking and novel strategies. We review here the development of a strategy based on targeting the combination of hydrophobic and electrostatic interactions that are key to the assembly and toxicity of amyloidogenic proteins using lysine (K)-specific "molecular tweezers" (MTs). Our discussion includes a survey of the literature demonstrating the important role of K residues in the assembly and toxicity of amyloidogenic proteins and the development of a lead MT derivative called CLR01, from an inhibitor of protein aggregation in vitro to a drug candidate showing effective amelioration of disease symptoms in animal models of Alzheimer's and Parkinson's diseases.

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloidogenic Proteins; Animals; Bridged-Ring Compounds; Clinical Trials as Topic; Humans; Hydrophobic and Hydrophilic Interactions; Models, Animal; Molecular Sequence Data; Organophosphates; Protein Aggregation, Pathological; Protein Multimerization; Static Electricity; Synapses

Although α-synuclein protein (αS) aggregates from a monomer to assemblies such as oligomer, protofibril and mature fibril, the early intermediate aggregate, that is, oligomer has been considered to be most toxic species in recent reports. While it was reported that αS concentration in cerebrospinal fluid was decreased significantly in the patients with Parkinson's disease (PD) and dementia with Lewy bodies, there were reports that αS oligomer concentration was elevated in cerebrospinal fluid of PD patients. Moreover, it was supposed that αS oligomer concentration was also elevated in blood of PD patients. Further studies of αS in cerebrospinal fluid and blood would lead to establishment of the significance of αS as a biomarker for α-synucleinopathies including PD.

Topics: alpha-Synuclein; Biomarkers; Early Diagnosis; Humans; Lewy Body Disease; Parkinsonian Disorders; Protein Aggregates; Protein Aggregation, Pathological

Amyloid aggregation of α-synuclein (α-syn) in Lewy bodies (LBs) is the pathological hallmark of Parkinson's disease (PD). Iron, especially Fe

Topics: alpha-Synuclein; Amyloid; Cryoelectron Microscopy; Humans; Iron; Mutation; Parkinson Disease; Protein Aggregation, Pathological

Mutations in the SNCA gene, which encodes the protein α-synuclein, have been linked with early onset Parkinson's disease. The exact nature of this association, however, is still poorly understood. To investigate this problem, we started from the observation that α-synuclein is constitutively N-terminally acetylated, a post-translational modification that alters the charge and structure of α-synuclein molecules and affects their interaction with lipid membranes, as well as their aggregation process. We thus studied five N-terminal acetylated familial variants (A30P, E46K, H50Q, G51D and A53T) of α-synuclein through a wide range of biophysical assays to probe the microscopic steps in their aggregation process and the structures of the resulting aggregates. Our results reveal a great complexity in the combined effects of the disease-related mutations with N-terminal acetylation on the aggregation of α-synuclein, which underscores the great sensitivity to even relatively small perturbations of the behaviour of this protein.

Topics: Acetylation; alpha-Synuclein; Humans; Parkinson Disease; Protein Aggregation, Pathological; Protein Processing, Post-Translational

α-Synuclein (α-syn) has been shown to form various conformational fibrils associated with different synucleinopathies. But whether the conformation of α-syn fibrils changes during disease progression is unclear. Here, we amplified α-syn aggregates from the cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) staged in preclinical PD (pre-PD), middle- to late-stage PD (mid-PD), and late-stage PD (late-PD). Our results show that α-syn fibrils derived from the late-PD patient are most potent in inducing endogenous α-syn aggregation in primary neurons, followed by the mid-PD and pre-PD fibrils. By using cryo-electron microscopy, we further determined the high-resolution structures of the CSF-amplified fibrils. The structures exhibit remarkable differences in a minor but significant population of conformational species in different staged samples. Our work demonstrates structural and pathological differences between α-syn fibrils derived from PD patients at a spectrum of clinical stages, which suggests potential conformational transition of α-syn fibrils during the progression of PD.

Topics: alpha-Synuclein; Amyloid; Cryoelectron Microscopy; Humans; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation

The self-association of amylogenic proteins to the fibril form is considered a pivotal factor in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). PD causes unintended or uncontrollable movements in its common symptoms. α-synuclein is the major cause of PD development and thus has been the main target of numerous studies to suppress and sequester its expression or effectively degrade it. Nonetheless, to date, there are no efficient and proven ways to prevent pathological protein aggregation. Recent investigations proposed applying an external electric field to interrupt the fibrils. This method is a non-invasive approach that has a certain benefit over others. We performed molecular dynamics (MD) simulations by applying an electric field on highly toxic fibrils of α-synuclein to gain a molecular-level insight into fibril disruption mechanisms. The results revealed that the applied external electric field induces substantial changes in the conformation of the α-synuclein fibrils. Furthermore, we show the threshold value for electric field strength required to completely disrupt the α-synuclein fibrils by opening the hydrophobic core of the fibril. Thus, our findings might serve as a valuable foundation to better understand molecular-level mechanisms of the α-synuclein fibrils disaggregation process under an applied external electric field.

Topics: alpha-Synuclein; Amyloid; Humans; Molecular Dynamics Simulation; Parkinson Disease; Protein Aggregation, Pathological

Anomalous aggregation of α-synuclein (α-Syn) is a pathological hallmark of many degenerative synucleinopathies including Lewy body dementia (LBD) and Parkinson's disease (PD). Despite its strong link to disease, the precise molecular mechanisms that link α-Syn aggregation to neurodegeneration have yet to be elucidated. Here, we find that elevated α-Syn leads to an increase in the plasma membrane (PM) phosphoinositide PI(4,5)P

Topics: alpha-Synuclein; Humans; Neurons; Parkinson Disease; Phosphatidylinositol 4,5-Diphosphate; Phosphotransferases (Alcohol Group Acceptor); Protein Aggregation, Pathological; Signal Transduction

Alpha synuclein has a key role in the pathogenesis of Parkinson's disease (PD), Dementia with Lewy Bodies (LBD) and Multiple System Atrophy (MSA). Immunotherapies aiming at neutralising toxic αSyn species are being investigated in the clinic as potential disease modifying therapies for PD and other synucleinopathies. In this study, the effects of active immunisation against αSyn with the UB-312 vaccine were investigated in the Thy1SNCA/15 mouse model of PD. Young transgenic and wild-type mice received an immunisation regimen over a period of 6 weeks, then observed for an additional 9 weeks. Behavioural assessment was conducted before immunisation and at 15 weeks after the first dose. UB-312 immunisation prevented the development of motor impairment in the wire test and challenging beam test, which was associated with reduced levels of αSyn oligomers in the cerebral cortex, hippocampus and striatum of Thy1SNCA/15 mice. UB-312 immunotherapy resulted in a significant reduction of theαSyn load in the colon, accompanied by a reduction in enteric glial cell reactivity in the colonic ganglia. Our results demonstrate that immunisation with UB-312 prevents functional deficits and both central and peripheral pathology in Thy1SNCA/15 mice.

Topics: alpha-Synuclein; Animals; Brain; Disease Models, Animal; Humans; Intestines; Mice; Mice, Transgenic; Parkinsonian Disorders; Protein Aggregation, Pathological; Vaccination; Vaccines, Subunit

Neurodegenerative disorders are characterized by a collapse in proteostasis, as shown by the accumulation of insoluble protein aggregates in the brain. Proteostasis involves a balance of protein synthesis, folding, trafficking, and degradation, but how aggregates perturb these pathways is unknown. Using Parkinson's disease (PD) patient midbrain cultures, we find that aggregated α-synuclein induces endoplasmic reticulum (ER) fragmentation and compromises ER protein folding capacity, leading to misfolding and aggregation of immature lysosomal β-glucocerebrosidase. Despite this, PD neurons fail to initiate the unfolded protein response, indicating perturbations in sensing or transducing protein misfolding signals in the ER. Small molecule enhancement of ER proteostasis machinery promotes β-glucocerebrosidase solubility, while simultaneous enhancement of trafficking improves ER morphology, lysosomal function, and reduces α-synuclein. Our studies suggest that aggregated α-synuclein perturbs the ability of neurons to respond to misfolded proteins in the ER, and that synergistic enhancement of multiple proteostasis branches may provide therapeutic benefit in PD.

Topics: alpha-Synuclein; Endoplasmic Reticulum; Humans; Mesencephalon; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Protein Folding; Protein Transport; Proteostasis

Parkinson's disease is a neurodegenerative disease characterized by the formation of neuronal inclusions of α-synuclein in patient brains. As the disease progresses, toxic α-synuclein aggregates transmit throughout the nervous system. No effective disease-modifying therapy has been established, and preventing α-synuclein aggregation is thought to be one of the most promising approaches to ameliorate the disease. In this study, we performed a two-step screening using the thioflavin T assay and a cell-based assay to identify α-synuclein aggregation inhibitors. The first screening, thioflavin T assay, allowed the identification of 30 molecules, among a total of 1262 FDA-approved small compounds, which showed inhibitory effects on α-synuclein fibrilization. In the second screening, a cell-based aggregation assay, seven out of these 30 candidates were found to prevent α-synuclein aggregation without causing substantial toxicity. Of the seven final candidates, tannic acid was the most promising compound. The robustness of our screening method was validated by a primary neuronal cell model and a Caenorhabditis elegans model, which demonstrated the effect of tannic acid against α-synuclein aggregation. In conclusion, our two-step screening system is a powerful method for the identification of α-synuclein aggregation inhibitors, and tannic acid is a promising candidate as a disease-modifying drug for Parkinson's disease.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Antiparkinson Agents; Benzothiazoles; Biological Assay; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; Drug Repositioning; HeLa Cells; High-Throughput Screening Assays; Humans; Mice, Inbred C57BL; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Spectrometry, Fluorescence; Tannins

For disordered proteins, including α-synuclein (Syn), the aggregation of which is implicated in Parkinson's disease, it is known that at mild acidic and at the pI solution conditions the use of either strong or weak electrolytes minimized Syn aggregation. The mechanism is driven by electrostatic forces but remains, however, poorly understood. To address this issue, we used two biological buffers as weak electrolytes, at a low concentration (10 mM) and monitored the aggregation of Syn solutions from pH 7 to pH 2, by means of light scattering techniques. When the citrate buffer was used, in which there is buffering capacity in the pH range studied, the maximum of Syn aggregation was very close to the isoelectric point (pI = 4.7). When using tris-HCl, in which there is almost no buffering capacity in the pH range studied, it was for the first time observed a slow transition of the pI (of ca. 1 h) from 4.7 to 4-3, for a 33.5 μM protein concentration, as an example. We also observed in the protein solutions (in tris-HCl) the very early formation of large Syn aggregates. When there is buffering capacity, such as pH 7, these early large Syn aggregates dissociate, followed by association/aggregation. When there is no buffering capacity, such as pH 3, the referred early large Syn aggregates only dissociate. Overall, early large Syn aggregates dissociation can cause entropy in the protein solutions and Syn aggregation is only restored by the altered electrostatic forces due to the existing buffering capacity. Finally, by using an innovative strategy based in the ANS dye fluorescence intensity variation, we determined of the occurrence of the liquid-liquid phase separation process at pH 7 Syn solutions.

Topics: alpha-Synuclein; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Static Electricity

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of β-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure-function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.

Topics: alpha-Synuclein; Humans; Mutation; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization; Spectrum Analysis

Glycation is a nonenzymatic posttranslational modification (PTM) known to be increased in the brains of hyperglycemic patients. Alpha-synuclein (αSN), a central player in the etiology of Parkinson's disease, can be glycated at lysine residues, thereby reducing αSN fibril formation in vitro and modulating αSN aggregation in cells. However, the molecular basis for these effects is unclear. To elucidate this, we investigated the aggregation of αSN modified by eight glycating agents, namely the dicarbonyl compound methylglyoxal (MGO) and the sugars ribose, fructose, mannose, glucose, galactose, sucrose, and lactose. We found that MGO and ribose modify αSN to the greatest extent, and these glycation products are the most efficient inhibitors of fibril formation. We show glycation primarily inhibits elongation rather than nucleation of αSN and has only a modest effect on the level of oligomerization. Furthermore, glycated αSN is not significantly incorporated into fibrils. For both MGO and ribose, we discovered that a level of ∼5 modifications per αSN is optimal for inhibition of elongation. The remaining sugars showed a weak but optimal inhibition at ∼2 modifications per αSN. We propose that this optimal level balances the affinity for the growing ends of the fibril (which decreases with the extent of modification) with the ability to block incorporation of subsequent αSN subunits (which increases with modification). Our results are not only relevant for other αSN PTMs but also for understanding PTMs affecting other fibrillogenic proteins and may thus open novel avenues for therapeutic intervention in protein aggregation disorders.

Topics: alpha-Synuclein; Humans; Kinetics; Monosaccharides; Protein Aggregates; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Pyruvaldehyde

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials.

Topics: alpha-Synuclein; Amyloid; Humans; Lewy Body Disease; Parkinson Disease; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Serine

Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previous reports showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. This paper reports quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is unavailable, binding poses were predicted by molecular docking of MMP1 with aSyn using ClusPro. MMP1 dynamics were simulated using predicted binding poses and compared with the experimental interdomain dynamics to identify an appropriate pose. The selected aSyn-MMP1 binding pose near aSyn residue K45 was simulated and analyzed to define conformational changes at the catalytic site. Allosteric residues in aSyn-bound MMP1 exhibiting strong correlations with the catalytic motif residues were compared with allosteric residues in free MMP1, and aSyn-specific residues were identified. The allosteric residues in aSyn-bound MMP1 are K281, T283, G292, G327, L328, E329, R337, F343, G345, N346, Y348, G353, Q354, D363, Y365, S366, S367, F368, P371, R372, V374, K375, A379, F391, A394, R399, M414, F419, V426, and C466. Shannon entropy was defined to quantify MMP1 dynamics. Virtual screening was performed against a site on selected aSyn-MMP1 binding poses, which showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for potential substrate-specific control of MMP1 activity in the future. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Topics: alpha-Synuclein; Catalytic Domain; Matrix Metalloproteinase 1; Molecular Docking Simulation; Protein Aggregation, Pathological

Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, as well as the accumulation of intraneuronal proteinaceous inclusions known as Lewy bodies and Lewy neurites. The major protein component of Lewy inclusions is the intrinsically disordered protein α-synuclein (α-Syn), which can adopt diverse amyloid structures. Different conformational strains of α-Syn have been proposed to be related to the onset of distinct synucleinopathies; however, how specific amyloid fibrils cause distinctive pathological traits is not clear. Here, we generated three different α-Syn amyloid conformations at different pH and salt concentrations and analyzed the activity of SynuClean-D (SC-D), a small aromatic molecule, on these strains. We show that incubation of α-Syn with SC-D reduced the formation of aggregates and the seeded polymerization of α-Syn in all cases. Moreover, we found that SC-D exhibited a general fibril disaggregation activity. Finally, we demonstrate that treatment with SC-D also reduced strain-specific intracellular accumulation of phosphorylated α-Syn inclusions. Taken together, we conclude that SC-D may be a promising hit compound to inhibit polymorphic α-Syn aggregation.

Topics: alpha-Synuclein; Amyloid; Humans; Lewy Bodies; Neuroprotective Agents; Parkinson Disease; Polymerization; Protein Aggregation, Pathological; Pyridines; Synucleinopathies

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson's disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.

Topics: alpha-Synuclein; Amyloid; Animals; Escherichia coli; Escherichia coli Proteins; Humans; Mice; Microbiota; Parkinson Disease; Protein Aggregation, Pathological

Topics: alpha-Synuclein; Animals; Calcium; EF Hand Motifs; Heat-Shock Response; HEK293 Cells; Humans; Insulin; Insulin-Secreting Cells; Molecular Chaperones; Oxidative Stress; Protein Aggregation, Pathological; Protein Folding; Proteostasis Deficiencies; Rats; Secretagogins

α-Synuclein (α-Syn) amyloids in synucleinopathies are suggested to be structurally and functionally diverse, reminiscent of prion-like strains. The mechanism of how the aggregation of the same precursor protein results in the formation of fibril polymorphs remains elusive. Here, we demonstrate the structure-function relationship of two polymorphs, pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs), based on α-Syn aggregation intermediates. These polymorphs display the structural differences as demonstrated by solid-state NMR and mass spectrometry studies and also possess different cellular activities such as seeding, internalization, and cell-to-cell transfer of aggregates. HMFs, with a compact core structure, exhibit low seeding potency but readily internalize and transfer from one cell to another. The less structured PMFs lack transcellular transfer ability but induce abundant α-Syn pathology and trigger the formation of aggresomes in cells. Overall, the study highlights that the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior.

Topics: alpha-Synuclein; Amyloid; Humans; Inclusion Bodies; Magnetic Resonance Spectroscopy; Prions; Protein Aggregation, Pathological

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-β (Aβ) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aβ, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Amyloidosis; Humans; Parkinson Disease; Protein Aggregation, Pathological; tau Proteins

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.

Topics: alpha-Synuclein; Cell Line; Cell Survival; Humans; Multiple System Atrophy; Nerve Tissue Proteins; Oligodendroglia; Phosphorylation; Prolyl Oligopeptidases; Protein Aggregates; Protein Aggregation, Pathological

The misfolding of host-encoded proteins into pathological prion conformations is a defining characteristic of many neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and Lewy body dementia. A current area of intense study is the way in which the pathological deposition of these proteins might influence each other, as various combinations of co-pathology between prion-capable proteins are associated with exacerbation of disease. A spectrum of pathological, genetic and biochemical evidence provides credence to the notion that amyloid β (Aβ) accumulation can induce and promote α-synuclein pathology, driving neurodegeneration.. To assess the interplay between α-synuclein and Aβ on protein aggregation kinetics, we crossed mice expressing human α-synuclein (M20) with APPswe/PS1dE9 transgenic mice (L85) to generate M20/L85 mice. We then injected α-synuclein preformed fibrils (PFFs) unilaterally into the hippocampus of 6-month-old mice, harvesting 2 or 4 months later.. Immunohistochemical analysis of M20/L85 mice revealed that pre-existing Aβ plaques exacerbate the spread and deposition of induced α-synuclein pathology. This process was associated with increased neuroinflammation. Unexpectedly, the injection of α-synuclein PFFs in L85 mice enhanced the deposition of Aβ; whereas the level of Aβ deposition in M20/L85 bigenic mice, injected with α-synuclein PFFs, did not differ from that of mice injected with PBS.. These studies reveal novel and unexpected interplays between α-synuclein pathology, Aβ and neuroinflammation in mice that recapitulate the pathology of Alzheimer's disease and Lewy body dementia.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Astrocytes; Cerebral Cortex; Crosses, Genetic; Dementia; Disease Models, Animal; Gliosis; Hippocampus; Humans; Injections; Lewy Body Disease; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Neuroinflammatory Diseases; Parkinson Disease; Prions; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Proteins

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a "pathological package" capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein-ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.

Topics: alpha-Synuclein; Ceramides; Extracellular Vesicles; Glucosylceramidase; Humans; Mutation; Parkinsonian Disorders; Protein Aggregation, Pathological

Glycation of α-synuclein (αSyn), as occurs with aging, has been linked to the progression of Parkinson's disease (PD) through the promotion of advanced glycation end-products and the formation of toxic oligomers that cannot be properly cleared from neurons. DJ-1, an antioxidative protein that plays a critical role in PD pathology, has been proposed to repair glycation in proteins, yet a mechanism has not been elucidated. In this study, we integrate solution nuclear magnetic resonance (NMR) spectroscopy and liquid atomic force microscopy (AFM) techniques to characterize glycated N-terminally acetylated-αSyn (glyc-ac-αSyn) and its interaction with DJ-1. Glycation of ac-αSyn by methylglyoxal increases oligomer formation, as visualized by AFM in solution, resulting in decreased dynamics of the monomer amide backbone around the Lys residues, as measured using NMR. Upon addition of DJ-1, this NMR signature of glyc-ac-αSyn monomers reverts to a native ac-αSyn-like character. This phenomenon is reversible upon removal of DJ-1 from the solution. Using relaxation-based NMR, we have identified the binding site on DJ-1 for glycated and native ac-αSyn as the catalytic pocket and established that the oxidation state of the catalytic cysteine is imperative for binding. Based on our results, we propose a novel mechanism by which DJ-1 scavenges glyc-ac-αSyn oligomers without chemical deglycation, suppresses glyc-ac-αSyn monomer-oligomer interactions, and releases free glyc-ac-αSyn monomers in solution. The interference of DJ-1 with ac-αSyn oligomers may promote free ac-αSyn monomer in solution and suppress the propagation of toxic oligomer and fibril species. These results expand the understanding of the role of DJ-1 in PD pathology by acting as a scavenger for aggregated αSyn.

Topics: Acetylation; alpha-Synuclein; Cysteine; Glycation End Products, Advanced; Humans; Magnetic Resonance Spectroscopy; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Protein Deglycase DJ-1; Protein Multimerization

Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson's disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak's seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD.

Topics: alpha-Synuclein; Amyloid; Cell Line; HEK293 Cells; Humans; Parkinson Disease; Phenols; Phosphorylation; Protein Aggregation, Pathological; Staphylococcus aureus

The capacity of a cell to maintain proteostasis progressively declines during aging. Virtually all age-associated neurodegenerative disorders associated with aggregation of neurotoxic proteins are linked to defects in the cellular proteostasis network, including insufficient lysosomal hydrolysis. Here, we report that proteotoxicity in yeast and Drosophila models for Parkinson's disease can be prevented by increasing the bioavailability of Ca2+, which adjusts intracellular Ca2+ handling and boosts lysosomal proteolysis. Heterologous expression of human α-synuclein (αSyn), a protein critically linked to Parkinson's disease, selectively increases total cellular Ca2+ content, while the levels of manganese and iron remain unchanged. Disrupted Ca2+ homeostasis results in inhibition of the lysosomal protease cathepsin D and triggers premature cellular and organismal death. External administration of Ca2+ reduces αSyn oligomerization, stimulates cathepsin D activity and in consequence restores survival, which critically depends on the Ca2+/calmodulin-dependent phosphatase calcineurin. In flies, increasing the availability of Ca2+ discloses a neuroprotective role of αSyn upon manganese overload. In sum, we establish a molecular interplay between cathepsin D and calcineurin that can be activated by Ca2+ administration to counteract αSyn proteotoxicity.

Topics: Aging; alpha-Synuclein; Animals; Animals, Genetically Modified; Calcineurin; Calcium; Cathepsin D; Cell Death; Drosophila melanogaster; Gene Expression Regulation; Humans; Lysosomes; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Proteolysis; Saccharomyces cerevisiae

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Humans; Lipid Droplets; Lipid Metabolism; Lipolysis; Membrane Transport Proteins; Neuroblastoma; Neurons; Parkinson Disease; Perilipin-2; Protein Aggregation, Pathological; Proteolysis

Protein aggregate formation is linked with multiple amyloidoses, including Alzheimer's and Parkinson's diseases. Currently, the understanding of such fibrillar structure formation and propagation is still not sufficient, the outcome of which is a lack of potent, anti-amyloid drugs. The environmental conditions used during in vitro protein aggregation assays play an important role in determining both the aggregation kinetic parameters, as well as resulting fibril structure. In the case of alpha-synuclein, ionic strength has been shown as a crucial factor in its amyloid aggregation. In this work, we examine a large sample size of alpha-synuclein aggregation reactions under thirty different ionic strength and protein concentration combinations and determine the resulting fibril structural variations using their dye-binding properties, secondary structure and morphology. We show that both ionic strength and protein concentration determine the structural variability of alpha-synuclein amyloid fibrils and that sometimes even identical conditions can result in up to four distinct types of aggregates.

Topics: alpha-Synuclein; Amyloid; In Vitro Techniques; Kinetics; Osmolar Concentration; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Structure, Secondary

Parkinson's disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or β-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that β-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl β-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that β-sheet structures comprise the assembly of the fibrils.

Topics: alpha-Synuclein; Amyloid; Cetrimonium; Circular Dichroism; Galactosides; Humans; Lewy Bodies; Neurodegenerative Diseases; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation; Protein Conformation, beta-Strand; Protein Folding; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Spectroscopy, Fourier Transform Infrared

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.

Topics: alpha-Synuclein; Amyloid; Calpain; HSC70 Heat-Shock Proteins; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Molecular Chaperones; Protein Aggregates; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Proteolysis; Synucleinopathies

Neuroinflammation and mitochondrial impairment play important roles in the neuropathogenesis of Parkinson's disease (PD). The activation of NLRP3 inflammasome and the accumulation of α-synuclein (α-Syn) are strictly correlated to neuroinflammation. Therefore, the regulation of NLRP3 inflammasome activation and α-Syn aggregation might have therapeutic potential. It has been indicated that Dl-3-n-butylphthalide (NBP) produces neuroprotection against some neurological diseases such as ischemic stroke. We here intended to explore whether NBP suppressed NLRP3 inflammasome activation and reduced α-Syn aggregation, thus protecting dopaminergic neurons against neuroinflammation.. In our study, we established a MPTP-induced mouse model and 6-OHDA-induced SH-SY5Y cell model to examine the neuroprotective actions of NBP. We then performed behavioral tests to examine motor dysfunction in MPTP-exposed mice after NBP treatment. Western blotting, immunofluorescence staining, flow cytometry and RT-qPCR were conducted to investigate the expression of NLRP3 inflammasomes, neuroinflammatory cytokines, PARP1, p-α-Syn, and markers of microgliosis and astrogliosis.. The results showed that NBP exerts a neuroprotective effect on experimental PD models.. In summary, NBP rescued dopaminergic neurons by reducing NLRP3 inflammasome activation and ameliorating mitochondrial impairments and increases in p-α-Syn levels. This current study may provide novel neuroprotective mechanisms of NBP as a potential therapeutic agent.

Topics: alpha-Synuclein; Animals; Apoptosis; Benzofurans; Cell Line; Disease Models, Animal; Dopaminergic Neurons; Humans; Inflammasomes; Mice; Mitochondria; Neuroprotective Agents; NLR Family, Pyrin Domain-Containing 3 Protein; Parkinson Disease; Protein Aggregation, Pathological

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.

Topics: alpha-Synuclein; Apoptosis; Autophagy; Cell Line, Tumor; Dopaminergic Neurons; Ellagic Acid; Humans; Lewy Bodies; Neurodegenerative Diseases; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Topics: alpha-Synuclein; Animals; Brain; Brain Ischemia; Down-Regulation; Endosomes; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Models, Animal; Nedd4 Ubiquitin Protein Ligases; Neurodegenerative Diseases; Neurons; Neuroprotection; Parkinson Disease; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Stroke; Ubiquitin-Protein Ligases; Ubiquitination; Up-Regulation

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that (1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; (2) provide a structural characterization of the oligomers and aggregates formed by the BiFC. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple biochemical approaches and techniques. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that (1) the C-terminal-Venus fused to α-syn (α-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to α-syn (Vn-α-syn); (2) Vn-α-syn exhibits a high propensity to form oligomers and higher-order aggregates; and (3) the expression of either or both fragments does not result in the formation of α-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-α-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of α-syn-Vc in Vn-α-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission based on the α-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

Topics: alpha-Synuclein; Animals; HEK293 Cells; Humans; Models, Animal; Optical Imaging; Protein Aggregates; Protein Aggregation, Pathological

The interaction of α-synuclein with mitochondria in both typical and atypical Parkinson's disease is a critical component of degeneration. The mechanism of cell-to-cell propagation of pathological α-synuclein in synucleinopathies is unclear. Intercellular exchange of mitochondria along tunnelling nanotubes has been described in other diseases, such as cancer; however, its role in synucleinopathies is unknown. Pathological α-synuclein species have been demonstrated previously to move from cell to cell via tunnelling nanotubes. This process was further explored using co-culture and monoculture systems to determine if α-synuclein binds to migrating mitochondria within tunnelling nanotubes. Super-resolution analysis via stimulated emission depletion microscopy showed interaction between α-synuclein with the mitochondrial outer membrane and the presence of alpha-synuclein associated with mitochondria in tunnelling nanotubes between 1321N1, differentiated THP-1 and SH-SY5Y cell types. siRNA knockdown of Miro1, a critical protein-bridging mitochondria to the motor adaptor complex, had no effect on mitochondrial density or α-synuclein association with mitochondria in tunnelling nanotubes. The results show that α-synuclein aggregates associate with mitochondria in intercellular tunnelling nanotubes, suggesting that mitochondria-mediated α-synuclein transfer between cells may contribute to cell-to-cell spread of α-synuclein aggregates and disease propagation.

Topics: alpha-Synuclein; Cell Line, Tumor; Coculture Techniques; Humans; Mitochondria; Nanotubes; Protein Aggregation, Pathological

Alpha-synuclein (αsyn) is the key component of proteinaceous aggregates termed Lewy Bodies that pathologically define a group of disorders known as synucleinopathies, including Parkinson's Disease (PD) and Dementia with Lewy Bodies. αSyn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of αsyn necessitates the release of misfolded αsyn from one cell and the uptake of that αsyn by another, in which it can template the misfolding of endogenous αsyn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3θ acts as a chaperone to reduce αsyn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on αsyn toxicity in the in vivo αsyn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, αsyn aggregation, and neuronal counts in the PFF model. 14-3-3θ transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3θ overexpression was associated with delayed αsyn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated αsyn aggregation and reduction in NECAB1-positive neuron counts induced by PFFs. 14-3-3θ overexpression by AAV in the substantia nigra (SN) also delayed αsyn aggregation in the SN and partially rescued PFF-induced reduction in tyrosine hydroxylase (TH)-positive dopaminergic cells in the SN. 14-3-3 inhibition in the SN accelerated nigral αsyn aggregation and enhanced PFF-induced reduction in TH-positive dopaminergic cells. These data indicate a neuroprotective role for 14-3-3θ against αsyn toxicity in vivo.

Topics: 14-3-3 Proteins; alpha-Synuclein; Amygdala; Animals; Behavior, Animal; Cerebral Cortex; Disease Models, Animal; Dopaminergic Neurons; Gene Knock-In Techniques; Mice; Mice, Transgenic; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Proteins; Social Dominance; Substantia Nigra

In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the central nervous system. Recent evidences collected in non-human primates challenge however the hypothesis of a transmission of α-synuclein (α-syn) pathology through the vagus nerve. Would the hypothesis whereby the bloodstream acts as a route for long-distance transmission of pathological α-syn hold true, an inter-individual transmission of synucleinopathy could occur via blood contact. Here, we used a parabiosis approach to join the circulatory systems of wild type and GFP transgenic C57BL/6 J mice, for which one of the partners parabiont received a stereotaxic intranigral injection of patient-derived α-syn aggregates. While the Lewy Body-receiving mice exhibited a loss of dopamine neurons and an increase in nigral S129 phosphorylated α-syn immunoreactivity, their parabiotic bloodstream-sharing partners did not show any trend for a lesion or change in S129 phosphorylated-α-syn levels. Altogether, our study suggests that, in the patient-derived α-synuclein aggregates-injected mouse model and within the selected time frame, the disease is not "transmitted" through the bloodstream.

Topics: alpha-Synuclein; Animals; Lewy Bodies; Mice; Mice, Transgenic; Neostriatum; Neurons; Parabiosis; Protein Aggregates; Protein Aggregation, Pathological; Stereotaxic Techniques; Substantia Nigra

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders characterized by the misfolding and aggregation of alpha-synuclein (aSyn). Doxycycline, a tetracyclic antibiotic shows neuroprotective effects, initially proposed to be due to its anti-inflammatory properties. More recently, an additional mechanism by which doxycycline may exert its neuroprotective effects has been proposed as it has been shown that it inhibits amyloid aggregation. Here, we studied the effects of doxycycline on aSyn aggregation in vivo, in vitro and in a cell free system using real-time quaking induced conversion (RT-QuiC). Using H4, SH-SY5Y and HEK293 cells, we found that doxycycline decreases the number and size of aSyn aggregates in cells. In addition, doxycycline inhibits the aggregation and seeding of recombinant aSyn, and attenuates the production of mitochondrial-derived reactive oxygen species. Finally, we found that doxycycline induces a cellular redistribution of aggregates in a C.elegans animal model of PD, an effect that is associated with a recovery of dopaminergic function. In summary, we provide strong evidence that doxycycline treatment may be an effective strategy against synucleinopathies.

Topics: alpha-Synuclein; Animals; Caenorhabditis elegans; Cell Line; Doxycycline; Humans; Inclusion Bodies; Neuroprotective Agents; Protein Aggregation, Pathological; Synucleinopathies

The accumulation of α-synuclein amyloid fibrils in the brain is linked to Parkinson's disease and other synucleinopathies. The intermediate species in the early aggregation phase of α-synuclein are involved in the emergence of amyloid toxicity and considered to be the most neurotoxic. The N-terminal region flanking the non-amyloid-β component domain of α-synuclein has been implicated in modulating its aggregation. Herein, we report the development of a SUMO1-derived peptide inhibitor (SUMO1(15-55)), which targets two SUMO-interacting motifs (SIMs) within this aggregation-regulating region and suppresses α-synuclein aggregation. Molecular modeling, site-directed mutagenesis, and binding studies are used to elucidate the mode of interaction, namely, via the binding of either of the two SIM sequences on α-synuclein to a putative hydrophobic binding groove on SUMO1(15-55). Subsequent studies show that SUMO1(15-55) also reduces α-synuclein-induced cytotoxicity in cell-based and Drosophila disease models.

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Drosophila; Drug Discovery; Humans; Parkinson Disease; Peptides; Protein Aggregates; Protein Aggregation, Pathological; Protein Interaction Maps; SUMO-1 Protein

Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.

Topics: alpha-Synuclein; Animals; Biological Assay; Cell Line; Endopeptidases; Green Fluorescent Proteins; Mice; Neurons; Protein Aggregation, Pathological

The thermodynamic hypothesis of protein folding, known as the "Anfinsen's dogma" states that the native structure of a protein represents a free energy minimum determined by the amino acid sequence. However, inconsistent with the Anfinsen's dogma, globular proteins can misfold to form amyloid fibrils, which are ordered aggregates associated with diseases such as Alzheimer's and Parkinson's diseases. Here, we present a general concept for the link between folding and misfolding. We tested the accessibility of the amyloid state for various proteins upon heating and agitation. Many of them showed Anfinsen-like reversible unfolding upon heating, but formed amyloid fibrils upon agitation at high temperatures. We show that folding and amyloid formation are separated by the supersaturation barrier of a protein. Its breakdown is required to shift the protein to the amyloid pathway. Thus, the breakdown of supersaturation links the Anfinsen's intramolecular folding universe and the intermolecular misfolding universe.

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloid; Amyloidosis; Chemical Precipitation; DNA-Binding Proteins; Humans; Intrinsically Disordered Proteins; Islet Amyloid Polypeptide; Osmolar Concentration; Protein Aggregation, Pathological; Protein Conformation; Protein Folding; Protein Multimerization; tau Proteins; Thermodynamics

The propagation of conformational strains by templated seeding is central to the prion concept. Seeded assembly of α-synuclein into filaments is believed to underlie the prion-like spreading of protein inclusions in a number of human neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We previously determined the atomic structures of α-synuclein filaments from the putamen of five individuals with MSA. Here, we used filament preparations from three of these brains for the in vitro seeded assembly of recombinant human α-synuclein. We find that the structures of the seeded assemblies differ from those of the seeds, suggesting that additional, as yet unknown, factors play a role in the propagation of the seeds. Identification of these factors will be essential for understanding the prion-like spreading of α-synuclein proteinopathies.

Topics: alpha-Synuclein; Amyloid; Brain; Humans; Molecular Structure; Multiple System Atrophy; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation

Abnormal aggregation of the α-synuclein protein is a key molecular feature of Parkinson's disease and other neurodegenerative diseases. The precise mechanisms that trigger α-synuclein aggregation are unclear, and it is not known what role aggregation plays in disease pathogenesis. Here we use an in vivo zebrafish model to express several different forms of human α-synuclein and measure its aggregation in presynaptic terminals. We show that human α-synuclein tagged with GFP can be expressed in zebrafish neurons, localizing normally to presynaptic terminals and undergoing phosphorylation at serine-129, as in mammalian neurons. The visual advantages of the zebrafish system allow for dynamic in vivo imaging to study α-synuclein, including the use of fluorescence recovery after photobleaching (FRAP) techniques to probe protein mobility. These experiments reveal three distinct terminal pools of α-synuclein with varying mobility, likely representing different subpopulations of aggregated and non-aggregated protein. Human α-synuclein is phosphorylated by an endogenous zebrafish Polo-like kinase activity, and there is a heterogeneous population of neurons containing either very little or extensive phosphorylation throughout the axonal arbor. Both pharmacological and genetic manipulations of serine-129 show that phosphorylation of α-synuclein at this site does not significantly affect its mobility. This suggests that serine-129 phosphorylation alone does not promote α-synuclein aggregation. Together our results show that human α-synuclein can be expressed and measured quantitatively in zebrafish, and that disease-relevant post-translational modifications occur within neurons. The zebrafish model provides a powerful in vivo system for measuring and manipulating α-synuclein function and aggregation, and for developing new treatments for neurodegenerative disease.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Disease Models, Animal; Humans; Parkinson Disease; Phosphorylation; Presynaptic Terminals; Protein Aggregation, Pathological; Serine; Zebrafish

α-Synuclein is a neuronal protein involved in synaptic vesicle trafficking. During the course of Parkinson's disease, it aggregates, forming amyloid fibrils that accumulate in the midbrain. This pathological fibrillization process is strongly modulated by physiological interactions of α-synuclein with lipid membranes. However, the detailed mechanism of this effect remains unclear. In this work, we used environment-sensitive fluorescent dyes to study the influence of model lipid membranes on the kinetics of α-synuclein fibrillization. We observed that formation of the fibrils from α-synuclein monomers is strongly delayed even by small amounts of lipids. Furthermore, we found that membrane-bound α-synuclein monomers are not involved in fibril elongation. Hence, presence of lipids slows down fibril growth proportionally to the fraction of membrane-bound protein.

Topics: alpha-Synuclein; Amyloid; Humans; Kinetics; Lipids; Parkinson Disease; Protein Aggregation, Pathological

Parkinson's disease is a neurodegenerative disorder associated with misfolding and aggregation of α-synuclein as a hallmark protein. Two yeast strain collections comprising conditional alleles of essential genes were screened for the ability of each allele to reduce or improve yeast growth upon α-synuclein expression. The resulting 98 novel modulators of α-synuclein toxicity clustered in several major categories including transcription, rRNA processing and ribosome biogenesis, RNA metabolism and protein degradation. Furthermore, expression of α-synuclein caused alterations in pre-rRNA transcript levels in yeast and in human cells. We identified the nucleolar DEAD-box helicase Dbp4 as a prominent modulator of α-synuclein toxicity. Downregulation of DBP4 rescued cells from α-synuclein toxicity, whereas overexpression led to a synthetic lethal phenotype. We discovered that α-synuclein interacts with Dbp4 or its human ortholog DDX10, sequesters the protein outside the nucleolus in yeast and in human cells, and stabilizes a fraction of α-synuclein oligomeric species. These findings provide a novel link between nucleolar processes and α-synuclein mediated toxicity with DDX10 emerging as a promising drug target.

Topics: alpha-Synuclein; Amyloid; DEAD-box RNA Helicases; Gene Expression Regulation; Humans; Inclusion Bodies; Models, Biological; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization; Protein Transport; Yeasts

In many scientific disputes, research evidence may support one side or the other of a working hypothesis, and even in case of largely coherent hypotheses, arguments may be in favor of discrepant points of view. In the case of α-synuclein pathology and specific mechanisms of disease progression, various animal and cellular models have been established to pinpoint the physiological and pathological mechanisms. In the present 'Editorial controversy', two well-reputed researchers, Hilal Lashuel and Tiago F. Outeiro, discuss-with view to findings from their own and others' groups in the context of current status of knowledge-the question of how well models on α-synuclein pathology can reflect actual pathomechanisms, and derive recommendations for future research from it that shall help advance our understanding not only of α-synuclein-related pathologies and its role in the formation of Lewy bodies in particular, but of cellular or animal models in general.

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Humans; Protein Aggregation, Pathological; Synucleinopathies

The accumulation of proteins into insoluble aggregates is a common feature of several neurodegenerative diseases. Aggregated α-synuclein is a major component of Lewy bodies that pathologically define Parkinson's disease (PD). Here, we present methods for the detection of pathogenic conformations of α-synuclein in induced pluripotent stem cell (iPSC) patient-derived neuron models and brain tissue. These methods can be applied to studies of PD pathogenesis and the discovery of novel therapeutics that restore physiological α-synuclein. For complete details on the use and execution of this protocol, please refer to Cuddy et al. (2019) and Zunke et al. (2018).

Topics: alpha-Synuclein; Chromatography, Gel; Fluorescent Antibody Technique; Humans; Induced Pluripotent Stem Cells; Neurons; Protein Aggregation, Pathological

The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson's disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.

Topics: alpha-Synuclein; Amyloid; Animals; Calcium; Cell Line, Tumor; Cells, Cultured; Humans; Inclusion Bodies; Kinetics; Microscopy, Confocal; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Protein Multimerization; Rats, Sprague-Dawley

Intracellular aggregates are a common pathological hallmark of neurodegenerative diseases such as polyglutamine (polyQ) diseases, amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and multiple system atrophy (MSA). Aggregates are mainly formed by aberrant disease-specific proteins and are accompanied by accumulation of other aggregate-interacting proteins. Although aggregate-interacting proteins have been considered to modulate the formation of aggregates and to be involved in molecular mechanisms of disease progression, the components of aggregate-interacting proteins remain unknown. In this study, we showed that small glutamine-rich tetratricopeptide repeat-containing protein alfa (SGTA) is an aggregate-interacting protein in neurodegenerative diseases. Immunohistochemistry showed that SGTA interacted with intracellular aggregates in Huntington disease (HD) cell models and neurons of HD model mice. We also revealed that SGTA colocalized with intracellular aggregates in postmortem brains of patients with polyQ diseases including spinocerebellar ataxia (SCA)1, SCA2, SCA3, and dentatorubral-pallidoluysian atrophy. In addition, SGTA colocalized with glial cytoplasmic inclusions in the brains of MSA patients, whereas no accumulation of SGTA was observed in neurons of PD and ALS patients. In vitro study showed that SGTA bound to polyQ aggregates through its C-terminal domain and SGTA overexpression reduced intracellular aggregates. These results suggest that SGTA may play a role in the formation of aggregates and may act as potential modifier of molecular pathological mechanisms of polyQ diseases and MSA.

Topics: alpha-Synuclein; Animals; Autopsy; Brain; Brain Chemistry; Cell Line, Tumor; Humans; Huntingtin Protein; Inclusion Bodies; Mice; Mice, Transgenic; Molecular Chaperones; Nerve Tissue Proteins; Neuroblastoma; Neurodegenerative Diseases; Peptide Fragments; Peptides; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Proteins; Solubility; Subcellular Fractions; Transfection

α-Synuclein aggregation at the synapse is an early event in Parkinson's disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and α-synuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like α-synuclein, chaperones the SNARE complex assembly and controls neurotransmitter release. α-Synuclein can rescue neurodegeneration in CSPαKO mice. However, whether α-synuclein aggregation alters CSPα expression and function is unknown. Here we show that α-synuclein aggregation at the synapse is associated with a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with α-synuclein aggregates and in vivo reduces synaptic α-synuclein aggregates increasing monomeric α-synuclein and restoring normal dopamine release in 1-120hαSyn mice. These novel findings reveal a mechanism by which α-synuclein aggregation alters CSPα at the synapse, and show that CSPα rescues α-synuclein aggregation-related phenotype in 1-120hαSyn mice similar to the effect of α-synuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage Parkinson's disease.

Topics: alpha-Synuclein; Animals; Corpus Striatum; Dopamine; HSP40 Heat-Shock Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Parkinson Disease; Protein Aggregation, Pathological; Synapses

The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration. Protein aggregation in brain can be considered as one of the important reasons behind the neurodegeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aβ, α-synuclein, tau, prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 protein might assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain. The results will help us to prevent future outcomes of neurodegeneration by targeting this binding and aggregation process.

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Brain; COVID-19; DNA-Binding Proteins; Heparin; Humans; Molecular Docking Simulation; Neurodegenerative Diseases; Prions; Protein Aggregation, Pathological; Protein Binding; SARS-CoV-2; Spike Glycoprotein, Coronavirus; tau Proteins

Neuronal aggregates of misfolded alpha-synuclein protein are found in the brain and periphery of patients with Parkinson's disease. Braak and colleagues have hypothesized that the initial formation of misfolded alpha-synuclein may start in the gut, and then spread to the brain via peripheral autonomic nerves hereby affecting several organs, including the heart and intestine. Age is considered the greatest risk factor for Parkinson's disease, but the effect of age on the formation of pathology and its propagation has not been studied in detail. We aimed to investigate whether propagation of alpha-synuclein pathology from the gut to the brain is more efficient in old versus young wild-type rats, upon gastrointestinal injection of aggregated alpha-synuclein. Our results demonstrate a robust age-dependent gut-to-brain and brain-to-gut spread of alpha-synuclein pathology along the sympathetic and parasympathetic nerves, resulting in age-dependent dysfunction of the heart and stomach, as observed in patients with Parkinson's disease. Moreover, alpha-synuclein pathology is more densely packed and resistant to enzymatic digestion in old rats, indicating an age-dependent maturation of alpha-synuclein aggregates. Our study is the first to provide a detailed investigation of alpha-synuclein pathology in several organs within one animal model, including the brain, skin, heart, intestine, spinal cord and autonomic ganglia. Taken together, our findings suggest that age is a crucial factor for alpha-synuclein aggregation and complete propagation to heart, stomach and skin, similar to patients. Given that age is the greatest risk factor for human Parkinson's disease, it seems likely that older experimental animals will yield the most relevant and reliable findings. These results have important implications for future research to optimize diagnostics and therapeutics in Parkinson's disease and other age-associated synucleinopathies. Increased emphasis should be placed on using aged animals in preclinical studies and to elucidate the nature of age-dependent interactions.

Topics: Aging; alpha-Synuclein; Animals; Autonomic Nervous System; Brain; Duodenum; Kidney; Muscle, Skeletal; Myocardium; Parkinson Disease; Primary Dysautonomias; Protein Aggregation, Pathological; Rats, Inbred F344; Skin; Spinal Cord; Stomach

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson's diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

Topics: alpha-Synuclein; Amyloid; Animals; Cryoelectron Microscopy; Disease Models, Animal; Humans; Male; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Atomic Force; Microscopy, Confocal; Motor Activity; Mutation, Missense; Nervous System Diseases; Parkinson Disease; Protein Aggregation, Pathological

Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aβ) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aβ/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear.. The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aβ pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aβ and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized "close-culture" chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space.. Our data show that intracellular deposits of αSYN and Aβ are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the "close-culture" chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte.. Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aβ/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Astrocytes; Brain; Cell Membrane Structures; Cells, Cultured; Coculture Techniques; Humans; Induced Pluripotent Stem Cells; Microglia; Microscopy, Confocal; Nanotubes; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Proteolysis

α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson's disease and related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into a high anti-aggregation potency and the ability to abrogate oligomer-induced cell damage. Using a structure-guided search we identify a human peptide expressed in the brain and the gastrointestinal tract with analogous binding, anti-aggregation, and detoxifying properties. The chemical entities we describe here may represent a therapeutic avenue for the synucleinopathies and are promising tools to assist diagnosis by discriminating between native and toxic α-synuclein species.

Topics: alpha-Synuclein; Amyloid; Brain; Gastrointestinal Tract; Humans; Parkinson Disease; Protein Aggregation, Pathological

α-Synuclein is critical in the pathogenesis of Parkinson's disease and related disorders, yet it remains unclear how its aggregation causes degeneration of human dopaminergic neurons. In this study, we induced α-synuclein aggregation in human iPSC-derived dopaminergic neurons using fibrils generated de novo or amplified in the presence of brain homogenates from Parkinson's disease or multiple system atrophy. Increased α-synuclein monomer levels promote seeded aggregation in a dose and time-dependent manner, which is associated with a further increase in α-synuclein gene expression. Progressive neuronal death is observed with brain-amplified fibrils and reversed by reduction of intraneuronal α-synuclein abundance. We identified 56 proteins differentially interacting with aggregates triggered by brain-amplified fibrils, including evasion of Parkinson's disease-associated deglycase DJ-1. Knockout of DJ-1 in iPSC-derived dopaminergic neurons enhance fibril-induced aggregation and neuronal death. Taken together, our results show that the toxicity of α-synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our study demonstrates how Parkinson's disease-associated genes influence the phenotypic manifestation of strains in human neurons.

Topics: alpha-Synuclein; Brain; Cell Death; Dopaminergic Neurons; Humans; Induced Pluripotent Stem Cells; Multiple System Atrophy; Parkinson Disease; Phenotype; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Protein Deglycase DJ-1; Protein Interaction Mapping

Fibril formation and aggregation of α-synuclein are important for the pathogenesis of neurodegenerative disorders including Parkinson's disease. In familial Parkinson's disease, the G51D mutation of α-synuclein causes severe symptoms and rapid progression. α-Synuclein, an intrinsically disordered protein, was shown to adopt an α-helical tetrameric state that resists fibrillation and aggregation. Here, we isolated the stable dimeric state of recombinant wild-type (WT) α-synuclein and G51D α-synuclein protein. Using circular dichroism spectroscopy, we determined that the α-synuclein dimer and monomer structures were unfolded. The WT α-synuclein dimer was more resistant to fibril formation than the monomer. However, the fibril formation rate of the G51D α-synuclein dimer was similar to that of the G51D α-synuclein monomer. The fibril morphology and properties of the G51D α-synuclein monomer were different from those of the WT α-synuclein monomer and dimer and G51D α-synuclein dimer. Additionally, G51D α-synuclein monomer fibrils were more cytotoxic than other fibrils. Our findings indicate that the structural differences between G51D α-synuclein monomer fibrils and other fibrils are critically responsible for its severe neurotoxicity in familial Parkinson's disease.

Topics: alpha-Synuclein; Humans; Mutation; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization; Recombinant Proteins

Parkinson's disease (PD) is linked to aggregation of the protein α-synuclein (aS) into amyloid fibers. aS is proposed to regulate synaptic activity and may also play a role in gene regulation via interaction with DNA in the cell nucleus. Here, we address the role of the negatively-charged C-terminus in the interaction between aS and DNA using single-molecule techniques. Using nanofluidic channels, we demonstrate that truncation of the C-terminus of aS induces differential effects on DNA depending on the extent of the truncation. The DNA extension increases for full-length aS and the (1-119)aS variant, but decreases about 25% upon binding to the (1-97)aS variant. Atomic force microscopy imaging showed full protein coverage of the DNA at high aS concentration. The characterization of biophysical properties of DNA when in complex with aS variants may provide important insights into the role of such interactions in PD, especially since C-terminal aS truncations have been found in clinical samples from PD patients.

Topics: alpha-Synuclein; Amino Acid Sequence; DNA; Humans; Nucleic Acid Conformation; Parkinson Disease; Protein Aggregation, Pathological; Protein Domains

The pathological hallmark of multiple system atrophy (MSA) is fibrillary aggregates of α-synuclein (α-Syn) in the cytoplasm and nucleus of both oligodendrocytes and neurons. In neurons, α-Syn localizes to the cytosolic and membrane compartments, including the synaptic vesicles, mitochondria, and endoplasmic reticulum (ER). α-Syn binds to vesicle-associated membrane protein-binding protein B (VAPB) in the ER membrane. Overexpression of wild-type and familial Parkinson's disease mutant α-Syn perturbs the association between the ER and mitochondria, leading to ER stress and ultimately neurodegeneration. We examined brains from MSA patients (n = 7) and control subjects (n = 5) using immunohistochemistry and immunoelectron microscopy with antibodies against VAPB and phosphorylated α-Syn. In controls, the cytoplasm of neurons and glial cells was positive for VAPB, whereas in MSA lesions VAPB immunoreactivity was decreased. The proportion of VAPB-negative neurons in the pontine nucleus was significantly higher in MSA (13.6%) than in controls (0.6%). The incidence of cytoplasmic inclusions in VAPB-negative neurons was significantly higher (42.2%) than that in VAPB-positive neurons (3.6%); 67.2% of inclusion-bearing oligodendrocytes and 51.1% of inclusion-containing neurons were negative for VAPB. Immunoelectron microscopy revealed that α-Syn and VAPB were localized to granulofilamentous structures in the cytoplasm of oligodendrocytes and neurons. Many vesicular structures labeled with anti-α-Syn were also observed within the granulofilamentous structures in the cytoplasm and nucleus of both oligodendrocytes and neurons. These findings suggest that, in MSA, reduction of VAPB is involved in the disease process and that vesicular structures are associated with inclusion formation.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Endoplasmic Reticulum Stress; Female; Humans; Inclusion Bodies; Male; Middle Aged; Mitochondria; Multiple System Atrophy; Neurons; Phosphorylation; Protein Aggregation, Pathological; Vesicular Transport Proteins

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

Topics: 3,4-Dihydroxyphenylacetic Acid; alpha-Synuclein; Amyloid; Cell Proliferation; Dopamine; Dopaminergic Neurons; Humans; Oxidative Stress; Parkinson Disease; Phenylethyl Alcohol; Protein Aggregation, Pathological; Protein Multimerization

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs β-synuclein (βS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-β component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in βS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that βS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased βS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Topics: alpha-Synuclein; Amino Acid Sequence; beta-Synuclein; Cell Membrane; Conserved Sequence; gamma-Synuclein; Humans; Inclusion Bodies; Mutagenesis; Protein Aggregation, Pathological; Protein Binding; Protein Interaction Domains and Motifs; Protein Multimerization; Solubility

Aggregation of α-synuclein is associated with neurodegeneration and a hallmark pathology in synucleinopathies. These aggregates are thought to function as prion-like particles where the conformation of misfolded α-synuclein determines the traits of the induced pathology, similar to prion diseases. Still, little is known about the molecular targets facilitating the conformation-specific biological effects, but their identification could form the basis for new therapeutic interventions. High-throughput screening of annotated compound libraries could facilitate mechanistic investigation by identifying targets with impact on α-synuclein aggregation. To this end, we developed a FRET-based cellular reporter in HEK293T cells, with sensitivity down to 6.5 nM α-synuclein seeds. Using this model system, we identified GF109203X, SB202190, and SB203580 as inhibitors capable of preventing induction of α-synuclein aggregation via inhibition of p38 MAPK and PKC, respectively. We further investigated the mechanisms underlying the protective effects and found alterations in the endo-lysosomal system to be likely candidates of the protection. We found the changes did not stem from a reduction in uptake but rather alteration of lysosomal abundance and degradative capacity. Our findings highlight the value high-throughput screening brings to the mechanistic investigation of α-synuclein aggregation while simultaneously identifying novel therapeutic compounds.

Topics: alpha-Synuclein; Cells, Cultured; Drug Delivery Systems; Enzyme Inhibitors; Fluorescence Resonance Energy Transfer; HEK293 Cells; Humans; Imidazoles; p38 Mitogen-Activated Protein Kinases; Protein Aggregation, Pathological; Protein Kinase C; Proteome; Pyridines

Cell-to-cell transmission of α-synuclein (α-syn) pathology is considered to underlie the spread of neurodegeneration in Parkinson's disease (PD). Previous studies have demonstrated that α-syn is secreted under physiological conditions in neuronal cell lines and primary neurons. However, the molecular mechanisms that regulate extracellular α-syn secretion remain unclear. In this study, we found that inhibition of monoamine oxidase-B (MAO-B) enzymatic activity facilitated α-syn secretion in human neuroblastoma SH-SY5Y cells. Both inhibition of MAO-B by selegiline or rasagiline and siRNA-mediated knock-down of MAO-B facilitated α-syn secretion. However, TVP-1022, the S-isomer of rasagiline that is 1000 times less active, failed to facilitate α-syn secretion. Additionally, the MAO-B inhibition-induced increase in α-syn secretion was unaffected by brefeldin A, which inhibits endoplasmic reticulum (ER)/Golgi transport, but was blocked by probenecid and glyburide, which inhibit ATP-binding cassette (ABC) transporter function. MAO-B inhibition preferentially facilitated the secretion of detergent-insoluble α-syn protein and decreased its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Moreover, in a rat model (male Sprague Dawley rats) generated by injecting recombinant adeno-associated virus (rAAV)-A53T α-syn, subcutaneous administration of selegiline delayed the striatal formation of Ser129-phosphorylated α-syn aggregates, and mitigated loss of nigrostriatal dopaminergic neurons. Selegiline also delayed α-syn aggregation and dopaminergic neuronal loss in a cell-to-cell transmission rat model (male Sprague Dawley rats) generated by injecting rAAV-wild-type α-syn and externally inoculating α-syn fibrils into the striatum. These findings suggest that MAO-B inhibition modulates the intracellular clearance of detergent-insoluble α-syn via the ABC transporter-mediated non-classical secretion pathway, and temporarily suppresses the formation and transmission of α-syn aggregates.

Topics: alpha-Synuclein; Animals; ATP-Binding Cassette Transporters; Cell Death; Cell Line, Tumor; Corpus Striatum; Culture Media, Conditioned; Dopaminergic Neurons; Gene Knockdown Techniques; Genetic Vectors; Humans; Indans; Injections; Lysosomes; Male; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Mutation, Missense; Neuroblastoma; Parkinsonian Disorders; Protein Aggregation, Pathological; Protein Transport; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Small Interfering; Selegiline; Substantia Nigra

Spreading of aggregate pathology across brain regions acts as a driver of disease progression in Tau-related neurodegeneration, including Alzheimer's disease (AD) and frontotemporal dementia. Aggregate seeds released from affected cells are internalized by naïve cells and induce the prion-like templating of soluble Tau into neurotoxic aggregates. Here we show in a cellular model system and in neurons that Clusterin, an abundant extracellular chaperone, strongly enhances Tau aggregate seeding. Upon interaction with Tau aggregates, Clusterin stabilizes highly potent, soluble seed species. Tau/Clusterin complexes enter recipient cells via endocytosis and compromise the endolysosomal compartment, allowing transfer to the cytosol where they propagate aggregation of endogenous Tau. Thus, upregulation of Clusterin, as observed in AD patients, may enhance Tau seeding and possibly accelerate the spreading of Tau pathology.

Topics: alpha-Synuclein; Animals; Clusterin; Disease Progression; Endocytosis; Humans; Mice; Neurodegenerative Diseases; Neurons; Protein Aggregation, Pathological; Protein Binding; tau Proteins

Parkinson's disease is a common neurodegenerative disease. The differential expression of alpha-synuclein within Lewy Bodies leads to this disease. Some missense mutations of alpha-synuclein may resultant in functional aberrations. In this study, our objective is to verify the functional adaptation due to early and late-onset mutation which can trigger or control the rate of alpha-synuclein aggregation. In this regard, we have proposed a computational model to study the difference and similarities among the Wild type alpha-synuclein and mutants i.e., A30P, A53T, G51D, E46K, and H50Q. Evolutionary sequence space analysis is also performed in this experiment. Subsequently, a comparative study has been performed between structural information and sequence space outcomes. The study shows the structural variability among the selected subtypes. This information assists inter pathway modeling due to mutational aberrations. Based on the structural variability, we have identified the protein-protein interaction partners for each protein that helps to increase the robustness of the inter-pathway connectivity. Finally, few pathways have been identified from 12 semantic networks based on their association with mitochondrial dysfunction and dopaminergic pathways.

Topics: alpha-Synuclein; Dopamine; Humans; Mitochondria; Mitochondrial Diseases; Mutation; Parkinson Disease; Protein Aggregation, Pathological; Signal Transduction

Liquid-liquid phase separation (LLPS) is a crucial phenomenon for the formation of functional membraneless organelles. However, LLPS is also responsible for protein aggregation in various neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease (PD). Recently, several reports, including ours, have shown that α-synuclein (α-Syn) undergoes LLPS and a subsequent liquid-to-solid phase transition, which leads to amyloid fibril formation. However, how the environmental (and experimental) parameters modulate the α-Syn LLPS remains elusive. Here, we show that in vitro α-Syn LLPS is strongly dependent on the presence of salts, which allows charge neutralization at both terminal segments of protein and therefore promotes hydrophobic interactions supportive for LLPS. Using various purification methods and experimental conditions, we showed, depending upon conditions, α-Syn undergoes either spontaneous (instantaneous) or delayed LLPS. Furthermore, we delineate that the kinetics of liquid droplet formation (i.e., the critical concentration and critical time) is relative and can be modulated by the salt/counterion concentration, pH, presence of surface, PD-associated multivalent cations, and N-terminal acetylation, which are all known to regulate α-Syn aggregation in vitro. Together, our observations suggest that α-Syn LLPS and subsequent liquid-to-solid phase transition could be pathological, which can be triggered only under disease-associated conditions (high critical concentration and/or conditions promoting α-Syn self-assembly). This study will significantly improve our understanding of the molecular mechanisms of α-Syn LLPS and the liquid-to-solid transition.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyotrophic Lateral Sclerosis; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Parkinson Disease; Phase Transition; Protein Aggregation, Pathological

Aggregation of amyloidogenic proteins is an abnormal biological process implicated in neurodegenerative disorders. Whereas the aggregation process of amyloid-forming proteins has been studied extensively, the mechanism of aggregate removal is poorly understood. We recently demonstrated that proteasomes could fragment filamentous aggregates into smaller entities, restricting aggregate size [1]. Here, we show in vitro that UBE2W can modify the N-terminus of both α-synuclein and a tau tetra-repeat domain with a single ubiquitin. We demonstrate that an engineered N-terminal ubiquitin modification changes the aggregation process of both proteins, resulting in the formation of structurally distinct aggregates. Single-molecule approaches further reveal that the proteasome can target soluble oligomers assembled from ubiquitin-modified proteins independently of its peptidase activity, consistent with our recently reported fibril-fragmenting activity. Based on these results, we propose that proteasomes are able to target oligomers assembled from N-terminally ubiquitinated proteins. Our data suggest a possible disassembly mechanism by which N-terminal ubiquitination and the proteasome may together impede aggregate formation.

Topics: alpha-Synuclein; Amyloidogenic Proteins; Cytoplasm; Holoenzymes; Humans; Neurodegenerative Diseases; Proteasome Endopeptidase Complex; Protein Aggregation, Pathological; Protein Domains; Protein Multimerization; tau Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination

The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells.

Topics: alpha-Synuclein; Cell Line, Tumor; Escherichia coli; Humans; Microscopy, Electron, Transmission; Protein Aggregation, Pathological; Synucleinopathies

α-Synuclein (α-Syn) is a key pathogenic protein in α-synucleinopathies including Parkinson disease and dementia with Lewy bodies. Accumulating evidence has shown that misfolded fibrillar α-Syn is transmitted from cell-to-cell, a phenomenon that correlates with clinical progression of the disease. We previously showed that deleting the MAP3 kinase apoptosis signal-regulating kinase 1 (ASK1), which is a central player linking oxidative stress with neuroinflammation, mitigates the phenotype of α-Syn transgenic mice. However, whether ASK1 impacts pathology and disease progression induced by recombinant α-Syn pre-formed fibrils (PFF) remains unknown. Here, we compared the neuropathological and behavioral phenotype of ASK1 knock-out mice with that of wild-type mice following intrastriatal injections of α-Syn PFF. At 6 months post-injections, ASK1 null mice exhibited reduced amount of phosphorylated α-Syn aggregates in the striatum and cortex, and less pronounced degeneration of the nigrostriatal pathway. Additionally, the neuroinflammatory reaction to α-Syn PFF injection and propagation seen in wild-type mice was attenuated in ASK1 knock-out animals. These neuropathological markers were associated with better behavioral performance. These data suggest that ASK1 plays an important role in pathological α-Syn fibril transmission and, consequently, may impact disease progression. These findings collectively support inhibiting ASK1 as a disease modifying therapeutic strategy for Parkinson disease and related α-synucleinopathies.

Topics: alpha-Synuclein; Animals; Apoptosis; Inflammation; MAP Kinase Kinase Kinase 5; Mice, Inbred C57BL; Mice, Knockout; Parkinson Disease; Protein Aggregation, Pathological; Signal Transduction

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the elderly people. To date, drugs able to reverse the disease are not available; the gold standard is levodopa that only relieves clinical symptoms, yet with severe side effects after prolonged administration. Many efforts are underway to find alternative targets for PD prevention or treatment, the most promising being α-synuclein (Syn). Recently, we reported that oleuropein aglycone (OleA) interferes with amyloid aggregation of Syn both stabilizing its monomeric state and inducing the formation of harmless, off-pathway oligomers. This study is focused at describing the interaction between Syn and hydroxytyrosol (HT), the phenolic moiety and main metabolite of OleA, and the interferences with Syn aggregation by using biophysical and biological techniques. Our results show that HT dose-dependently inhibits Syn aggregation and that covalent and non-covalent binding mediate HT-Syn interaction. HT does not modify the natively unfolded structure of Syn, rather, it stabilizes specific regions of the molecule leading to inhibition of protein fibrillation. Cellular assays showed that HT reduces the toxicity of Syn aggregates. Moreover, Syn aggregates interaction with the cell membrane, an important factor for prion-like properties of Syn on-pathway oligomers, was reduced in cells exposed to Syn aggregates grown in the presence of HT.

Topics: Acetates; alpha-Synuclein; Antioxidants; Antiparkinson Agents; Cell Line, Tumor; Cell Survival; Cyclopentane Monoterpenes; Humans; Levodopa; Molecular Structure; Parkinson Disease; Phenylethyl Alcohol; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Proteolysis; Pyrans

Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

Topics: Aged; alpha-Synuclein; Animals; Brain; Endopeptidase K; Female; Humans; Male; Mice; Middle Aged; Protein Aggregates; Protein Aggregation, Pathological; Synucleinopathies

The clinical and pathological differences between synucleinopathies such as Parkinson's disease and multiple system atrophy have been postulated to stem from unique strains of α-synuclein aggregates, akin to what occurs in prion diseases. Here we demonstrate that inoculation of transgenic mice with different strains of recombinant or brain-derived α-synuclein aggregates produces clinically and pathologically distinct diseases. Strain-specific differences were observed in the signs of neurological illness, time to disease onset, morphology of cerebral α-synuclein deposits and the conformational properties of the induced aggregates. Moreover, different strains targeted distinct cellular populations and cell types within the brain, recapitulating the selective targeting observed among human synucleinopathies. Strain-specific clinical, pathological and biochemical differences were faithfully maintained after serial passaging, which implies that α-synuclein propagates via prion-like conformational templating. Thus, pathogenic α-synuclein exhibits key hallmarks of prion strains, which provides evidence that disease heterogeneity among the synucleinopathies is caused by distinct α-synuclein strains.

Topics: alpha-Synuclein; Animals; Brain; Mice; Mice, Transgenic; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Proteins; Synucleinopathies

Aggregations of β-amyloid (Aβ) and α-synuclein (αS) into oligomeric and fibrillar assemblies are the pathological hallmarks of Alzheimer's and Parkinson's diseases, respectively. Although Aβ and αS affect different regions of the brain and are separated at the cellular level, there is evidence of their eventual interaction in the pathology of both disorders. Characterization of interactions of Aβ and αS at various stages of their aggregation pathways could reveal mechanisms and therapeutic targets for the prevention and cure of these neurodegenerative diseases. In this study, we comprehensively examined the interactions and their molecular manifestations using an array of characterization tools. We show for the first time that αS monomers and oligomers, but not αS fibrils, inhibit Aβ fibrillization while promoting oligomerization of Aβ monomers and stabilizing preformed Aβ oligomers via coassembly, as judged by Thioflavin T fluorescence, transmission electron microscopy, and SDS- and native-PAGE with fluorescently labeled peptides/proteins. In contrast, soluble Aβ species, such as monomers and oligomers, aggregate into fibrils, when incubated alone under the otherwise same condition. Our study provides evidence that the interactions with αS soluble species, responsible for the effects, are mediated primarily by the C-terminus of Aβ, when judged by competitive immunoassays using antibodies recognizing various fragments of Aβ. We also show that the C-terminus of Aβ is a primary site for its interaction with αS fibrils. Collectively, these data demonstrate aggregation state-specific interactions between αS and Aβ and offer insight into a molecular basis of synergistic biological effects between the two polypeptides.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Benzothiazoles; Brain; Electrophoresis, Polyacrylamide Gel; Humans; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Neurodegenerative Diseases; Parkinson Disease; Peptide Fragments; Protein Aggregation, Pathological

Seeding, in the context of amyloid disease, is the sequential transfer of pathogenic protein aggregates from cell-to-cell within affected tissues. The structure of pathogenic seeds provides the molecular basis and enables rapid conversion of soluble protein into fibrils. To date, there are no inhibitors that specifically target seeding of Parkinson's disease (PD)-associated α-synuclein (α-syn) fibrils, in part, due to lack of information of the structural properties of pathological seeds. Here we design small peptidic inhibitors based on the atomic structure of the core of α-syn fibrils. The inhibitors prevent α-syn aggregation in vitro and in cell culture models with binding affinities of 0.5 μM to α-syn fibril seeds. The inhibitors also show efficacy in preventing seeding by human patient-derived α-syn fibrils. Our results suggest that pathogenic seeds of α-syn contain steric zippers and suggest a therapeutic approach targeted at the spread and progression that may be applicable for PD and related synucleinopathies.

Topics: alpha-Synuclein; HEK293 Cells; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Mutations in the GBA1 gene are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GBA1 encodes the lysosomal lipid hydrolase glucocerebrosidase (GCase), and its activity has been linked to accumulation of α-synuclein. The current study systematically examines the relationship between GCase activity and both pathogenic and non-pathogenic forms of α-synuclein in primary hippocampal, cortical, and midbrain neuron and astrocyte cultures, as well as in transgenic mice and a non-transgenic mouse model of PD. We find that reduced GCase activity does not result in aggregation of α-synuclein. However, in the context of extant misfolded α-synuclein, GCase activity modulates neuronal susceptibility to pathology. Furthermore, this modulation does not depend on neuron type but rather is driven by the level of pathological α-synuclein seeds. This study has implications for understanding how GBA1 mutations influence PD pathogenesis and provides a platform for testing novel therapeutics.

Topics: alpha-Synuclein; Animals; Astrocytes; Cerebral Cortex; Disease Susceptibility; Genetic Predisposition to Disease; Glucosylceramidase; HEK293 Cells; Hippocampus; Humans; Lewy Body Disease; Mesencephalon; Mice; Mice, Transgenic; Neurons; Parkinson Disease; Parkinsonian Disorders; Primary Cell Culture; Protein Aggregation, Pathological; Synucleinopathies

Increasing evidence suggests that the process of alpha-synuclein (α-syn) aggregation from monomers into amyloid fibrils and Lewy bodies, via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies (DLB). However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying and targeting toxic species along the pathway of α-syn fibrillization. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different α-syn species, many of the fundamental questions regarding the role of α-syn aggregation in PD remain unanswered, and no therapeutic compounds targeting α-syn aggregates have passed clinical trials. Several factors have contributed to this slow progress, including the complexity of the aggregation pathways and the heterogeneity and dynamic nature of α-syn aggregates. In the majority of experiment, the α-syn samples used contain mixtures of α-syn species that exist in equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different α-syn species in different studies has contributed not only to experimental irreproducibility but also to misinterpretation of results and misdirection of valuable resources. Towards addressing these challenges and improving experimental reproducibility in Parkinson's research, we describe here a simple centrifugation-based filtration protocol for the isolation, quantification and assessment of the distribution of α-syn monomers, oligomers and fibrils, in heterogeneous α-syn samples of increasing complexity. The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. Here, we present and discuss several examples that illustrate the applications of this protocol and how it could contribute to improving the reproducibility of experiments aimed at elucidating the structural basis of α-syn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid-forming proteins.

Topics: alpha-Synuclein; Amyloid; Biomedical Research; Centrifugation; Filtration; Freeze Drying; Humans; Lewy Bodies; Parkinson Disease; Protein Aggregation, Pathological; Reproducibility of Results

The aggregation of the protein alpha synuclein (α-Syn), a known contributor in Parkinson's disease (PD) pathogenesis is triggered by transition metal ions through occupational exposure and disrupted metal ion homeostasis. Naturally occurring small molecules such as polyphenols have emerged as promising inhibitors of α-Syn fibrillation and toxicity and could be potential therapeutic agents against PD. Here, using an array of biophysical tools combined with cellular assays, we demonstrate that the novel polyphenolic compound scutellarin efficiently inhibits the uninduced and metal-induced fibrillation of α-Syn by acting at the nucleation stage and stabilizes a partially folded intermediate of α-Syn to form SDS-resistant, higher-order oligomers (∼680 kDa) and also disaggregates preformed fibrils of α-Syn into similar type of higher-order oligomers. ANS binding assay, fluorescence lifetime measurements and cell-toxicity experiments reveal scutellarin-generated oligomers as compact, low hydrophobicity structures with modulated surface properties and significantly reduced cytotoxicity than the fibrillation intermediates of α-Syn control. Fluorescence spectroscopy and isothermal titration calorimetry establish the binding between scutellarin and α-Syn to be non-covalent in nature and of moderate affinity (Ka ∼ 105 M-1). Molecular docking approaches suggest binding of scutellarin to the residues present in the NAC region and C-terminus of monomeric α-Syn and the C-terminal residues of fibrillar α-Syn, demonstrating inhibition of fibrillation upon binding to these residues and possible stabilization of the autoinhibitory conformation of α-Syn. These findings reveal interesting insights into the mechanism of scutellarin action and establish it as an efficient modulator of uninduced as well as metal-induced α-Syn fibrillation and toxicity.

Topics: alpha-Synuclein; Amyloid; Apigenin; Glucuronates; Humans; Molecular Docking Simulation; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Topics: alpha-Synuclein; Humans; Integrin alphaXbeta2; Macrophages; Parkinson Disease; Phagosomes; Protein Aggregation, Pathological; Protein Structure, Quaternary

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.

Topics: Acetylation; alpha-Synuclein; Cell Death; Humans; Mutation; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Protein Stability

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aβ(1-42) and pGlu-Aβ(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aβ(1-42) and pGlu-Aβ(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aβ in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aβ and pGlu-Aβ, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aβ species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Animals; Cell Survival; Fluorescent Antibody Technique; Kinetics; Lewy Bodies; Mice; Neurons; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Proteins

Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucleinopathies. The existence of various intracellular and extracellular αSyn species, including cleaved αSyn, complicates the quest for an appropriate therapeutic target. Hence, to develop efficient disease-modifying strategies, it is fundamental to achieve a deeper understanding of the relevant spreading and toxic αSyn species. Here, we describe comparative and proof-of-principle approaches to determine the involvement of αSyn fragments in intercellular spreading. We demonstrate that two different αSyn fragments (1-95 and 61-140) fulfill the criteria of spreading species. They efficiently instigate formation of proteinase-K-resistant aggregates from cell-endogenous full-length αSyn, and drive it into different aggregation pathways. The resulting aggregates induce cellular toxicity. Strikingly, these aggregates are only detectable by specific antibodies. Our results suggest that αSyn fragments might be relevant not only for spreading, but also for aggregation-fate determination and differential strain formation.

Topics: alpha-Synuclein; Cell Line; Extracellular Space; Gene Knockout Techniques; Humans; Neurons; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Domains; Protein Transport; Recombinant Proteins

Protein aggregation plays a central role in numerous neurodegenerative diseases. The key proteins in these diseases are of significant importance, but their investigation can be challenging due to unique properties of protein misfolding and oligomerization. Alpha-synuclein protein (α-Syn) is the predominant component of Lewy Bodies in Parkinson's disease (PD) and is a member of this class of proteins. Many α-Syn studies are limited by the inability to separate various monomeric, oligomeric, and fibrillar forms of the protein from heterogeneous mixtures. This Editorial Highlight summarizes the impact of a study published in the current issue of Journal of Neurochemistry, in which Lashuel and colleagues developed a simple, rapid centrifugation- and filter-based method for separating, isolating, and quantifying different forms of α-Syn. The researchers used electron microscopy, SDS-PAGE, circular dichroism, and protein assays to carefully validate the method and quantitate α-Syn yields and loss. The publication of this new method will not only aid in future studies of α-Syn, but will likely extend to other proteins that underlie a variety of neurodegenerative diseases.

Topics: alpha-Synuclein; Centrifugation; Filtration; Humans; Parkinson Disease; Protein Aggregation, Pathological; Reproducibility of Results

Parkinson's disease (PD) is the second most common neurodegenerative disease of the elderly. Current therapies are only symptomatic, and have no disease-modifying effect. Therefore, disease progresses continuously over time, presenting with both motor and non-motor features. The precise molecular basis for PD is still elusive, but the aggregation of the protein alpha-synuclein (α-syn) is a key pathological hallmark of the disease and is, therefore, a major focus of current research. Considering the intrinsic properties of cell-penetrating peptides (CPPs) for mediating drug delivery of neurotherapeutics across the blood brain barrier (BBB), these might open novel opportunities for the development of new solutions for the treatment of brain-related aspects of PD and other neurodegenerative disorders.. Here, we synthesized solid-phase CPPs using an amphipathic model peptide (MAP) conjugated with the drug Rasagiline (RAS), which we named RAS-MAP, and evaluated its effect on α-syn inclusion formation in a human cell-based model of synucleinopathy.. We found that treatment with RAS-MAP at low concentrations (1-3 µM) reduced α-syn aggregation in cells.. For the first time, we report that conjugation of a current drug used in the therapy of PD with CPP reduces α-syn aggregation, which might prove beneficial in PD and other synucleinopathies.

Topics: alpha-Synuclein; Blood-Brain Barrier; Cell Culture Techniques; Cell Line, Tumor; Cell-Penetrating Peptides; Drug Carriers; Drug Design; Drug Evaluation, Preclinical; Humans; Indans; Neuroprotective Agents; Parkinson Disease; Protein Aggregation, Pathological; Solid-Phase Synthesis Techniques

The main symptom of Parkinson's disease (PD) is motor dysfunction and remarkably approximately 30-40% of PD patients exhibit cognitive impairments. Recently, we have developed MF8, a heart-type fatty acid-binding protein (FABP3)-specific ligand, which can inhibit α-synuclein (α-syn) oligomerization induced by arachidonic acid in FABP3 overexpressing neuro2A cells. The present study aimed to determine whether MF8 attenuates dopaminergic neuronal death and motor and cognitive impairments in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model. MF8 can penetrate the blood-brain barrier and its peak brain concentration (21.5 ± 2.1 nM) was achieved 6 h after the oral administration (1.0 mg/kg). We also compared its effects and pharmacological action with those of L-DOPA (3,4-dihydroxy-l-phenylalanine). PD model mice were developed by administering MPTP (25 mg/kg, i.p.) once a day for five consecutive days. Twenty-four hours after the final MPTP injection, mice were administered MF8 (0.3, 1.0 mg/kg, p.o.) or L-DOPA (25 mg/kg, i.p.) once a day for 28 consecutive days and subjected to behavioral and histochemical studies. MF8 (1.0 mg/kg, p.o.), but not L-DOPA, inhibited the dopaminergic neuronal death in the ventral tegmental area and the substantia nigra pars compacta region of the MPTP-treated mice. MF8 also improved both, motor and cognitive functions, while L-DOPA ameliorated only motor dysfunction. Taken together, our results showed that MF8 attenuated the MPTP-induced dopaminergic neuronal death associated with PD pathology. We present MF8 as a novel disease-modifying therapeutic molecule for PD, which acts via a mechanism different from that of L-DOPA.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; alpha-Synuclein; Animals; Antiparkinson Agents; Blood-Brain Barrier; Cell Death; Cognitive Dysfunction; Disease Models, Animal; Dopaminergic Neurons; Fatty Acid Binding Protein 3; Levodopa; Ligands; Male; Mice; Mice, Inbred C57BL; Motor Activity; Neuroprotective Agents; Parkinson Disease; Protein Aggregation, Pathological

Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies.. Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process.. Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated α-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking β-sheet content after subjecting them to proteinase K digestion.. Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.

Topics: alpha-Synuclein; Animals; Benzofurans; Drugs, Chinese Herbal; Flavonols; HEK293 Cells; Humans; Mice; Molecular Structure; Plant Extracts; Protein Aggregation, Pathological; Synucleinopathies

Alterations in metal ion homeostasis appear coupled to neurodegenerative disorders but mechanisms are unknown. Amyloid formation of the protein α-synuclein in brain cells is a hallmark of Parkinson's disease. α-Synuclein can bind several metal ions in vitro and such interactions may affect the assembly process. Here we used biophysical methods to study the effects of micromolar concentrations of Cu

Topics: alpha-Synuclein; Amyloid; Copper; Dose-Response Relationship, Drug; Ferric Compounds; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation

The notion that nanoscale surfaces influence protein conformational transitions stimulates the investigation of fibrillogenic polypeptides adsorbing to nanomaterials. Alpha-synuclein (αS) is a prototypical amyloidogenic protein whose aggregation is associated with severe neurodegenerative disorders. We explored the interaction of αS with silica nanoparticles (SNPs) in diverse solution conditions, ranging from protein-free to protein-rich media. We found that the SNP-binding region of αS, determined by site-resolved NMR spectroscopy, was similar in simple buffer and blood serum. Competition binding experiments with isotopic homologues and different proteins showed that cosolutes elicited molecular exchange in a protein-specific manner. The interaction of an oxidized, fibrillation-resistant protein form with SNPs was similar to that of unmodified αS. SNPs, however, did not stimulate fibrillation of the oxidized protein, which remained fibrillation incompetent. CD experiments revealed SNP-induced perturbations of the structural properties of oxidized and non-oxidized αS. Thus, while αS binding to SNPs is essentially orthogonal to fibril formation, the interaction perturbs the distribution of conformational states populated by the protein in the colloidal suspension. This study sheds light on the dynamic nature of αS interactions with NPs, an aspect that crucially impacts on our ability to control aggregation of αS.

Topics: alpha-Synuclein; Humans; Nanoparticles; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Protein Folding; Recombinant Proteins; Silicon Dioxide

The amyloid fibril formation by α -synuclein is a hallmark of various neurodegenerative disorders, most notably Parkinson's disease. Epigallocatechin gallate (EGCG) has been reported to be an efficient inhibitor of amyloid formation by numerous proteins, among them α -synuclein. Here, we show that this applies only to a small region of the relevant parameter space, in particular to solution conditions where EGCG readily oxidizes, and we find that the oxidation product is a much more potent inhibitor compared to the unmodified EGCG. In addition to its inhibitory effects, EGCG and its oxidation products can under some conditions even accelerate α -synuclein amyloid fibril formation through facilitating its heterogeneous primary nucleation. Furthermore, we show through quantitative seeding experiments that, contrary to previous reports, EGCG is not able to re-model α -synuclein amyloid fibrils into seeding-incompetent structures. Taken together, our results paint a complex picture of EGCG as a compound that can under some conditions inhibit the amyloid fibril formation of α -synuclein, but the inhibitory action is not robust against various physiologically relevant changes in experimental conditions. Our results are important for the development of strategies to identify and characterize promising amyloid inhibitors.

Topics: alpha-Synuclein; Amyloid; Catechin; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

More than 20 unique diseases such as diabetes, Alzheimer's disease, Parkinson's disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, β-amyloid (Aβ), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Catalase; Cell Line; Diabetes Mellitus; Humans; Insulin; Islet Amyloid Polypeptide; Models, Molecular; Muramidase; Parkinson Disease; Pepsin A; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary; Serum Albumin, Bovine; Superoxide Dismutase

Synucleinopathies are neurological diseases that are characterized by the accumulation of aggregates of a cytosolic protein, α-synuclein, at the plasma membrane. Even though the pathological role of the protein is established, the mechanism by which it damages neurons remains unclear due to the difficulty to correctly mimic the plasma membrane in vitro. Using a microfluidic setup in which the composition of the plasma membrane, including the asymmetry of the two leaflets, is recapitulated, we demonstrate a triple action of α-synuclein on the membrane. First, it changes membrane topology by inducing pores of discrete sizes, likely nucleated from membrane-bound proteins and subsequently enlarged by proteins in solution. Second, protein binding to the cytosolic leaflet increases the membrane capacitance by thinning it and/or changing its relative permittivity. Third, α-synuclein insertion inside the membrane hydrophobic core immobilizes the lipids in both leaflets, including the opposing protein-free extracellular one.

Topics: alpha-Synuclein; Cell Membrane; Electric Capacitance; Fluorescence Recovery After Photobleaching; Hydrophobic and Hydrophilic Interactions; Lab-On-A-Chip Devices; Membrane Fluidity; Membrane Lipids; Membrane Potentials; Membranes, Artificial; Microfluidic Analytical Techniques; Neurons; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Structure-Activity Relationship; Synucleinopathies

The pathological hallmark of synucleinopathies, including Parkinson's disease (PD), is the aggregation of α-synuclein (α-Syn) protein. Even so, tau protein pathology is abundantly found in these diseases. Both α-Syn and tau can exist as polymorphic aggregates, a phenomenon that has been widely studied, mostly in their fibrillar assemblies. We have previously discovered that in addition to α-Syn oligomers, oligomeric tau is also present in the brain tissues of patients with PD and dementia with Lewy bodies (DLB). However, the effect of interaction between polymorphic α-Syn oligomers and tau has not been scrupulously studied. Here, we have explored the structural and functional diversity of distinct α-Syn oligomers, prepared by modifying the protein with dopamine (DA) and docosahexaenoic acid (DHA). The two α-Syn oligomers differed in aggregate size, conformation, sensitivity to proteinase K digestion, tryptic digestion, and toxicity, suggesting them as distinct α-Syn oligomeric strains. We examined their internalization mechanisms in primary neurons and seeding propensity in inducing α-Syn aggregation. Using a combined approach of molecular and cellular techniques, we observed that the tau aggregates cross-seeded with the individual α-Syn oligomeric strains differed in their biochemical and biological properties, suggesting two distinct tau strains. The tau aggregate cross-seeded with the DA-modified α-Syn oligomeric strain possessed a potent intracellular tau seeding propensity. This study provides a comprehensive analysis of unique strain-specific interaction between oligomeric α-Syn and tau. Furthermore, this study allows us to speculate that distinct α-Syn-tau interactions inducing tau aggregation might be an underlying mechanism of neurodegeneration in PD.

Topics: alpha-Synuclein; Cell Line, Tumor; Cell Survival; Docosahexaenoic Acids; Dopamine; Humans; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; tau Proteins

The α-synuclein fibrils are a pathological hallmark of Parkinson's disease (PD) and are abundant in the brains of PD patients. These amyloid fibrils can aggregate into distinct polymorphism under different physical conditions. Therefore, these different fibril polymorph formations should be considered in drug design studies targeting amyloid fibrils. Recently, the atomic structures of two small fibril segments of α-synuclein, named NACore (68-78) and SubNACore (69-77), have been crystallized. These segments are critical for cytotoxicity and fibril formation. Therefore, elucidation of interface interactions between pair sheets of the NACore and SubNACore is significant for the clarification of the mechanism of fibril formation in PD. In this context, molecular dynamics (MD) simulation technique is a convenient tool to investigate interface interactions of these segments at the atomic level. However, the accuracy of these simulations depends on the utilized force fields. Therefore, we have tested the dependence of interface interactions and stabilities of these small amyloid fibrils on various force fields. From the results of triple long (100 ns) MD simulations, we inferred for the stability investigations of the NACore and SubNACore that CHARMM27 and GROMOS53A6 are the most convenient force fields whereas AMBER99SB-ILDN is the most unfavorable one. Consequently, it is expected that our findings will guide the selection of the appropriate force field for simulations between these segments and possible inhibitors of this disease.

Topics: alpha-Synuclein; Amyloid; Humans; Molecular Dynamics Simulation; Parkinson Disease; Protein Aggregation, Pathological

Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the α-synuclein (α-syn)-preformed fibril (PFF) model, which better reflects the α-syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to α-syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal α-syn PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated α-syn aggregation (pSyn) in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific exacerbation of synucleinopathy-induced behavioral and biochemical deficits. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD.

Topics: alpha-Synuclein; Animals; Dieldrin; Dopamine; Female; Male; Mice, Inbred C57BL; Motor Activity; Parkinsonian Disorders; Pesticides; Protein Aggregation, Pathological; Sex Factors; Substantia Nigra

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; DNA-Binding Proteins; Dopaminergic Neurons; Gene Knockout Techniques; Humans; Lewy Body Disease; Locomotion; Longevity; Protein Aggregation, Pathological; RNA-Binding Proteins

Neurodegenerative diseases are characterized by the accumulation of specific phosphorylated protein aggregates in the brain, such as hyperphosphorylated tau (hp-tau) in tauopathies and phosphorylated α-synuclein (p-αSyn) in α-synucleinopathies. The simultaneous accumulation of different proteins is a common event in many neurodegenerative diseases. We herein describe the detection of the phosphorylation and dimerization of αSyn and activation of GSK-3β, a major kinase known to phosphorylate tau and αSyn, in the brains of rTg4510 mice that overexpress human P301L mutant tau. Immunohistochemistry showed p-αSyn aggregates in rTg4510 mice, which were suppressed by doxycycline-mediated decreases in mutant tau expression levels. A semi-quantitative analysis revealed a regional correlation between hp-tau and p-αSyn accumulation in rTg4510 mice. Furthermore, proteinase K-resistant αSyn aggregates were found in the region with excessive hp-tau accumulation in rTg4510 mice, and these aggregates were morphologically different from proteinase K-susceptible p-αSyn aggregates. Western blotting revealed decreases in p-αSyn monomers in TBS- and sarkosyl-soluble fractions and increases in ubiquitinated p-αSyn dimers in sarkosyl-soluble and insoluble fractions in rTg4510 mice. Furthermore, an activated form of GSK-3β was immunohistochemically detected within cells containing both hp-tau and p-αSyn aggregates. A semi-quantitative analysis revealed that increased GSK-3β activity strongly correlated with hp-tau and p-αSyn accumulation in rTg4510 mice. Collectively, the present results suggest that the overexpression of human P301L mutant tau promoted the phosphorylation and dimerization of endogenous αSyn by activating GSK-3β in rTg4510 mice. This synergic effect between tau, αSyn, and GSK-3β may be involved in the pathophysiology of several neurodegenerative diseases that show the accumulation of both tau and αSyn.

Topics: alpha-Synuclein; Animals; Brain; Disease Models, Animal; Glycogen Synthase Kinase 3 beta; Humans; Mice, Inbred C57BL; Mice, Transgenic; Phosphorylation; Protein Aggregation, Pathological; tau Proteins; Tauopathies

The phosphorylation and aggregation of alpha-synuclein (α-Syn) play a key role in methamphetamine (METH)-induced dopaminergic neurotoxicity. The exact mechanism underlying the interaction between METH-induced neurotoxicity and α-Syn was poorly clarified. We aimed to figure out the role of serine 129 phosphorylation (pS129) of α-Syn on its aggregation and neurotoxicity in vitro and in vivo. In this study, we examined pS129 α-Syn expression in vitro and in vivo at the protein phosphorylation and genetic levels and evaluated its effect on METH-induced neurotoxicity. Here, we found that pS129 α-Syn was significantly increased after METH treatment; moreover, the neuronal α-Syn aggregation and apoptosis caused by METH exposure were significantly attenuated after inhibiting α-Syn phosphorylation. We demonstrate that pS129 α-Syn contributes to the aggregation of α-Syn, and that phosphorylated and aggregated forms of α-Syn play an important role in METH-induced neurotoxicity in dopaminergic neurons and SH-SY5Y cells, supporting a potential insight into the treatment of METH-induced neurotoxicity.

Topics: alpha-Synuclein; Animals; Cell Line; Central Nervous System Stimulants; Humans; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Neurons; Neurotoxicity Syndromes; Phosphorylation; Protein Aggregation, Pathological; Serine

Cyclophilin D (CypD) is a peptidyl-prolyl isomerase expressed in the nucleus and transported into the mitochondria where it is best associated with the regulation of the mitochondrial permeability transition pore (MPTP). There are, however, other possible roles of CypD in the mitochondria which may or may not be linked with the MPTP. Alpha synuclein (αSyn) is shown here to interact directly with CypD via its acidic proline-rich C-terminus region and binding at the putative ligand binding pocket of CypD. The study shows that CypD binding with soluble αSyn prevents its aggregation. Furthermore, the addition of CypD to preformed αSyn fibrils leads to the disassembly of these fibrils. Enzymatically-compromised mutants of CypD show reduced abilities to dissociate αSyn aggregates, suggesting that fibril disassembly is linked to the increased rate of peptidyl-prolyl isomerisation catalysed by CypD. Protein aggregation in the mitochondria is increasingly seen as the cause of neurodegeneration. However, protein aggregation is a reversible process but disaggregation requires help from other proteins such as isomerases and chaperones. The results here demonstrate a possible mechanism by which CypD achieves this and suggest that disaggregation could be one of the many functions of this protein.

Topics: alpha-Synuclein; Catalysis; Cyclohexane Monoterpenes; In Vitro Techniques; Mitochondria; Mitochondrial Transmembrane Permeability-Driven Necrosis; Molecular Chaperones; Peptidyl-Prolyl Isomerase F; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Solubility

Parkinson's disease has become one of the most common neurodegenerative diseases. Pathological changes typically manifest following dopaminergic neuron loss in the substantia nigra and abnormal alpha-synuclein (α-syn) aggregation in the neurons. α-Syn is the major component of Lewy bodies. However, research pertaining to the spread of abnormal α-syn aggregations, which results in specific damage to the brain structure and function, is lacking. In the present study, full-length human α-syn fibrils were injected into the medial forebrain bundle of rats, with an experimental endpoint of 6 months. Histological analysis was conducted to observe the pathological progress of abnormal endogenous α-syn aggregation and nerve fiber quality. Changes in gray and white matter integrity were quantitatively analyzed using voxel-based morphometry (VBM). Behavioral changes were observed over the 6-month period. Histological analysis showed reduced dopamine transporter levels in the striatum of the experimental rats; widespread abnormal endogenous α-syn accumulation; and damaged, sparse, and disordered nerve fibers in the experimental group. VBM showed that at 6 months after surgery, bilateral anterior limbic, bilateral inferior limbic, right hippocampal, and right cortical volumes had reduced, whereas thalamic volume had increased in the experimental group compared with that in the control group. Damage to the limbic and thalamic fiber structure may occur in the earlier stages of Parkinson's disease.

Topics: alpha-Synuclein; Animals; Brain; Disease Models, Animal; Male; Medial Forebrain Bundle; Parkinson Disease; Phosphorylation; Protein Aggregation, Pathological; Rats, Sprague-Dawley; Serine

Parkinson´s disease is characterized by the accumulation of proteinaceous aggregates in Lewy bodies and Lewy Neurites. The main component found in such aggregates is α-synuclein. Here, we investigate how bovine eye lens crystallin proteins influence the aggregation kinetics of α-synuclein at mildly acidic pH (5.5) where the underlying aggregation mechanism of this protein is dominated by secondary nucleation of monomers on fibril surface providing an autocatalytic amyloid amplification process. Bovine α-, βH- and γB-crystallins were found to display chaperone-like activity inhibiting α-synuclein aggregation. This effect was shown to be time-dependent, with early additions of α-crystallin capable of retarding and even inhibiting aggregation during the time frame of the experiment. The inhibitory nature of crystallins was further investigated using trap and seed kinetic experiments. We propose crystallins interact with mature α-synuclein fibrils, possibly binding along the surfaces and at fibril free ends, inhibiting both elongation and monomer-dependent secondary nucleation processes in a mechanism that may be generic to some chaperones that prevent the onset of protein misfolding related pathologies.

Topics: alpha-Crystallins; alpha-Synuclein; Amyloid; Animals; beta-Crystallins; Cattle; Cloning, Molecular; Escherichia coli; gamma-Crystallins; Humans; Kinetics; Lens, Crystalline; Parkinson Disease; Protein Aggregation, Pathological; Protein Binding

Protein fibrils drive the onset and progression of many diseases in a prion-like manner, i.e. they transcellular propagate through the extracellular space to health cells to initiate toxic aggregation as seeds. The conversion of native α-synuclein into filamentous aggregates in Lewy bodies is a hallmark of Parkinson's disease (PD). Copper and iron ions accumulate in PD brains, however, whether they influence the prion-like propagation of α-synuclein remain unclear. Here, we reported that copper/iron ions accelerate prion-like propagation of α-synuclein fibrils by promoting cellular internalization of α-synuclein fibrils, intracellular α-synuclein aggregation and the subsequent release of mature fibrils to the extracellular space to induce further propagation. Mechanistically, copper/iron ions enhanced α-synuclein fibrils internalization was mediated by negatively charged membrane heparan sulfate proteoglycans (HSPGs). α-Synuclein fibrils formed in the presence of copper/iron ions were more cytotoxic, causing increased ROS production, cell apoptosis, and shortened the lifespan of a C. elegans PD model overexpressing human α-synuclein. Notably, these deleterious effects were ameliorated by two clinically used chelators, triethylenetetramine and deferiprone. Together, our results suggest a new role for heavy metal ions, e.g. copper and iron, in the pathogenesis of PD through accelerating prion-like propagation of α-synuclein fibrils.

Topics: alpha-Synuclein; Amyloid; Animals; Brain; Chelating Agents; Copper; Disease Susceptibility; Gene Expression; Genes, Reporter; Heparan Sulfate Proteoglycans; Humans; Iron; Parkinson Disease; Prions; Protein Aggregates; Protein Aggregation, Pathological

α-Synuclein fibrillation is now regarded as a major pathogenic process in Parkinson's disease and its proteinaceous deposits are also detected in other neurological disorders including Alzheimer's disease. Therefore anti-amyloidegenic compounds may delay or prevent the progression of synucleinopathies disease. Molecular chaperones are group of proteins which mediate correct folding of proteins by preventing unsuitable interactions which may lead to aggregation. The objective of this study was to investigate the anti-amyloidogenic effect of molecular chaperone artemin on α-synuclein. As the concentration of artemin was increased up to 4 μg/ml, a decrease in fibril formation of α-synuclein was observed using thioflavin T (ThT) fluorescence and congo red (CR) assay. Transmission electron microscopy (TEM) images also demonstrated a reduction in fibrils in the presence of artemin. The secondary structure of α-synuclein was similar to its native form prior to fibrillation when incubated with artemin. A cell-based assay has shown that artemin inhibits α-synuclein aggregation and reduce cytotoxicity, apoptosis and reactive oxygen species (ROS) production. Our results revealed that artemin has efficient chaperon activity for preventing α-synuclein fibril formation and toxicity.

Topics: alpha-Synuclein; Apoptosis; Cell Line, Tumor; Cell Survival; Humans; Molecular Chaperones; Nerve Tissue Proteins; Protein Aggregates; Protein Aggregation, Pathological; Reactive Oxygen Species

Parkinson's disease (PD) and Alzheimer's disease (AD) are common neurodegenerative disorders of the elderly and, therefore, affect a growing number of patients worldwide. Both diseases share, as a common hallmark, the accumulation of characteristic protein aggregates, known as Lewy bodies (LB) in PD, and neurofibrillary tangles in AD. LBs are primarily composed of misfolded α-synuclein (aSyn), and neurofibrillary tangles are primarily composed of tau protein. Importantly, upon pathological evaluation, most AD and PD/Lewy body dementia cases exhibit mixed pathology, with the co-occurrence of both LB and neurofibrillary tangles, among other protein inclusions. Recent studies suggest that both aSyn and tau pathology can spread and propagate through neuronal connections. Therefore, it is important to investigate the mechanisms underlying aggregation and propagation of these proteins for the development of novel therapeutic strategies. Here, we assessed the effects of different pharmacological interventions on the aggregation and internalization of tau and aSyn. We found that anle138b and fulvic acid decrease aSyn and tau aggregation, that epigallocatechin gallate decreases aSyn aggregation, and that dynasore reduces tau internalization. Establishing the effects of small molecules with different chemical properties on the aggregation and spreading of aSyn and tau will be important for the development of future therapeutic interventions.

Topics: alpha-Synuclein; Alzheimer Disease; Benzodioxoles; Benzopyrans; Brain; Catechin; Cells, Cultured; Humans; Hydrazones; Lewy Bodies; Molecular Targeted Therapy; Neurofibrillary Tangles; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Pyrazoles; tau Proteins

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor impairments. Most PD drugs act by improving motor impairments, whereas very few drugs that efficiently recover PD-related neuropathological features, particularly α-synuclein-related toxicity, have been developed. In this study, we found that papaverine (PAP) attenuated behavioral deficits and protected against nigrostriatal dopaminergic degeneration in the subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid (MPTP/P) mouse model of PD. Histological analysis of tissue dissected from mice sacrificed nearly 3 weeks after the completion of treatment revealed that PAP significantly ameliorated microglia/astrocyte activation in the striatum and substantia nigra of MPTP/P-treated mice. In addition, PAP diminished α-synuclein expression and aggregation in this model. Furthermore, PAP inhibited the phosphorylation of α-synuclein at serine 129, which may underlie the observed reduction in α-synuclein aggregation. PAP also reduced the expression of matrix metalloproteinase-3 (MMP-3), and the MMP3-positive area co-labeled with thioflavin-S. Taken together, our data suggest that PAP inhibits dopaminergic neuronal cell death and α-synuclein aggregation by suppressing neuroinflammation and MMP-3 expression in the subacute MPTP/P mouse model of PD. Accordingly, PAP may be a promising drug for the treatment of PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; alpha-Synuclein; Animals; Astrocytes; Disease Models, Animal; Dopaminergic Neurons; Male; Matrix Metalloproteinase 3; Mice, Inbred C57BL; Microglia; MPTP Poisoning; Neuroprotective Agents; Neurotoxins; Papaverine; Protein Aggregation, Pathological

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies.

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Humans; Inclusion Bodies; Mice; Mice, Transgenic; Microglia; Neurons; Protein Aggregation, Pathological; Protein Conformation; Synucleinopathies

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson's disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloid; Animals; Cell Line; HSC70 Heat-Shock Proteins; Humans; Mice; Models, Molecular; Neurons; Parkinson Disease; Peptides; Prions; Protein Aggregates; Protein Aggregation, Pathological; Sodium-Potassium-Exchanging ATPase

The present study was aimed to study the effect of kaempferol, on the transgenic Drosophila model of Parkinson's disease. Kaempferol was added in the diet at final concentration of 10, 20, 30 and 40 µM and the effect was studied on various cognitive and oxidative stress markers. The results of the study showed that kaempferol, delayed the loss of climbing ability as well as the activity of PD flies in a dose dependent manner compared to unexposed PD flies. A dose-dependent reduction in oxidative stress markers was also observed. Histopathological examination of fly brains using anti-tyrosine hydroxylase immunostaining has revealed a significant dose-dependent increase in the expression of tyrosine hydroxylase in PD flies exposed to kaempferol. Molecular docking results revealed that kaempferol binds to human alpha synuclein at specific sites that might results in the inhibition of alpha synuclein aggregation and prevents the formation of Lewy bodies.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila; Humans; Kaempferols; Lewy Bodies; Motor Activity; Parkinson Disease; Protein Aggregation, Pathological; Tyrosine 3-Monooxygenase

Topics: alpha-Synuclein; Bacterial Translocation; COVID-19; Dysbiosis; Enteric Nervous System; Gastrointestinal Microbiome; Humans; Inflammation; Lipopolysaccharides; Microglia; Models, Biological; Neuroimmunomodulation; Parkinson Disease; Protein Aggregation, Pathological; Risk Factors; SARS-CoV-2; Vagus Nerve

Protein aggregates are the pathogenic hallmarks of many different neurodegenerative diseases and include the accumulation of α-synuclein, the main component of Lewy bodies found in Parkinson's disease. Aggresomes are closely-related, cellular accumulations of misfolded proteins. They develop in a juxtanuclear position, adjacent to the centrosome, the microtubule organizing centre of the cell, and share some protein components. Despite the long-standing observation that aggresomes/Lewy bodies and the centrosome sit side-by-side in the cell, no studies have been done to see whether these protein accumulations impede organelle function. We investigated whether the formation of aggresomes affected key centrosome functions: its ability to organise the microtubule network and to promote cilia formation. We find that when aggresomes are present, neuronal cells are unable to organise their microtubule network. New microtubules are not nucleated and extended, and the cells fail to respond to polarity cues. Since neurons are polarised, ensuring correct localisation of organelles and the effective intracellular transport of neurotransmitter vesicles, loss of centrosome activity could contribute to functional deficits and neuronal cell death in Parkinson's disease. In addition, we provide evidence that many cell types, including dopaminergic neurons, cannot form cilia when aggresomes are present, which would affect their ability to receive extracellular signals.

Topics: alpha-Synuclein; Animals; Biomarkers; Cell Line; Cell Movement; Centrosome; Cilia; Humans; Lewy Bodies; Microtubules; Organogenesis; Parkinson Disease; Protein Aggregation, Pathological; Rats; Zebrafish

α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology.. Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test.. Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology.. Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

Topics: alpha-Synuclein; Animals; Humans; Mice; Neurons; Protein Aggregation, Pathological; Rats; Rats, Sprague-Dawley

Toxic abnormal aggregation of α-synuclein (α-Syn) is a feature of Parkinson's disease. Several biochemical and biophysical studies have demonstrated that many post-translational modifications (PTM) of α-Syn could distinctly alleviate its oligomerization-mediated toxicity. Recently, a compelling link is emerging between the PTM O-GlcNAcylation (O-GlcNAc) and protein aggregation, yet the underlying molecular mechanism remains unclear. Based on the all-atom molecular dynamics simulations, we found that O-GlcNAc modifications can suppress the process of oligomerization of α-Syn aggregates via a steric effect-the additional O-linked glycosyl group disrupts the formation of hydrogen bonds (H-bonds) between α-Syn monomers. Besides, we proposed a theoretical model to further capture the physical mechanism of α-Syn aggregation/disaggregation in the absence/presence of O-GlcNAc-modified α-Syn. Our findings unveil the molecular mechanism of the O-GlcNAc-induced inhibition of α-Syn oligomerization, which may help to understand how O-GlcNAc prevents the oligomerization of other proteins and provides the guideline for the development of O-GlcNAc-based therapeutic strategies in neurodegenerative diseases.

Topics: Acylation; alpha-Synuclein; Models, Biological; Neurodegenerative Diseases; Polymerization; Protein Aggregation, Pathological; Protein Processing, Post-Translational

Aggregation of α-synuclein (α-syn) is one of the central hypotheses for Parkinson's disease (PD), therefore, its inhibition and disaggregation is an optimistic approach for the treatment of PD. Here, we report design, synthesis and in-vitro efficacy studies of a series of diphenyl triazine hybrids as potential inhibitors of α-syn fibrillogenesis. From the docking studies, we concluded that compounds A1, A2, A4, A8 and A9 display promising binding affinity with the essential residues of α-syn with binding energy values: -6.0, -7.0, -6.3, -6.6 and -6.7 kcal/mol respectively. The target compounds were synthesized using multistep organic synthesis reactions. Compounds A1, A2 A4, A8 and A9 showed a significant lowering of the α-syn fibril formation during Thioflavin-T assay and fluorescence microscopy. In addition, these compounds A1, A2, A4, A8 and A9 also proved to be good disaggregators in the pre-aggregated form of α-syn. Most of the compounds exhibited no cytotoxicity in mouse embryonic fibroblast (MEF) and human adenocarcinomic alveolar basal epithelial cells (A549) except A2. Overall, diphenyl triazine-based compounds can be further investigated for the treatment of synucleinopathies and for Lewy body dementia in which α-syn is predominantly observed.

Topics: alpha-Synuclein; Amyloid; Biphenyl Compounds; Drug Design; Humans; Models, Molecular; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Triazines

Parkinson's Disease (PD) is a progressive neurodegenerative disease characterized by the presence of proteinaceous aggregates of αSynuclein (αSyn) in the dopaminergic neurons. Chaperones are key components of the proteostasis network that are able to counteract αSyn's aggregation, as well as its toxic effects. Clusterin (CLU), a molecular chaperone, was consistently found to interfere with Aβ aggregation in Alzheimer's Disease (AD). However, its role in PD pathogenesis has yet to be extensively investigated. In this study, we assessed the involvement of CLU in the αSyn aggregation process by using SH-SY5Y cells stably overexpressing αSyn (SH-Syn). First, we showed that αSyn overexpression caused a strong increase in CLU expression without affecting levels of Hsp27, Hsp70, and Hsp90, which are the chaperones widely recognized to counteract αSyn burden. Then, we demonstrated that αSyn aggregation, induced by proteasome inhibition, determines a strong increase of CLU in insoluble aggregates. Remarkably, we revealed that CLU down-regulation results in an increase of αSyn aggregates in SH-Syn without significantly affecting cell viability and the Unfolded Protein Response (UPR). Furthermore, we demonstrated the direct molecular interaction between CLU and αSyn via a co-immunoprecipitation (co-IP) assay. All together, these findings provide incontrovertible evidence that CLU is an important player in the response orchestrated by the cell to cope with αSyn burden.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Clusterin; Dopaminergic Neurons; Gene Expression Regulation; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Molecular Chaperones; Parkinson Disease; Protein Aggregation, Pathological; Unfolded Protein Response

Aggregation of α-synuclein (α-syn) is associated with the manifestation of various pathogenic synucleinopathies, including Parkinson's disease attributed to both genetic and environmental stress factors. The initial events triggering α-syn aggregation and disease initiation due to environmental stress factors are still largely unknown. Here, to understand the mechanism of misfolding and aggregation initiation, we induced α-syn aggregation with rotenone, an established chemical inducer of PD like symptoms. We found that rotenone accelerates the formation of structurally distinct oligomers and fibrils that act as templates and increase the formation of conformers capable of spreading to the neighboring neuronal cells. Molecular dynamics simulations and NMR studies revealed the involvement of NAC region and formation of helical conformations resulting in structural variations in oligomers and fibrils. These structural variations affect the cytotoxic potential of oligomers and fibrils, where, the beta sheet rich oligomers and fibrils alter the membrane potential of neuronal cells and lead to early apoptosis. Our results describe the initial mechanistic events in pathogenic protein aggregation, where initial structural alterations in response to external stress factors dictate the toxicity of resulting conformers. This information will further provide insights in the understanding of protein aggregation, disease progression and pathogenesis.

Topics: alpha-Synuclein; Biopolymers; Circular Dichroism; Environmental Pollutants; Humans; Kinetics; Microscopy, Electron, Transmission; Molecular Dynamics Simulation; Parkinson Disease; Protein Aggregation, Pathological; Protein Structure, Secondary; Risk Factors; Rotenone

The deposition of highly ordered fibrillar-type aggregates into inclusion bodies is a hallmark of neurodegenerative diseases such as Parkinson's disease. The high stability of such amyloid fibril aggregates makes them challenging substrates for the cellular protein quality-control machinery

Topics: Adenosine Triphosphate; alpha-Synuclein; Amyloid; Entropy; HSP110 Heat-Shock Proteins; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Hydrolysis; Models, Biological; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

The ubiquitous heat shock protein 70 (HSP70) family consists of ATP-dependent molecular chaperones, which perform numerous cellular functions that affect almost all aspects of the protein life cycle from synthesis to degradation

Topics: alpha-Synuclein; Amyloid; Binding Sites; Glycine; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Molecular Chaperones; Mutation; Phenylalanine; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Domains; Sequence Deletion; Substrate Specificity

The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. One of the main aspects prohibiting effective aggregation tracking is the environment's effect on amyloid-specific dyes, namely thioflavin-T (ThT). Currently, there are only a few studies hinting at ionic strength being one of the factors that modulate the dye's binding affinity and fluorescence intensity. In this work we explore this effect under a range of ionic strength conditions, using insulin, lysozyme, mouse prion protein, and α-synuclein fibrils. We show that ionic strength is an extremely important factor affecting both the binding affinity, as well as the fluorescence intensity of ThT.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Animals; Benzothiazoles; Binding Sites; Fluorescence; Humans; Insulin; Kinetics; Mice; Osmolar Concentration; Parkinson Disease; Prion Proteins; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in

Topics: alpha-Synuclein; Animals; Caenorhabditis elegans; Cell Line; DNA-Binding Proteins; Endopeptidase Clp; Escherichia coli; Genetic Variation; Heat-Shock Proteins; HEK293 Cells; Humans; Neurodegenerative Diseases; Protein Aggregation, Pathological; Protein Folding; Proteostasis Deficiencies; RNA-Binding Protein FUS; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Accumulation of α-synuclein (α-Syn) is a remarkable pathology for Parkinson's disease (PD), therefore clearing it is possibly a promising strategy for treating PD. Aberrant copper (Cu(II)) homeostasis and oxidative stress play critical roles in the abnormal aggregation of α-Syn in the progress of PD. It is reported that the polyphenol (-)-epi-gallocatechin gallate (EGCG) can inhibit α-Syn fibrillation and aggregation, disaggregate α-Syn mature fibrils, as well as protect α-Syn overexpressed-PC12 cells against damage. Also, previous studies have reported that EGCG can chelate many divalent metal ions. What we investigate here is whether EGCG can interfere with the Cu(II) induced fibrillation of α-Syn and protect the cell viability. In this work, on a molecular and cellulaire basis, we demonstrated that EGCG can form a Cu(II)/EGCG complex, leading to the inhibition of Cu(II)-induced conformation transition of α-Syn from random coil to β-sheet, which is a dominant structure in α-Syn fibrils and aggregates. Moreover, we found that the mixture of Cu(II) and EGCG in a molar ratio from 0.5 to 2 can efficiently inhibit this process. Furthermore, we demonstrated that in the α-Syn transduced-PC12 cells, EGCG can inhibit the overexpression and fibrillation of α-Syn in the cells, and reduce Cu(II)-induced reactive oxygen species (ROS), protecting the cells against Cu(II)-mediated toxicity.

Topics: alpha-Synuclein; Amyloidogenic Proteins; Animals; Catechin; Cell Line; Copper; Neuroprotective Agents; Nuclear Magnetic Resonance, Biomolecular; Oxidative Stress; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Rats; Reactive Oxygen Species

Parkinson's disease etiology involves amyloid formation by α-synuclein (αSyn).

Topics: Acetylation; alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Humans; Parkinson Disease; Polymorphism, Genetic; Protein Aggregation, Pathological; Protein Domains; Protein Processing, Post-Translational; Protein Structure, Secondary

α-synucleopathies are protein-misfolding disorders occur primarily due to aggregation and toxicity of α-synuclein. This study characterized the small molecule AGK2 as a modifier of α-synuclein mediated toxicity in an autophagy dependent manner in both yeast and mammalian cell line models. In yeast system, AGK2 enhances autophagy to clear toxic α-synuclein aggregates in an autophagy dependent manner. Autophagy flux analyses revealed that AGK2 induces autophagy especially autolysosomes. Importantly, AGK2 induces autophagy in an mTOR independent manner. These features enable AGK2 to exert cytoprotective potential against α-synuclein mediated toxicity in different model systems.

Topics: alpha-Synuclein; Autophagosomes; Autophagy; Cytoprotection; Drug Evaluation, Preclinical; Furans; HeLa Cells; Humans; Protein Aggregation, Pathological; Quinolines; Saccharomyces cerevisiae; Synucleinopathies

Secretagogin (SCGN) is a secreted calcium sensor that has emerged as a potential multifunctional protein of neuroendocrine cells. A significantly reduced level of expression of SCGN has been reported in the hippocampus of a mouse model of Alzheimer's disease (AD) and in Parkinson's patients, although the biochemical implications and mechanistic underpinnings of the altered SCGN expression in neurodegenerative diseases remain unknown. We have pursued the interaction of SCGN with α-synuclein that we discovered in impartial pull-down analyses to decode the SCGN interactome. SCGN physically binds α-synuclein and rescues it from detrimental fibrillation. Correspondingly, it is observed that a significant reduction in the cytotoxicity of α-synuclein fibrils is caused by SCGN. We map these antifibrillar attributes to the central region and C-terminal domain of SCGN, while the N-terminal domain is not essential for this activity. On the basis of these results, a broader neuroprotective function of SCGN by proficient chaperone action is proposed. An intriguing correlation of this interaction with a reduced level of expression of SCGN in neurodegenerative diseases shall inspire further studies of the physiological role of SCGN in precluding pathological protein aggregation.

Topics: alpha-Synuclein; Animals; Cell Line; Mice; Models, Molecular; Protein Aggregation, Pathological; Protein Binding; Protein Interaction Domains and Motifs; Secretagogins

Amyloid formation is a process involving interconverting protein species and results in toxic oligomers and fibrils. Aggregated alpha-synuclein (αS) participates in neurodegenerative maladies, but a closer understanding of the early αS polymerization stages and polymorphism of heritable αS variants is sparse still. Here, we distinguished αS oligomer and protofibril interconversions in Thioflavin T polymerization reactions. The results support a hypothesis reconciling the nucleation-polymerization and nucleation-conversion-polymerization models to explain the dissimilar behaviors of wild-type and the A53T mutant. Cryo-electron microscopy with a direct detector shows the polymorphic nature of αS fibrils formed by heritable A30P, E46K, and A53T point mutations. By showing that A53T rapidly nucleates competent species, continuously elongates fibrils in the presence of increasing amounts of seeds, and overcomes wild-type surface requirements for growth, our findings place A53T with features that may explain the early onset of familial Parkinson's disease cases bearing this mutation.

Topics: Age of Onset; alpha-Synuclein; Amyloid; Cryoelectron Microscopy; Humans; Kinetics; Microscopy, Electron, Transmission; Parkinson Disease; Point Mutation; Protein Aggregation, Pathological

Deposits of amyloid fibrils of α-synuclein are the histological hallmarks of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, with hereditary mutations in α-synuclein linked to the first two of these conditions. Seeing the changes to the structures of amyloid fibrils bearing these mutations may help to understand these diseases. To this end, we determined the cryo-EM structures of α-synuclein fibrils containing the H50Q hereditary mutation. We find that the H50Q mutation results in two previously unobserved polymorphs of α-synuclein: narrow and wide fibrils, formed from either one or two protofilaments, respectively. These structures recapitulate conserved features of the wild-type fold but reveal new structural elements, including a previously unobserved hydrogen-bond network and surprising new protofilament arrangements. The structures of the H50Q polymorphs help to rationalize the faster aggregation kinetics, higher seeding capacity in biosensor cells and greater cytotoxicity that we observe for H50Q compared to wild-type α-synuclein.

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloid; Cryoelectron Microscopy; HEK293 Cells; Humans; Models, Molecular; Parkinson Disease; Point Mutation; Protein Aggregation, Pathological; Protein Conformation

Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson's disease (PD) and related α-synucleopathies.. Using chemical biology, we identified a small molecule inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy.. In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation.. These protective mechanisms ensure the negation of Parkinson's disease related motor impairments. FUND: This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP.

Topics: alpha-Synuclein; Animals; Biomarkers; Cell Line; Cytokines; Disease Models, Animal; Humans; Immunohistochemistry; Interleukin-6; Lipopolysaccharides; Macroautophagy; Male; Mice; Microglia; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Oxidative Stress; Protein Aggregates; Protein Aggregation, Pathological; Pyridones; Pyrimidines

Alpha-synuclein (αS) fibrils are toxic to cells and contribute to the pathogenesis and progression of Parkinson's disease and other synucleinopathies. β-Synuclein (βS), which co-localizes with αS, has been shown to provide a neuroprotective effect, but the molecular mechanism by which this occurs remains elusive. Here we show that αS fibrils formed in the presence of βS are less cytotoxic, exhibit reduced cell seeding capacity and are more resistant to fibril shedding compared to αS fibrils alone. Using solid-state NMR, we found that the overall structure of the core of αS fibrils when co-incubated with βS is minimally perturbed, however, the dynamics of Lys and Thr residues, located primarily in the imperfect KTKEGV repeats of the αS N-terminus, are increased. Our results suggest that amyloid fibril dynamics may play a key role in modulating toxicity and seeding. Thus, enhancing the dynamics of amyloid fibrils may be a strategy for future therapeutic targeting of neurodegenerative diseases.

Topics: alpha-Synuclein; Amyloid; beta-Synuclein; Brain; Cell Line, Tumor; Humans; Magnetic Resonance Spectroscopy; Microscopy, Atomic Force; Microscopy, Confocal; Microscopy, Fluorescence; Protein Aggregation, Pathological

Parkinson's disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Brain; Female; Humans; Male; Models, Molecular; Multiple System Atrophy; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation; Synucleinopathies; Tissue Extracts

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.

Topics: alpha-Synuclein; Animals; Axons; Hippocampus; Mice, Inbred C57BL; Mice, Knockout; Neurons; Organ Culture Techniques; Protein Aggregation, Pathological; Synucleinopathies

Protein aggregation in neurons is a prominent pathological mark of neurodegeneration. In Parkinson's disease (PD), inclusions of the α-Synuclein (α-Syn) protein form the Lewy bodies in dopaminergic (DA) neurons. Ectopic expression of human α-Syn inDrosophila neurons leads to the protein accumulation, degeneration of DA neurons and locomotor deterioration, and therefore constitutes the present fly PD model. Yet, this model does not enable to study the role of genes, which are essential for normal development, in neurodegeneration.. Using the Gal80/Gal4/UAS system we optimized the current PD model, such that only the adult stage of the fly is affected by α-Syn expression in the brain.. The symptoms of neurodegeneration typifying the classic model, including reduced locomotor ability, shortened lifespan and the loss of DA neurons, are significantly demonstrated in the novel adult fly PD model.. The neurodegeneration symptoms exhibited by the innovative model are very similar to those manifested in the recognized one.. Specific expression of α-Syn in the adult fly brain enables the investigation of developmental genes involved in neurodegeneration, thereby deciphering gene functions and molecular mechanisms. It may further be used for addressing therapeutic targets and treatment platforms specifically during adult stages.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Behavior, Animal; Brain; Disease Models, Animal; Dopaminergic Neurons; Drosophila melanogaster; Female; Neurons; Parkinson Disease; Protein Aggregation, Pathological

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound. In this study, we demonstrated that RA could protect against the degeneration of the nigrostriatal dopaminergic system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease (PD). In addition, RA could inhibit MPTP-induced decrease of superoxide dismutase (SOD) and tyrosine hydroxylase (TH) and increase in nigral iron content. Further studies elucidated the effects of RA on iron-induced neurotoxicity and the possible underlying mechanisms in the SK-N-SH cells. Results showed that iron could induce a decrease in the mitochondrial transmembrane potential and result in α-synuclein aggregation in the SK-N-SH cells, which could be restored by RA pretreatment. Further results showed RA pretreatment could inhibit iron-induced α-synuclein aggregation by up-regulating hemeoxygenase-1 (HO-1). In addition, iron could increase the mRNA levels of α-synuclein via iron responsive element/iron regulatory protein (IRE/IRP) system. RA pretreatment could decrease the mRNA levels of α-synuclein by decreasing the protein levels of IRP1. These results indicated that RA protected against iron-induced α-synuclein aggregation by up-regulating HO-1 and inhibiting α-synuclein expression.

Topics: alpha-Synuclein; Animals; Antiparkinson Agents; Cell Line, Tumor; Cinnamates; Depsides; Dopaminergic Neurons; Humans; Iron; Male; Mice, Inbred C57BL; MPTP Poisoning; Neuroprotective Agents; Protein Aggregation, Pathological; Random Allocation; Rosmarinic Acid

It has been suggested that aggregation of α-synuclein (α-syn) into oligomers leads to neurodegeneration in Parkinson's disease (PD), but intravenous immunoglobulin (IVIG) which contains antibodies against α-syn monomers and oligomers fails to treat PD mouse model. The reason may be because IVIG contains much low level of antibodies against α-syn, and of which only a small part can penetrate the blood-brain barrier, resulting in an extremely low level of effective antibodies in the brain, and limiting the beneficial effect of IVIG on PD mice. Here, we first isolated naturally occurring autoantibodies against α-syn (NAbs-α-syn) from IVIG. Our further investigation results showed that NAbs-α-syn inhibited α-syn aggregation and attenuated α-syn-induced cytotoxicity in vitro. Compared with vehicles, NAbs-α-syn significantly attenuated the memory and motor deficits by reducing the levels of soluble α-syn, total human α-syn and α-syn oligomers, decreasing the intracellular p-α-syn

Topics: alpha-Synuclein; Animals; Autoantibodies; Brain; Disease Models, Animal; Immunization, Passive; Immunoglobulins, Intravenous; Mice, Transgenic; Microglia; Motor Activity; Parkinson Disease; Protein Aggregation, Pathological; Spatial Memory

The intestinal microbiota actively converts dietary flavanols into phenolic acids, some of which are bioavailable in vivo and may promote resilience to select neurological disorders by interfering with key pathologic mechanisms. Since every person harbors a unique set of gut bacteria, we investigated the influence of the gut microbiota's interpersonal heterogeneity on the production and bioavailability of flavonoid metabolites that may interfere with the misfolding of alpha (α)-synuclein, a process that plays a central role in Parkinson's disease and other α-synucleinopathies. We generated two experimental groups of humanized gnotobiotic mice with compositionally diverse gut bacteria and orally treated the mice with a flavanol-rich preparation (FRP). The two gnotobiotic mouse groups exhibited distinct differences in the generation and bioavailability of FRP-derived microbial phenolic acid metabolites that have bioactivity towards interfering with α-synuclein misfolding or inflammation. We also demonstrated that these bioactive phenolic acids are effective in modulating the development and progression of motor dysfunction in a Drosophila model of α-synucleinopathy. Lastly, through in vitro bacterial fermentation studies, we identified select bacteria that are capable of supporting the generation of these bioavailable and bioactive phenolic acids. Outcomes from our studies provide a better understanding of how interpersonal heterogeneity in the gut microbiota differentially modulates the efficacy of dietary flavanols to protect against select pathologic mechanisms. Collectively, our findings provide the basis for future developments of probiotic, prebiotic, or synbiotic approaches for modulating the onset and/or progression of α-synucleinopathies and other neurological disorders involving protein misfolding and/or inflammation.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Biological Availability; Brain; Disease Models, Animal; Drosophila; Female; Gastrointestinal Microbiome; Humans; Male; Mice, Inbred C57BL; Parkinson Disease; Polyphenols; Protein Aggregation, Pathological; Protein Folding; Specific Pathogen-Free Organisms; Synucleinopathies

Six α-synuclein (aSyn) point mutations are currently known to be associated with familial parkinsonism: A30P, E46K, H50Q, G51D, A53E, and A53T. We performed a comprehensive in vitro analysis to study the impact of all aSyn mutations on lipid binding and aggregation behavior. Markedly reduced lipid binding of A30P, moderately attenuated binding of G51D, and only very slightly reduced binding for the other mutants were observed. A30P was particularly prone to form metal ion induced oligomers, whereas A53T exhibited only weak tendencies to form oligomers. In turn, fibril formation occurred rapidly in H50Q, G51D, and A53T, but only slowly in A30P, suggesting mutants prone to form oligomers tend to form fibrils to a lesser extent. This was supported by the observation that fibril formation of wild type aSyn, A30P, and A53T was impaired in the presence of ferric iron. Additionally, we found the aggregation kinetics of mixtures of A30P or A53T and wt aSyn to be determined by the faster aggregating aSyn variant. Our results implicate differential mechanisms playing a role in aSyn pathology on the molecular level. This might contribute to a better understanding of Parkinson's disease pathogenesis and provide potential links to develop prevention strategies and disease-modifying therapy.

Topics: alpha-Synuclein; Humans; Lipids; Mutation; Parkinson Disease; Point Mutation; Protein Aggregates; Protein Aggregation, Pathological; Spectrometry, Fluorescence

Gaucher disease (GD) patients and carriers of GD mutations have a higher propensity to develop Parkinson's disease (PD) in comparison to the non-GD population. This implies that mutant GBA1 allele is a predisposing factor for the development of PD. One of the major characteristics of PD is the presence of oligomeric α-synuclein-positive inclusions known as Lewy bodies in the dopaminergic neurons localized to the substantia nigra pars compacta. In the present study we tested whether presence of human mutant GCase leads to accumulation and aggregation of α-synuclein in two models: in SHSY5Y neuroblastoma cells endogenously expressing α-synuclein and stably transfected with human GCase variants, and in Drosophila melanogaster co-expressing normal human α-synuclein and mutant human GCase. Our results showed that heterologous expression of mutant, but not WT, human GCase in SHSY5Y cells, led to a significant stabilization of α-synuclein and to its aggregation. In parallel, there was also a significant stabilization of mutant, but not WT, GCase. Co-expression of human α-synuclein and human mutant GCase in the dopaminergic cells of flies initiated α-synuclein aggregation, earlier death of these cells and significantly shorter life span, compared with flies expressing α-synuclein or mutant GCase alone. Taken together, our results strongly indicate that human mutant GCase contributes to accumulation and aggregation of α-synuclein. In the fly, this aggregation leads to development of more severe parkinsonian signs in comparison to flies expressing either mutant GCase or α-synuclein alone.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Disease Models, Animal; Dopaminergic Neurons; Drosophila melanogaster; Gaucher Disease; Gene Expression Regulation; Glucosylceramidase; Heterozygote; Humans; Lewy Bodies; Lysosomes; Mutation; Parkinson Disease; Pars Compacta; Protein Aggregation, Pathological

The aggregation of α-synuclein (α-Syn) has been implicated strongly in Parkinson's disease (PD). The intrinsically disordered nature of α-Syn makes this protein prone to self-association or heteroassociation with another protein or lipid. While conformational fluctuation and free radical chemistry have been shown to play important roles in its ability toward self- and heteroassociation, any systematic understanding of their contributions is missing. Here, we report an in vitro investigation of the interaction between α-Syn and cytochrome c in the oxidized (cyt c III) and reduced forms (cyt c II), in which cyt c III was found to induce a large compaction of α-Syn and inhibit the aggregation by favoring a hetero-dityrosine bond formation. In contrast, the presence of cyt c II did not result in any compaction and its presence was found to facilitate α-Syn aggregation. The variation in the charge distribution of the surface residues of cyt c III and cyt c II is expected to play a decisive role in their interaction with α-Syn.

Topics: alpha-Synuclein; Cytochromes c; Enzyme Inhibitors; Escherichia coli; Free Radicals; Humans; Imidazoles; Oxidation-Reduction; Protein Aggregation, Pathological; Protein Binding; Recombinant Proteins

A compelling link is emerging between the posttranslational modification O-GlcNAc and protein aggregation. A prime example is α-synuclein, which forms toxic aggregates that are associated with neurodegeneration in Parkinson's and related diseases. α-Synuclein has been shown to be O-GlcNAcylated at nine different positions in in vivo proteomics experiments from mouse and human tissues. This raises the possibility that O-GlcNAc may alter the aggregation of this protein and could be both an important biological mediator of neurodegeneration and also a therapeutic target. Here, we expand upon our previous research in this area through the chemical synthesis of six site-specifically O-GlcNAcylated variants of α-synuclein. We then use a variety of biochemical experiments to show that O-GlcNAc in general inhibits the aggregation of α-synuclein but can also alter the structure of α-synuclein aggregates in site-specific ways. Additionally, an α-synuclein protein bearing three O-GlcNAc modifications can inhibit the aggregation of unmodified protein. Primary cell culture experiments also show that several of the O-GlcNAc sites inhibit the toxicity of extracellular α-synuclein fibers that are likely culprits in the spread of Parkinson's disease. We also demonstrate that O-GlcNAcylation can inhibit the aggregation of an aggressive mutant of α-synuclein, indicating that therapies currently in development that increase this modification might be applied in animal models that rely on this mutant. Finally, we also show that the pan-selective antibody for O-GlcNAc does not generally recognize this modification on α-synuclein, potentially explaining why it remains understudied. These results support further development of O-GlcNAcylation tools and therapeutic strategies in neurodegenerative diseases.

Topics: Acetylglucosamine; Acylation; alpha-Synuclein; Animals; Cells, Cultured; Female; Mice; Mice, Inbred C57BL; Parkinson Disease; Pregnancy; Protein Aggregation, Pathological; Protein Processing, Post-Translational

Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets.

Topics: alpha-Synuclein; Animals; Autophagy; Cell Line, Tumor; Escherichia coli; Glioma; Humans; Lysosomes; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Rats; Rats, Wistar; Recombinant Proteins; Sirolimus; Trehalose

α-Synuclein is a protein that aggregates as amyloid fibrils in the brains of patients with Parkinson's disease and dementia with Lewy bodies. Small oligomers of α-synuclein are neurotoxic and are thought to be closely associated with disease. Whereas α-synuclein fibrillization and fibril morphologies have been studied extensively with various methods, the earliest stages of aggregation and the properties of oligomeric intermediates are less well understood because few methods are able to detect and characterize early-stage aggregates. We used fluorescence spectroscopy to investigate the early stages of aggregation by studying pairwise interactions between α-synuclein monomers, as well as between engineered tandem oligomers of various sizes (dimers, tetramers, and octamers). The hydrodynamic radii of these engineered α-synuclein species were first determined by fluorescence correlation spectroscopy and dynamic light scattering. The rate of pairwise aggregation between different species was then monitored using dual-color fluorescence cross-correlation spectroscopy, measuring the extent of association between species labelled with different dyes at various time points during the early aggregation process. The aggregation rate and extent increased with tandem oligomer size. Self-association of the tandem oligomers was found to be the preferred pathway to form larger aggregates: interactions between oligomers occurred faster and to a greater extent than interactions between oligomers and monomers, indicating that the oligomers were not as efficient in seeding further aggregation by addition of monomers. These results suggest that oligomer-oligomer interactions may play an important role in driving aggregation during its early stages.

Topics: alpha-Synuclein; Genetic Engineering; Humans; Kinetics; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization; Recombinant Proteins; Solubility

The presence of αSN fibrils indisputably associates with the development of synucleinopathies. However, while certain fibril morphologies have been linked to downstream pathological phenotypes, others appear less harmful, leading to the concept of fibril strains, originally described in relation to prion disease. Indeed, the presence of fibrils does not associate directly with neurotoxicity. Rather, it has been suggested that the toxic compounds are soluble amyloidogenic oligomers, potentially co-existing with fibrils. Here, combining synchrotron radiation circular dichroism, transmission electron microscopy and binding assays on native plasma membrane sheets, we reveal distinct biological and biophysical differences between initial and matured fibrils, transformed within the timespan of few days. Immature fibrils are reservoirs of membrane-binding species, which in response to even gentle experimental changes release into solution in a reversible manner. In contrast, mature fibrils, albeit macroscopically indistinguishable from their less mature counterparts, are structurally robust, shielding the solution from the membrane active soluble species. We thus show that particular biological activity resides transiently with the fibrillating sample, distinct for one, but not the other, spontaneously formed fibril polymorph. These results shed new light on the principles of fibril polymorphism with consequent impact on future design of assays and therapeutic development.

Topics: alpha-Synuclein; Amyloid; Cell Membrane; Humans; Protein Aggregation, Pathological; Protein Binding; Solubility; Structure-Activity Relationship; Thermodynamics

The aggregation of α-synuclein (α-syn) into amyloid fibrils is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The mechanisms underlying the structural transition of soluble and innocuous α-syn to aggregated neurotoxic forms remains largely unknown. The disordered nature of α-syn has hampered the use of structure-based protein engineering approaches to elucidate the molecular determinants of this transition. The recent 3D structure of a pathogenic α-syn fibril provides a template for this kind of studies. The structure supports the NAC domain being a critical element in fibril formation, since it constitutes the core of the fibril, delineating a Greek-key motif. Here, we stapled the ends of this motif with a designed disulfide bond and evaluated its impact on the conformation, aggregation and toxicity of α-syn in different environments. The new covalent link biases the native structural ensemble of α-syn toward compact conformations, reducing the population of fully unfolded species. This conformational bias results in a strongly reduced fibril formation propensity both in the absence and in the presence of lipids and impedes the formation of neurotoxic oligomers. Our study does not support the Greek-key motif being already imprinted in early α-syn assemblies, discarding it as a druggable interface to prevent the initiation of fibrillation. In contrast, it suggests the stabilization of native, compact ensembles as a potential therapeutic strategy to avoid the formation of toxic species and to target the early stages of PD.

Topics: alpha-Synuclein; Amino Acid Motifs; Amino Acid Sequence; Amyloid; Disulfides; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Lipid Metabolism; Magnetic Resonance Spectroscopy; Mutation; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Solubility

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Line; Endoplasmic Reticulum; Glutathione; Glutathione Reductase; Homeostasis; Humans; Maleates; Muscle Cells; Neurons; Oxidation-Reduction; Peptides; Phenotype; Protein Aggregation, Pathological; Proteolysis; Proteostasis; Saccharomyces cerevisiae; Sequestosome-1 Protein

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by intracellular inclusions composed mostly of α-synuclein (Baba et al., Am J Pathol 152:879-884, 1998). How inclusion formation impacts neuronal function prior to death is key to understanding disease progression and identifying therapeutic windows. In the α-synuclein fibril model, exposure of primary neurons to α-synuclein fibrils induces endogenously expressed α-synuclein to form inclusions which closely resembles pathologic mechanisms in humans with PD and DLB (Volpicelli-Daley et al., Neuron 72, 57-71, 2011). In this model, at 7 days after exposure of neurons to fibrils, when there is no neuron death, inclusions in the axon selectively impair axonal transport of endosomes carrying the TrkB receptor and LC3-positive autophagosomes (Volpicelli-Daley et al., Mol Biol Cell 25:4010-4023, 2014). In addition, the frequency and amplitude of spontaneous Ca

Topics: alpha-Synuclein; Animals; Axonal Transport; Calcium; Cells, Cultured; Female; Inclusion Bodies; Mice; Molecular Imaging; Neurons; Pregnancy; Protein Aggregates; Protein Aggregation, Pathological; Pyramidal Cells

Here, we describe a detailed protocol to set up the αS-PMCA assay using αS synthetic aggregates in buffer and to accurately detect endogenous αS aggregates from human CSF samples. Given the amplificative nature of the technique, minute amounts of misfolded protein aggregates circulating in human bodily fluids can be multiplied and thereafter detected by more conventional methods, such as immune assays or fluorescence. Following these principles, αS-PMCA was standardized for the highly sensitive and specific detection of αS misfolded aggregates in cerebrospinal fluid (CSF) of patients with synucleinopathies.

Topics: alpha-Synuclein; Data Analysis; Humans; Protein Aggregates; Protein Aggregation, Pathological; Protein Folding; Proteostasis Deficiencies

The accumulation of intraneuronal inclusions containing misfolded alpha-synuclein (aSyn) within the central nervous system (CNS) is a common feature found in several neurodegenerative disorders including Parkinson's disease (PD). Emerging evidence indicates that aSyn amyloid fibrils, a configuration that is present within these characteristic inclusions, are capable of self-replicating by templating the conversion of endogenously expressed aSyn in neurons. Stereotaxic administration of synthetic α-synuclein preformed fibrils (PFFs) into the mouse brain has been shown to seed the formation of intracellular aSyn pathology reminiscent of Lewy body (LB) inclusions present in human PD and related synucleinopathies. Moreover, pathology can be targeted to specific CNS regions. This experimental approach provides a versatile platform for investigating PD-like LB pathology in vivo. We focus here on procedures for initiating aSyn inclusion formation at various regions of the mouse brain using computer-assisted motorized stereotaxic microinjection of aSyn PFFs and discuss appropriate strategies for controls and analysis.

Topics: alpha-Synuclein; Amyloid; Animals; Biomarkers; Brain; Immunohistochemistry; Inclusion Bodies; Mice; Neurons; Protein Aggregates; Protein Aggregation, Pathological; Stereotaxic Techniques

Alpha-synuclein oligomers are thought to be toxic mediators of Parkinson's disease and other alpha-synucleinopathies, but their histological detection in situ in diseased brain has been a challenge in the field for some time. Here we describe a method, the alpha-synuclein proximity ligation assay (AS-PLA), to detect alpha-synuclein oligomers in paraffin-embedded brain sections. Using AS-PLA previously unobserved alpha-synuclein oligomeric pathology is revealed.

Topics: alpha-Synuclein; Brain; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization

Misfolding and aggregation of α-synuclein are linked to many neurodegenerative disorders, including Parkinson's and Alzheimer's disease. Despite intense research efforts, detailed structural characterization of early conformational transitions that initiate and drive α-synuclein aggregation remains elusive often due to the low sensitivity and ensemble averaging of commonly used techniques. Single-molecule Förster resonance energy transfer (smFRET) provides unique advantages in detecting minor conformations that initiate protein pathologic aggregation. In this chapter, we describe an smFRET-based method for characterizing early conformational conversions that are responsible for α-synuclein self-assembly and aggregation.

Topics: alpha-Synuclein; Fluorescence Resonance Energy Transfer; Intrinsically Disordered Proteins; Neurodegenerative Diseases; Protein Aggregation, Pathological; Protein Conformation; Protein Folding; Spectrum Analysis

Proteinaceous α-synuclein-containing inclusions are found in affected brain regions in patients with Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These appear in neurons as Lewy bodies in both PD and DLB and as glial cytoplasmic inclusions (GCIs) in oligodendrocytes in MSA. The role they play in the pathology of the diseases is unknown, and relatively little is still known about their composition. By purifying the inclusions from the surrounding tissue and comprehensively analysing their protein composition, vital clues to the formation mechanism and role in the disease process may be found. In this study, Lewy bodies were purified from postmortem brain tissue from DLB cases (n = 2) and GCIs were purified from MSA cases (n = 5) using a recently improved purification method, and the purified inclusions were analysed by mass spectrometry. Twenty-one percent of the proteins found consistently in the GCIs and LBs were synaptic-vesicle related. Identified proteins included those associated with exosomes (CD9), clathrin-mediated endocytosis (clathrin, AP-2 complex, dynamin), retrograde transport (dynein, dynactin, spectrin) and synaptic vesicle fusion (synaptosomal-associated protein 25, vesicle-associated membrane protein 2, syntaxin-1). This suggests that the misfolded or excess α-synuclein may be targeted to inclusions via vesicle-mediated transport, which also explains the presence of the neuronal protein α-synuclein within GCIs.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Female; Humans; Inclusion Bodies; Lewy Bodies; Lewy Body Disease; Male; Middle Aged; Multiple System Atrophy; Oligodendroglia; Protein Aggregation, Pathological; Synaptic Vesicles

Deposition of α-synuclein into Lewy bodies and Lewy neurites is the hallmark of Parkinson's disease (PD). It is hypothesized that α-synuclein pathology spreads by a "prion-like" mechanism (i.e., by seeded aggregation or templated misfolding). Therefore, various extracellular α-synuclein conformers and/or posttranslational modifications may serve as biomarkers of disease or potential targets for novel interventions. To explore whether the antibody repertoires of PD patients contain anti-α-synuclein antibodies that can potentially be used as markers or immunotherapy, we interrogated peripheral IgG

Topics: Aged; alpha-Synuclein; Antibodies; B-Lymphocytes; HEK293 Cells; Humans; Lewy Bodies; Mesencephalon; Middle Aged; Mutation; Parkinson Disease; Protein Aggregation, Pathological

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson's disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils. This conformational remodeling preserved the dynamic adoption of α-helical conformations, which are essential for physiological membrane interactions. Oxidation-dependent NDGA cyclization was required for the interaction with monomeric α-synuclein. NDGA analog-pretreated α-synuclein did not aggregate even without NDGA-analogs in the aggregation mixture. Strikingly, NDGA-pretreated α-synuclein suppressed aggregation of naïve untreated aggregation-competent monomeric α-synuclein. Further, cyclized NDGA reduced α-synuclein-driven neurodegeneration in Caenorhabditis elegans. The cyclized NDGA analogs may serve as a platform for the development of small molecules that stabilize aggregation-resistant α-synuclein monomers without interfering with functional conformations yielding potential therapies for PD and related disorders.

Topics: alpha-Synuclein; Amyloid; Animals; Caenorhabditis elegans; Cell Membrane; Humans; Masoprocol; Parkinson Disease; Phospholipids; Protein Aggregation, Pathological

The aggregation of α-synuclein (αSyn) is considered a key pathophysiological feature of certain neurodegenerative disorders, collectively termed synucleinopathies. Given that a prion-like, cell-to-cell transfer of misfolded αSyn has been recognized in the spreading of αSyn pathology in synucleinopathies, we investigated the biological mechanisms underlying the propagation of the disease with respect to environmental neurotoxic stress. Considering the potential role of the divalent metal manganese (Mn

Topics: alpha-Synuclein; Animals; Cell Line; Corpus Striatum; Disease Models, Animal; Dopaminergic Neurons; Exosomes; Manganese; Mice; Parkinson Disease, Secondary; Prions; Protein Aggregation, Pathological

Parkinson's disease is characterized by the deterioration of dopaminergic neurons of substantia nigra pars compacta along with a substantial loss of noradrenergic neurons of the locus coeruleus, which is the major source of noradrenaline (NA) in the brain. We have investigated the interaction of NA with α-synuclein (α-syn), the major protein constituent of Lewy bodies that are the pathological hallmark of Parkinson's disease (PD). It is expected that NA, like dopamine, could bind to α-syn and modulate its aggregation propensity and kinetics, which could also contribute to the onset of PD. We have, thus, evaluated the thermodynamic parameters of interaction of NA with α-syn monomer as well as species formed at different stages during its fibrillation pathway and have investigated the conformational and aggregation properties using various spectroscopic and calorimetric techniques. Binding isotherms of NA with α-syn species formed at different time points in the pathway have been observed to be exothermic in nature, suggesting hydrogen bonding interactions and weak affinity with binding constants in the millimolar range in all the cases. The interaction site of NA for α-syn was determined using Förster resonance energy transfer measurements that resulted in its binding in close proximity (23 Å) of an Alexa-labeled A90C mutant of α-syn. Docking studies further suggested binding of NA to the C-terminal as well as the non-amyloid-β component (NAC) region of α-syn. We have shown that α-syn oligomerization into sodium dodecyl sulfate resistant, higher-order, β-sheet-rich species is dependent on the oxidation of NA. Under non-reducing conditions, NA was also found to disaggregate the intermediates, populated during the fibrillation pathway, which are more cytotoxic compared to amyloid fibrils, as observed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide cytotoxicity assay using a human neuroblastoma cell line. On the basis of these and earlier data, we propose that NA-induced formation of α-syn oligomers may contribute to the progressive loss of the noradrenergic neuronal population and the pronounced Lewy body deposition observed in patients with PD.

Topics: alpha-Synuclein; Amyloid; Cell Line, Tumor; Humans; Neurons; Norepinephrine; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Progressive aggregation of the protein alpha-synuclein (α-syn) and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are key histopathological hallmarks of Parkinson's disease (PD). Accruing evidence suggests that α-syn pathology can propagate through neuronal circuits in the brain, contributing to the progressive nature of the disease. Thus, it is therapeutically pertinent to identify modifiers of α-syn transmission and aggregation as potential targets to slow down disease progression. A growing number of genetic mutations and risk factors has been identified in studies of familial and sporadic forms of PD. However, how these genes affect α-syn aggregation and pathological transmission, and whether they can be targeted for therapeutic interventions, remains unclear. We performed a targeted genetic screen of risk genes associated with PD and parkinsonism for modifiers of α-syn aggregation, using an α-syn preformed-fibril (PFF) induction assay. We found that decreased expression of Lrrk2 and Gba modulated α-syn aggregation in mouse primary neurons. Conversely, α-syn aggregation increased in primary neurons from mice expressing the PD-linked LRRK2 G2019S mutation. In vivo, using LRRK2 G2019S transgenic mice, we observed acceleration of α-syn aggregation and degeneration of dopaminergic neurons in the SNpc, exacerbated degeneration-associated neuroinflammation and behavioral deficits. To validate our findings in a human context, we established a novel human α-syn transmission model using induced pluripotent stem cell (iPS)-derived neurons (iNs), where human α-syn PFFs triggered aggregation of endogenous α-syn in a time-dependent manner. In PD subject-derived iNs, the G2019S mutation enhanced α-syn aggregation, whereas loss of LRRK2 decreased aggregation. Collectively, these findings establish a strong interaction between the PD risk gene LRRK2 and α-syn transmission across mouse and human models. Since clinical trials of LRRK2 inhibitors in PD are currently underway, our findings raise the possibility that these may be effective in PD broadly, beyond cases caused by LRRK2 mutations.

Topics: alpha-Synuclein; Amyloid; Animals; Cells, Cultured; Cerebral Cortex; Exploratory Behavior; Glucosylceramidase; Hippocampus; Humans; Induced Pluripotent Stem Cells; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation, Missense; Neurons; Parkinson Disease; Pars Compacta; Primary Cell Culture; Protein Aggregation, Pathological; Recombinant Proteins; RNA Interference; Rotarod Performance Test

Parkinson's disease (PD) is a synucleinopathy that has multiple neuropathological characteristics, with nigrostriatal dopamine system degeneration being a core feature. Current models of PD pathology typically fail to recapitulate several attributes of the pathogenic process and neuropathology. We aimed to define the effects of combining a mouse model exhibiting multiple PD-like changes with intrastriatal injections of α-synuclein (α-syn) pre-formed fibril (PFFs) aggregates. We employed the heterozygous Engrailed 1 (En1+/-) mouse that features several pathophysiological hallmarks of clinical PD.. To test the hypothesis that the neuropathological changes in the En1+/- mice will promote formation of α-syn aggregates following intrastriatal injections of pathogenic human α-syn PFFs.. We unilaterally injected PFFs into the striata of 1-month-old En1+/- and control wild-type mice and euthanized animals at 3 months for post-mortem analysis.. Using immunohistochemistry and unbiased stereology, we established that PFF-injected En1+/- mice exhibited a near-threefold increase in pS129-α-syn-positive neurons in the substantia nigra compared to PFF-injected wild-type mice. The PFF-injected En1+/- mice also displayed significant increases in pS129-α-syn-positive neurons in the amygdala and ventral tegmental area; regions of known PD pathology with projections to the striatum. Additionally, we observed amplified pS129-α-syn-positive aggregation in En1+/- mice in multiple cortical regions.. Following intrastriatal injection of PFFs, absence of an En1 allele leads to additional aggregation of pathological α-syn, potentially due to En1-loss mediated nigrostriatal impairment. We propose that further development of this double-hit model could result in a PD mouse model that predicts which experimental therapies will be effective in PD.

Topics: alpha-Synuclein; Amygdala; Animals; Brain; Cerebral Cortex; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Immunohistochemistry; Mice; Neostriatum; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Substantia Nigra; Synucleinopathies; Ventral Tegmental Area

Most of the Parkinson's disease (PD) cases are sporadic, although several genes are directly related to PD. Several pathways are central in PD pathogenesis: protein aggregation linked to proteasomal impairments, mitochondrial dysfunctions and impairment in dopamine (DA) release. Here we studied the close crossing of mitochondrial dysfunction and aggregation of α-synuclein (α-syn) and in the extension in the dopaminergic neuronal death. Here, using rat primary cultures of mesencephalic neurons, we induced the mitochondrial impairments using "DA-toxins" (MPP+, 6OHDA, rotenone). We showed that the DA-Toxins induced dopaminergic cell death through different pathways: caspase-dependent cell death for 6OHDA; MPP+ stimulated caspase-independent cell death, and rotenone activated both pathways. In addition, a decrease in energy production and/or a development of oxidative stress were observed and were linked to α-syn aggregation with generation of Lewy body-like inclusions (found inside and outside the dopaminergic neurons). We demonstrated that any of induced mitochondrial disturbances and processes of death led to α-syn protein aggregation and finally to cell death. Our study depicts the cell death mechanisms taking place in in vitro models of Parkinson's disease and how mitochondrial dysfunctions is at the cross road of the pathologies of this disease.

Topics: 1-Methyl-4-phenylpyridinium; alpha-Synuclein; Animals; Apoptosis; Cells, Cultured; Dopaminergic Neurons; Embryo, Mammalian; Energy Metabolism; Female; Humans; Mesencephalon; Mitochondria; Necroptosis; Necrosis; Neurotoxins; Oxidative Stress; Oxidopamine; Parkinson Disease; Primary Cell Culture; Protein Aggregation, Pathological; Rats; Rotenone

Evidence is accumulating to suggest that viral infections and consequent viral-mediated neuroinflammation may contribute to the etiology of idiopathic Parkinson's disease. Moreover, viruses have been shown to influence α-synuclein oligomerization as well as the autophagic clearance of abnormal intra-cellular proteins aggregations, both of which are key neuropathological events in Parkinson's disease pathogenesis. To further investigate the interaction between viral-mediated neuroinflammation and α-synuclein aggregation in the context of Parkinson's disease, this study sought to determine the impact of viral neuroinflammatory priming on α-synuclein aggregate-induced neuroinflammation and neurotoxicity in the rat nigrostriatal pathway. To do so, male Sprague-Dawley rats were intra-nigrally injected with a synthetic mimetic of viral dsRNA (poly I:C) followed two weeks later by a peptidomimetic small molecule which accelerates α-synuclein fibril formation (FN075). The impact of the viral priming on α-synuclein aggregation-induced neuroinflammation, neurodegeneration and motor dysfunction was assessed. We found that prior administration of the viral mimetic poly I:C significantly exacerbated or precipitated the α-synuclein aggregate induced neuropathological and behavioral effects. Specifically, sequential exposure to the two challenges caused a significant increase in nigral microgliosis (p < 0.001) and astrocytosis (p < 0.01); precipitated a significant degeneration of the nigrostriatal cell bodies (p < 0.05); and precipitated a significant impairment in forelimb kinesis (p < 0.01) and sensorimotor integration (p < 0.01). The enhanced sensitivity of the nigrostriatal neurons to pathological α-synuclein aggregation after viral neuroinflammatory priming further suggests that viral infections may contribute to the etiology and pathogenesis of Parkinson's disease.

Topics: alpha-Synuclein; Animals; Biomimetic Materials; Corpus Striatum; Dependovirus; Disease Models, Animal; Genetic Vectors; Gliosis; Male; Motor Activity; Neurodegenerative Diseases; Neuroimmunomodulation; Neurons; Parkinson Disease; Poly I-C; Protein Aggregation, Pathological; Rats; Rats, Sprague-Dawley; Substantia Nigra; Tyrosine 3-Monooxygenase

Synucleinopathies are characterized by the accumulation of insoluble α-synuclein (αSyn). To test whether αSyn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing αSyn pathology. We demonstrated that preformed fibrils (PFFs) generated with recombinant human αSyn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of αSyn pathology in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of αSyn PFFs from whole-cell patch-clamp pipette decreased mEPSC frequency within 10 min followed by induction of αSyn pathology within 1 d. Moreover, by direct addition of αSyn PFFs into culture medium, the formation of misfolded αSyn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with αSyn PFFs. Our data indicate that intraneuronal αSyn fibrils impaired the initiation of synaptogenesis and their physiological functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.

Topics: alpha-Synuclein; Animals; Cell Survival; Hippocampus; Mice; Mice, Knockout; Neurons; Protein Aggregates; Protein Aggregation, Pathological; Synapses

The self-assembly of proteins into structured fibrillar aggregates is associated with a range of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, in which an important cytotoxic role is thought to be played by small soluble oligomers accumulating during the aggregation process or released by mature fibrils. As the structural characteristics of such species and their links with toxicity are still not fully defined, we have compared six examples of preformed misfolded protein oligomers with different β-sheet content, as determined using Fourier transform infrared spectroscopy, and with different toxicity, as determined by three cellular readouts of cell viability. The results show the absence of any measurable correlation between the nature of their secondary structure and their cellular toxicity, both when comparing the six types of oligomers as a group and when comparing species in subgroups characterized by either the same size or the same exposure of hydrophobic moieties.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Carboxyl and Carbamoyl Transferases; Cell Line; Cell Survival; Escherichia coli; Escherichia coli Proteins; Humans; Parkinson Disease; Protein Aggregation, Pathological; Protein Folding; Protein Structure, Secondary; Proteostasis Deficiencies

A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids-long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic

Topics: alpha-Synuclein; Amyloid; Animals; Cathepsin B; Cathepsin L; Cysteine; Dopaminergic Neurons; Humans; Lysosomes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Parkinson Disease; Protein Aggregation, Pathological; Rats

The aggregation of disordered α-synuclein protein is pathogenically connected with Parkinson's disease. Therefore, discovering molecules that can inhibit the misfolding and aggregation of α-synuclein is an active research area in PD drug development. A key property of such required therapeutic agents is specific binding to the target protein. Mass spectrometry allows rapid detection of direct interactions between molecules and proteins and is an ideal technique for discovering specific α-synuclein binders. Here, by setting up an automated mass spectrometry-based screening system, we were able to screen over 2500 compounds and identify a new α-synuclein inhibitor, 3-[(3-methoxyphenyl)carbamoyl]-7-[( E)-2-phenylethenyl]-4,7-dihydropyrazolo [1,5- a]pyrimidine-5-carboxylic acid (compound 2). This compound not only significantly inhibits the misfolding and aggregation of α-synuclein and protects neuroblastoma cells from α-synuclein toxicity, but also has a more specific binding site compared with positive controls. Our work for the first time reports the inhibition of compound 2 on α-synuclein aggregation and also consolidates the capability of mass spectrometry to discover α-synuclein aggregation inhibitors.

Topics: alpha-Synuclein; Carboxylic Acids; Cell Line, Tumor; Drug Evaluation, Preclinical; Humans; Mass Spectrometry; Protein Aggregation, Pathological; Protein Folding; Pyrimidines

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD.

Topics: alpha-Synuclein; Animals; Cell Death; Disease Models, Animal; Dopaminergic Neurons; Gait; Mice; Mice, Transgenic; Motor Activity; Parkinson Disease; Protein Aggregation, Pathological; Substantia Nigra; Tyrosine 3-Monooxygenase

α-Synucleinopathies are neurodegenerative diseases that are characterized pathologically by α-synuclein inclusions in neurons and glia. The pathologic contribution of glial α-synuclein in these diseases is not well understood. Glial α-synuclein may be of particular importance in multiple system atrophy (MSA), which is defined pathologically by glial cytoplasmic α-synuclein inclusions. We have previously described Drosophila models of neuronal α-synucleinopathy, which recapitulate key features of the human disorders. We have now expanded our model to express human α-synuclein in glia. We demonstrate that expression of α-synuclein in glia alone results in α-synuclein aggregation, death of dopaminergic neurons, impaired locomotor function, and autonomic dysfunction. Furthermore, co-expression of α-synuclein in both neurons and glia worsens these phenotypes as compared to expression of α-synuclein in neurons alone. We identify unique transcriptomic signatures induced by glial as opposed to neuronal α-synuclein. These results suggest that glial α-synuclein may contribute to the burden of pathology in the α-synucleinopathies through a cell type-specific transcriptional program. This new Drosophila model system enables further mechanistic studies dissecting the contribution of glial and neuronal α-synuclein in vivo, potentially shedding light on mechanisms of disease that are especially relevant in MSA but also the α-synucleinopathies more broadly.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Cell Death; Constipation; Disease Models, Animal; Dopaminergic Neurons; Drosophila; Humans; Movement Disorders; Nerve Degeneration; Neurodegenerative Diseases; Neuroglia; Protein Aggregation, Pathological; Transcription, Genetic; Transcriptome

Mutations in lysosomal genes increase the risk of neurodegenerative diseases, as is the case for Parkinson's disease. Here, we found that pathogenic and protective mutations in arylsulfatase A (ARSA), a gene responsible for metachromatic leukodystrophy, a lysosomal storage disorder, are linked to Parkinson's disease. Plasma ARSA protein levels were changed in Parkinson's disease patients. ARSA deficiency caused increases in α-synuclein aggregation and secretion, and increases in α-synuclein propagation in cells and nematodes. Despite being a lysosomal protein, ARSA directly interacts with α-synuclein in the cytosol. The interaction was more extensive with protective ARSA variant and less with pathogenic ARSA variant than wild-type. ARSA inhibited the in vitro fibrillation of α-synuclein in a dose-dependent manner. Ectopic expression of ARSA reversed the α-synuclein phenotypes in both cell and fly models of synucleinopathy, the effects correlating with the extent of the physical interaction between these molecules. Collectively, these results suggest that ARSA is a genetic modifier of Parkinson's disease pathogenesis, acting as a molecular chaperone for α-synuclein.

Topics: Adult; Aged; alpha-Synuclein; Animals; Animals, Genetically Modified; Brain; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cells, Cultured; Cerebroside-Sulfatase; Dementia; Drosophila melanogaster; Drosophila Proteins; Female; Gene Knockout Techniques; Genes, Dominant; Humans; Male; Middle Aged; Molecular Chaperones; Mutation, Missense; Parkinson Disease; Pedigree; Point Mutation; Protein Aggregation, Pathological; Protein Interaction Mapping; Recombinant Proteins

Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development.

Topics: alpha-Synuclein; Animals; Brain; Bridged-Ring Compounds; Cell Line; Disease Models, Animal; Dopaminergic Neurons; Humans; Male; Mice; Multiple System Atrophy; Neuroprotective Agents; Organophosphates; Protein Aggregation, Pathological

α-Synuclein (α-syn) protein aggregation is associated with several neurodegenerative disorders collectively referred to as synucleinopathies, including Parkinson's disease. We used protein misfolding cyclic amplification (PMCA) to study α-syn aggregation in brain homogenates of wild-type or transgenic mice expressing normal (D line) or A53T mutant (M83 line) human α-syn. We found that sonication-incubation cycles of M83 mouse brain gradually produce large quantities of SDS-resistant α-syn aggregates, involving both human and mouse proteins. These PMCA products, containing partially proteinase K-resistant α-syn species, are competent to accelerate the onset of neurologic symptoms after intracerebral inoculation to young M83 mice and to seed aggregate formation of α-syn following PMCA, including in D and wild-type mouse brain substrates. PMCA seeding activity in the M83 diseased brain correlates positively with regions mostly targeted by the α-syn pathology in this model. Our data indicate that similar to prions, PMCA can reproduce some characteristics of α-syn aggregation and seeded propagation

Topics: alpha-Synuclein; Animals; Brain; Gene Amplification; Humans; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mutation; Protein Aggregates; Protein Aggregation, Pathological; Proteostasis Deficiencies

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates. In flies, AS69 reduced [Formula: see text]-synuclein aggregates and the locomotor deficit resulting from [Formula: see text]-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-[Formula: see text]-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes.

Topics: alpha-Synuclein; Amyloid; HEK293 Cells; Humans; Protein Aggregation, Pathological; Protein Multimerization; Recombinant Proteins

The aggregation of α-synuclein is linked directly to the histopathology of Parkinson's disease (PD). However, several missense mutations present in the α-synuclein gene (SNCA) have been known to be associated with PD. Several studies have highlighted the effect of SNCA mutations on the α-synuclein aggregation, but their pathological roles are not completely established. In this study, we have focused on the effects of the recently discovered α-synuclein missense mutants (H50Q and G51D) on the aggregation using computational approaches. We performed all atom molecular dynamics (MD) simulation on these mutants and compared their conformational dynamics with Wild-Type (WT) α-synuclein. We noticed the solvent accessible surface area (SASA), radius of gyration, atomic fluctuations, and beta strand content to be higher in H50Q than G51D and WT. Using PDBSum online server; we analyzed the inter-molecular interactions that drive the association of monomeric units of H50Q, WT, and G51D in forming the respective homo-dimer. We noticed the interface area, number of interacting residues and binding free energy to be higher for H50Q homo-dimer than the WT and G51D homo-dimers. Our findings in this study suggest that in comparison to WT and G51D, H50Q mutation to have a positive effect on increasing the α-synuclein aggregation propensity. Hence, we see that H50Q and G51D mutation show conflicting effect on the aggregation propensity of α-synuclein.

Topics: alpha-Synuclein; Amino Acid Substitution; Humans; Models, Molecular; Mutation; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization; Quantitative Structure-Activity Relationship

Soluble epoxide hydrolase (sEH) is widely expressed in the mammalian brain and possesses dual enzymatic activities, including C-terminal epoxide hydrolase (C-EH) which degrades epoxyeicosatrienoic acid (EET), a beneficial arachidonic acid metabolite. In the present study, the neuroprotective effect of sEH inhibition on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration of nigrostriatal dopaminergic system was investigated using genetic and pharmacological approaches. MPTP (15 mg/kg) was intraperitoneally injected in sEH knockout (KO) mice and C57BL/6J mice as wild-type (WT) mice. Compared with the MPTP-treated WT mice, MPTP-induced reductions in striatal dopamine content and nigral tyrosine hydroxylase level (TH, a biomarker of dopaminergic neurons) were less significant in the treated sEH mice. Furthermore, MPTP-induced HO-1 elevation (a redox-regulated protein), α-synuclein aggregation, and caspase 12 activation (a hallmark of ER stress) were less prominent in sEH KO mice than in WT mice. These data indicate that sEH KO mice are more resistant to MPTP-induced neurotoxicity. The pharmacological effect of N-[1-(1-oxopropyl)-4-piperidinyl]-N0-[4-(trifluoromethoxy)phenyl)-urea (TPPU, an sEH inhibitor) on MPTP-induced neurotoxicity was investigated in WT mice. TPPU (1 mg/kg, i.p.) attenuated MPTP-induced reduction in striatal dopamine content, TH-positive cell numbers, TH, and pro-caspase 9 protein levels (an initiator caspase of apoptosis) in mouse SN. Moreover, TPPU reduced MPTP-induced HO-1 elevation, α-synuclein aggregation and caspase 12 activation, indicating that TPPU is effective in attenuating MPTP-induced oxidative stress, apoptosis, protein aggregation, and ER stress. In conclusion, our study suggests that sEH is a potential target for developing therapies for parkinsonism. Furthermore, sEH inhibitors may be of clinical significance for treating CNS neurodegenerative diseases.

Topics: alpha-Synuclein; Animals; Corpus Striatum; Dopaminergic Neurons; Endoplasmic Reticulum Stress; Epoxide Hydrolases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; MPTP Poisoning; Neuroprotective Agents; Phenylurea Compounds; Piperidines; Protein Aggregation, Pathological; Substantia Nigra

Deposition of presynaptic protein α-synuclein in Lewy bodies and Lewy neurites in the substantia nigra region of brain has been linked with the clinical symptoms of the Parkinson's disease (PD). Proteotoxic stress conditions and mutations that cause abnormal aggregation of α-synuclein have close association with onset of PD and its progression. Therefore, studies pertaining to α-synuclein mutations play important roles in mechanistic understanding of aggregation behavior of the protein and subsequent pathology. Herein, guided by this fact, we have studied the aggregation kinetics, morphology, and neurotoxic effects of the two newly discovered sporadic PD associated mutants A18T and A29S of α-synuclein. Our studies demonstrate that both of the mutants are aggregation prone and undergo rapid aggregation compared to wild-type α-synuclein. Further, it was found that A18T mutant followed faster aggregation kinetics compared to A29S substitution. Additionally, we have designed three point mutations of α-synuclein for better understanding of the effects of substitutions on protein aggregation and demonstrated that substitution of alanine at the 18th position is highly sensitive compared to adjacent positions. Our results provide better understanding of the effects of α-synuclein mutations on its aggregation behavior that may be important in development of PD pathology.

Topics: alpha-Synuclein; Cell Death; Cell Line, Tumor; Circular Dichroism; Escherichia coli; Humans; Kinetics; Microscopy, Atomic Force; Microscopy, Confocal; Nuclear Magnetic Resonance, Biomolecular; Parkinson Disease; Point Mutation; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary

Parkinson's disease is characterized by degeneration of substantia nigra dopamine neurons and by intraneuronal aggregates, primarily composed of misfolded α-synuclein. The α-synuclein aggregates in Parkinson's patients are suggested to first appear in the olfactory bulb and enteric nerves and then propagate, following a stereotypic pattern, via neural pathways to numerous regions across the brain. We recently demonstrated that after injection of either mouse or human α-synuclein fibrils into the olfactory bulb of wild-type mice, α-synuclein fibrils recruited endogenous α-synuclein into pathological aggregates that spread transneuronally to over 40 other brain regions and subregions, over 12 months. We previously reported the progressive spreading of α-synuclein aggregates, between 1 and 12 months following α-synuclein fibril injections, and now report how far the pathology has spread 18- and 23-month post-injection in this model. Our data show that between 12 and 18 months, there is a further increase in the number of brain regions exhibiting pathology after human, and to a lesser extent mouse, α-synuclein fibril injections. At both 18 and 23 months after injection of mouse and human α-synuclein fibrils, we observed a reduction in the density of α-synuclein aggregates in some brain regions compared to others at 12 months. At 23 months, no additional brain regions exhibited α-synuclein aggregates compared to earlier time points. In addition, we also demonstrate that the induced α-synucleinopathy triggered a significant early neuron loss in the anterior olfactory nucleus. By contrast, there was no loss of mitral neurons in the olfactory bulb, even at 18 month post-injection. We speculate that the lack of continued progression of α-synuclein pathology is due to compromise of the neural circuitry, consequential to neuron loss and possibly to the activation of proteolytic mechanisms in resilient neurons of wild-type mice that counterbalances the spread and seeding by degrading pathogenic α-synuclein.

Topics: alpha-Synuclein; Animals; Biological Transport; Brain; Cell Death; Disease Models, Animal; Disease Progression; DNA-Binding Proteins; Female; Humans; Mice, Inbred C57BL; Neurodegenerative Diseases; Neurons; Olfactory Bulb; Protein Aggregation, Pathological; Recombinant Proteins; tau Proteins

Numerous long non-coding RNAs (lncRNAs) have been identified as aberrantly expressed in Parkinson's disease (PD). However, limited knowledge is available concerning the roles of dysregulated lncRNAs and the underlying molecular regulatory mechanism in the pathological process of PD. In this study, we found that lncRNA small nucleolar RNA host gene 1 (SNHG1) and seven in absentia homolog 1 (SIAH1) were upregulated, but microRNA-15b-5p (miR-15b-5p) was downregulated in SH-SY5Y cells pretreated with MPP+, as well as in MPTP-induced mouse model of PD. Overexpression of SIAH1 enhanced cellular toxicity of α-synuclein in SH-SY5Y cells, as indicated by the reduction of cell viability and elevation of LDH release. The percentage of α-synuclein aggregate-positive cells and the number of α-synuclein aggregates per cell were increased in SH-SY5Y cells transfected with pcDNA-SIAH1, while decreased after transfection with short interfering RNA specific for SIAH1 (si-SIAH1). Bioinformatics and luciferase reporter assay revealed that SIAH1 was a direct target of miR-15b-5p. We also found that SNHG1 could directly bind to miR-15-5p and repress miR-15-5p expression. Upregulation of miR-15b-5p alleviated α-synuclein aggregation and apoptosis by targeting SIAH1 in SH-SY5Y cells overexpressing α-synuclein. Overexpression of SNHG1 enhanced, whereas SNHG1 knockdown inhibited α-synuclein aggregation and α-synuclein-induced apoptosis. Moreover, the neuroprotective effect of si-SNHG1 was abrogated by downregulation of miR-15b-5p. In summary, our data suggest that SNHG1, as a pathogenic factor, promotes α-synuclein aggregation and toxicity by targeting the miR-15b-5p/SIAH1 axis, contributing to a better understanding of the mechanisms of Lewy body formation and loss of dopaminergic neurons in PD.

Topics: alpha-Synuclein; Animals; Cell Line, Tumor; Gene Expression Regulation; Male; Mice, Inbred C57BL; MicroRNAs; Nuclear Proteins; Parkinson Disease; Parkinsonian Disorders; Protein Aggregation, Pathological; RNA, Long Noncoding; Ubiquitin-Protein Ligases

α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of αS oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled in vitro, the in vivo situation might be more complex and subcellular locations where αS species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that αS oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble αS oligomers at the ER/M level precedes neuronal degeneration in a mouse model of α-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of αS species associated with the ER/M membrane. We found that by comparison with non-microsomal associated αS (P10), the ER/M-associated αS pool is a unique population of oligomers and aggregates with specific biochemical traits such as increased aggregation, N- and C-terminal truncations and phosphorylation at serine 129. Moreover, when administered to murine primary neurons, ER/M-associated αS species isolated from diseased A53T human αS transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated αS species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous αS aggregates along the neurites, occasionally covering the entire process or localizing at the soma level. By comparison treatment with P10 fractions from the same diseased mice resulted in the formation of scarce and small puncta only when administered at high amount. Moreover, increasing the amount of P100/M fractions obtained from diseased and, more surprisingly, from presymptomatic mice induced a significant level of neuronal death that was prevented when neurons were treated with ER/M fractions immunodepleted of αS high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of αS HMW species in vivo, putting the spotlight on the association to ER/M membrane

Topics: alpha-Synuclein; Animals; Apoptosis; Cell Line, Tumor; Cerebral Cortex; Disease Models, Animal; Endoplasmic Reticulum; Humans; Mice, Transgenic; Microsomes; Molecular Weight; Nerve Degeneration; Neurodegenerative Diseases; Neurons; Primary Cell Culture; Protein Aggregation, Pathological

Mitochondrial complex I deficiency occurs in the substantia nigra of individuals with Parkinson's disease. It is generally believed that this phenomenon is caused by accumulating mitochondrial DNA damage in neurons and that it contributes to the process of neurodegeneration. We hypothesized that if these theories are correct, complex I deficiency should extend beyond the substantia nigra to other affected brain regions in Parkinson's disease and correlate tightly with neuronal mitochondrial DNA damage. To test our hypothesis, we employed a combination of semiquantitative immunohistochemical analyses, Western blot and activity measurements, to assess complex I quantity and function in multiple brain regions from an extensively characterized population-based cohort of idiopathic Parkinson's disease (n = 18) and gender and age matched healthy controls (n = 11). Mitochondrial DNA was assessed in single neurons from the same areas by real-time PCR. Immunohistochemistry showed that neuronal complex I deficiency occurs throughout the Parkinson's disease brain, including areas spared by the neurodegenerative process such as the cerebellum. Activity measurements in brain homogenate confirmed a moderate decrease of complex I function, whereas Western blot was less sensitive, detecting only a mild reduction, which did not reach statistical significance at the group level. With the exception of the substantia nigra, neuronal complex I loss showed no correlation with the load of somatic mitochondrial DNA damage. Interestingly, α-synuclein aggregation was less common in complex I deficient neurons in the substantia nigra. We show that neuronal complex I deficiency is a widespread phenomenon in the Parkinson's disease brain which, contrary to mainstream theory, does not follow the anatomical distribution of neurodegeneration and is not associated with the neuronal load of mitochondrial DNA mutation. Our findings suggest that complex I deficiency in Parkinson's disease can occur independently of mitochondrial DNA damage and may not have a pathogenic role in the neurodegenerative process.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Brain; DNA Damage; DNA, Mitochondrial; Electron Transport Complex I; Female; Humans; Male; Middle Aged; Mitochondria; Mitochondrial Diseases; Nerve Degeneration; Neurons; Parkinson Disease; Prospective Studies; Protein Aggregation, Pathological

Aggregation of α-synuclein (α-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson's disease (PD). Recent studies have indicated that oligomeric species of α-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted α-Syn ligands, based on the most aggregation-prone sequence of α-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate α-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with α-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of α-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of α-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of α-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of α-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as α-Syn aggregation modulators.

Topics: alpha-Synuclein; Amino Acid Substitution; Amyloid; Arginine; Cell Line, Tumor; Drug Design; Humans; Hydrophobic and Hydrophilic Interactions; Ligands; Molecular Dynamics Simulation; Neuroblastoma; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Domains; Surface Plasmon Resonance

α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson's disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of βS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of βS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that βS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants-A30P and G51D-as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of βS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with βS.

Topics: alpha-Synuclein; beta-Synuclein; Cell-Free System; Fluorescent Antibody Technique; Gene Expression; Genes, Reporter; Humans; Mutation; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization

Topics: alpha-Synuclein; Animals; Autophagosomes; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Communication; Cell Line; Humans; Iron; Lysosomes; Mice; Protein Aggregation, Pathological

Topics: alpha-Synuclein; Attention Deficit Disorder with Hyperactivity; Biogenic Amines; Diagnosis, Differential; Dystonia; Homozygote; Humans; Intellectual Disability; Molecular Chaperones; Parkinsonian Disorders; Phenylketonurias; Protein Aggregation, Pathological; Proteostasis; Repressor Proteins

In Parkinson's disease (PD) there is widespread accumulation in the brain of abnormal α-synuclein aggregates forming intraneuronal Lewy bodies (LB). It is now well established that LB-type α-synuclein aggregates also occur in the peripheral autonomic nervous system in PD, from where it has been speculated they may progressively spread to the central nervous system through synaptically-connected brain networks and reach the substantia nigra to trigger herein dopaminergic dysfunction/degeneration and subsequent parkinsonism. Supporting a pathogenic role for α-synuclein aggregates we have previously shown that LB purified from postmortem PD brains promote α-synuclein pathology and dopaminergic neurodegeneration when intracerebrally inoculated into wild-type mice. However, the pathogenic capacity of PD-derived peripheral α-synuclein aggregates remains unknown. Here we addressed this question using purified LB-type α-synuclein aggregates from postmortem PD stellate ganglia (SG), a paravertebral sympathetic ganglion that exhibits consistent and conspicuous Lewy pathology in all PD patients. In contrast to our previous findings using nigral LB extracts, intracerebral inoculation of SG-derived LB into mice did not trigger long-term nigrostriatal neurodegeneration nor α-synuclein pathology. The differential pathogenic capacities of central- and peripheral-derived α-synuclein aggregates appear independent of the absolute amount and basic biochemical properties of α-synuclein within these aggregates and may rely instead on differences in α-synuclein conformation and/or yet unrecognized brain region-specific intrinsic factors. Our results argue against a putative pathogenic capacity of peripheral α-synuclein aggregates to promote α-synuclein pathology in the brain, propagate between neuronal networks or induce neurodegeneration.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Alzheimer Disease; Animals; Brain; Female; Humans; Lewy Bodies; Male; Mice, Inbred C57BL; Parkinson Disease; Protein Aggregation, Pathological; Stellate Ganglion

The accumulation of misfolded α-synuclein (aSyn) and neuron loss define several neurodegenerative disorders including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the precise relationship between pathology and neurotoxicity and why these processes disproportionately affect certain neuron subpopulations are poorly understood. We show here that Math2-expressing neurons in the hippocampal Cornu ammonis (CA), a region significantly affected by aSyn pathology in advanced PD and DLB, are highly susceptible to pathological seeding with pre-formed fibrils (PFFs), in contrast to dentate gyrus neurons, which are relatively spared. Math2

Topics: alpha-Synuclein; Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Death; Cells, Cultured; Female; Gene Knockdown Techniques; Hippocampus; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Neurons; Primary Cell Culture; Protein Aggregation, Pathological; Proteostasis Deficiencies

Amyloid aggregates of the protein α-synuclein (αS) called Lewy Bodies (LB) and Lewy Neurites (LN) are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. We have previously shown that high extracellular αS concentrations can be toxic to cells and that neurons take up αS. Here we aimed to get more insight into the toxicity mechanism associated with high extracellular αS concentrations (50-100 μM). High extracellular αS concentrations resulted in a reduction of the firing rate of the neuronal network by disrupting synaptic transmission, while the neuronal ability to fire action potentials was still intact. Furthermore, many cells developed αS deposits larger than 500 nm within five days, but otherwise appeared healthy. Synaptic dysfunction clearly occurred before the establishment of large intracellular deposits and neuronal death, suggesting that an excessive extracellular αS concentration caused synaptic failure and which later possibly contributed to neuronal death.

Topics: Action Potentials; alpha-Synuclein; Animals; Cell Survival; Cells, Cultured; Cerebral Cortex; Extracellular Space; Humans; Intracellular Space; Neurons; Protein Aggregation, Pathological; Rats, Wistar; Recombinant Proteins; Synapses; Synaptic Transmission

Aggregation of α-synuclein is closely connected to the pathology of Parkinson's disease. The phenomenon involves multiple steps, commenced by partial misfolding and eventually leading to mature amyloid fibril formation. Trehalose, a widely accepted osmolyte, has been shown previously to inhibit aggregation of various globular proteins owing to its ability to prevent the initial unfolding of protein. In this study, we have examined if it behaves in a similar fashion with intrinsically disordered protein α-synuclein and possesses the potential to act as therapeutic agent against Parkinson's disease. It was observed experimentally that samples coincubated with trehalose fibrillate faster compared to the case in its absence. Molecular dynamics simulations suggested that this initial acceleration is manifestation of trehalose's tendency to perturb the conformational transitions between different conformers of monomeric protein. It stabilizes the aggregation prone "extended" conformer of α-synuclein, by binding to its exposed acidic residues of the C terminus. It also favors the β-rich oligomers once formed. Interestingly, the total fibrils formed are still promisingly less since it accelerates the competing pathway toward formation of amorphous aggregates.

Topics: alpha-Synuclein; Amyloid; Humans; Molecular Dynamics Simulation; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation; Trehalose

α-Synuclein (α-syn) is a major component of Lewy bodies found in synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Under the pathological conditions, α-syn tends to generate a diverse form of aggregates showing toxicity to neuronal cells and able to transmit across cells. However, mechanisms by which α-syn aggregates affect cytotoxicity in neurons have not been fully elucidated. Here we report that α-syn aggregates preferentially sequester specific synaptic proteins such as vesicle-associated membrane protein 2 (VAMP2) and synaptosomal-associated protein 25 (SNAP25) through direct binding which is resistant to SDS. The sequestration effect of α-syn aggregates was shown in a cell-free system, cultured primary neurons, and PD mouse model. Furthermore, we identified a specific blocking peptide derived from VAMP2 which partially inhibited the sequestration by α-syn aggregates and contributed to reduced neurotoxicity. These results provide a mechanism of neurotoxicity mediated by α-syn aggregates and suggest that the blocking peptide interfering with the pathological role of α-syn aggregates could be useful for designing a potential therapeutic drug for the treatment of PD.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Brain; Cell Survival; Cells, Cultured; Disease Models, Animal; Escherichia coli; Humans; Mice, Inbred C3H; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Neurons; Neuroprotective Agents; Parkinson Disease; Peptides; Protein Aggregation, Pathological; Protein Binding; Rats, Sprague-Dawley; Recombinant Proteins; Vesicle-Associated Membrane Protein 2

Seipin gene is originally found in type 2 congenital generalized lipodystrophy (CGL2) to involve lipid droplet formation. Recently, decrease of seipin expression is reported in substantia nigra of Parkinson's disease patients. Dopaminergic neurons in substantia nigra pars compacta expressed the seipin protein. The objective of this study is to investigate influence of the seipin deficiency on dopaminergic neurons and motor behaviors. Neuronal seipin knockout (seipin-nKO) mice (3-12 months of age) displayed an age-related deficit in motor coordination. The number of dopaminergic neurons in seipin-nKO mice was age dependently reduced with increase in cleaved caspase-3. The levels of αSyn oligomers and oligomer phosphorylation (S129), but not αSyn monomers, were elevated in dopaminergic neurons and substantia nigra of seipin-nKO mice. The PPARγ expression in seipin-nKO mice was reduced. In seipin-nKO mice, the phosphorylation of GSK3β was increased at Tyr216 and was reduced at Ser9, which was corrected by the PPARγ agonist rosiglitazone. The increased IL-6 level in seipin-nKO mice was sensitive to rosiglitazone and GSK3β inhibitor AR-A014418. The enhanced phosphorylation of αSyn was prevented by rosiglitazone and AR-A014418, while the increase in αSyn oligomers was corrected only by rosiglitazone. The treatment of seipin-nKO mice with rosiglitazone and AR-A014418 rescued the death of dopaminergic neurons, which was accompanied by the improvement of motor coordination. Therefore, the results indicate that seipin deficiency causes an age-related loss of dopaminergic neurons and impairment of motor coordination through reducing PPARγ to enhance aggregation and phosphorylation of αSyn and neuroinflammation.

Topics: alpha-Synuclein; Animals; Caspase 3; Dopaminergic Neurons; Glycogen Synthase Kinase 3 beta; GTP-Binding Protein gamma Subunits; Heterotrimeric GTP-Binding Proteins; Inflammation; Mice; Mice, Knockout; Phosphorylation; PPAR gamma; Protein Aggregation, Pathological; Rosiglitazone

Alpha-Synuclein (α-Syn) is an important protein in the pathogenesis of Parkinson disease (PD) as it accumulates as fibrillar inclusions in affected brain regions including dopaminergic neurons in the substantia nigra. Elevated levels of α-Syn seem to be crucial in mediating its toxicity. Thus, detailed information regarding the regulatory mechanism of α-Syn expression in several layers such as transcription, post-transcription and post-translation is needed in order to devise therapeutic interventions for PD. Previously, we reported that expression of α-Syn is repressed by microRNA-7 (miR-7) through its effect on the 3'-untranslated region (UTR) of α-Syn mRNA. Here, we show that miR-7 also accelerates the clearance of α-Syn and its aggregates by promoting autophagy in differentiated ReNcell VM cells. Further, miR-7 facilitates the degradation of pre-formed fibrils of α-Syn transported from outside the cells. This additional mechanism for reducing α-Syn levels show miR-7 to be an important molecular target for PD and other alpha-synucleinopathies.

Topics: alpha-Synuclein; Autophagy; Cells, Cultured; Dopaminergic Neurons; HEK293 Cells; Humans; MicroRNAs; Neural Stem Cells; Protein Aggregation, Pathological; Proteolysis

Accumulation of aggregated α-synuclein into Lewy bodies is thought to contribute to the onset and progression of dopaminergic neuron degeneration in Parkinson's disease (PD) and related disorders. Although protein aggregation is associated with perturbation of proteostasis, how α-synuclein aggregation affects the brain proteome and signaling remains uncertain. In a mouse model of α-synuclein aggregation, 6% of 6215 proteins and 1.6% of 8183 phosphopeptides changed in abundance, indicating conservation of proteostasis and phosphorylation signaling. The proteomic analysis confirmed changes in abundance of proteins that regulate dopamine synthesis and transport, synaptic activity and integrity, and unearthed changes in mRNA binding, processing and protein translation. Phosphorylation signaling changes centered on axonal and synaptic cytoskeletal organization and structural integrity. Proteostatic responses included a significant increase in the levels of Lmp7, a component of the immunoproteasome. Increased Lmp7 levels and activity were also quantified in postmortem human brains with PD and dementia with Lewy bodies. Functionally, the immunoproteasome degrades α-synuclein aggregates and generates potentially antigenic peptides. Expression and activity of the immunoproteasome may represent testable targets to induce adaptive responses that maintain proteome integrity and modulate immune responses in protein aggregation disorders.

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Female; Mice; Mice, Knockout; Parkinson Disease; Proteasome Endopeptidase Complex; Protein Aggregation, Pathological; Proteostasis

Proteostasis is critical to maintain organismal viability, a process counteracted by aging-dependent protein aggregation. Chaperones of the heat shock protein (HSP) family help control proteostasis by reducing the burden of unfolded proteins. They also oversee the formation of protein aggregates. Here, we explore how AMPylation, a posttranslational protein modification that has emerged as a powerful modulator of HSP70 activity, influences the dynamics of protein aggregation. We find that adjustments of cellular AMPylation levels in

Topics: Adenosine Monophosphate; alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; HSP70 Heat-Shock Proteins; Molecular Chaperones; Neurodegenerative Diseases; Nucleotidyltransferases; Peptides; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Proteostasis

The intrinsically disordered protein β-synuclein is known to inhibit the aggregation of its intrinsically disordered homolog, α-synuclein, which is implicated in Parkinson's disease. While β-synuclein itself does not form fibrils at the cytoplasmic pH 7.4, alteration of pH and other environmental perturbations are known to induce its fibrilization. However, the sequence and structural determinants of β-synuclein inhibition and self-aggregation are not well understood. We have utilized a series of domain-swapped chimeras of α-synuclein and β-synuclein to probe the relative contributions of the N-terminal, C-terminal, and the central non-amyloid-β component domains to the inhibition of α-synuclein aggregation. Changes in the rates of α-synuclein fibril formation in the presence of the chimeras indicate that the non-amyloid-β component domain is the primary determinant of self-association leading to fibril formation, while the N- and C-terminal domains play critical roles in the fibril inhibition process. Our data provide evidence that all three domains of β-synuclein together contribute to providing effective inhibition, and support a model of transient, multi-pronged interactions between IDP chains in both processes. Inclusion of such multi-site inhibitory interactions spread over the length of synuclein chains may be critical for the development of therapeutics that are designed to mimic the inhibitory effects of β-synuclein.

Topics: alpha-Synuclein; beta-Synuclein; Binding Sites; Cytoplasm; Humans; Hydrogen-Ion Concentration; Protein Aggregation, Pathological; Protein Binding; Spectrometry, Mass, Electrospray Ionization

The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson's disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments' internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly.

Topics: alpha-Synuclein; Amyloid; Humans; Microscopy, Atomic Force; Parkinson Disease; Protein Aggregation, Pathological; Protein Unfolding

The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders.

Topics: alpha-Synuclein; Animals; Caenorhabditis elegans; Cell Line; Cholestanes; Disease Models, Animal; Humans; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Spermine

Parkinson's disease (PD) and other synucleinopathies are characterized by accumulation of misfolded aggregates of α-synuclein (α-syn). The normal function of α-syn is still under investigation, but it has been generally linked to synaptic plasticity, neurotransmitter release and the maintenance of the synaptic pool. α-Syn localizes at synaptic terminals where it can bind to synaptic vesicles as well as to other cellular membranes. It has become clear that these interactions have an impact on both α-syn functional role and its propensity to aggregate. In this study, we investigated the aggregation process of α-syn covalently modified with 4-hydroxy-2-nonenal (HNE). HNE is a product of lipid peroxidation and has been implicated in the pathogenesis of different neurodegenerative diseases by modifying the kinetics of soluble toxic oligomers. Although HNE-modified α-syn has been reported to assemble into stable oligomers, we found that slightly acidic conditions promoted further protein aggregation. Lipid vesicles delayed the aggregation process in a concentration-dependent manner, an effect that was observed only when they were added at the beginning of the aggregation process. Co-aggregation of lipid vesicles with HNE-modified α-syn also induced cytotoxic effects on differentiated SHSY-5Y cells. Under conditions in which the aggregation process was delayed cell viability was reduced. By exploring the behavior and potential cytotoxic effects of HNE-α-syn under acidic conditions in relation to protein-lipid interactions our study gives a framework to examine a possible pathway leading from a physiological setting to the pathological outcome of PD.

Topics: Aldehydes; alpha-Synuclein; Cell Line, Tumor; Cell Survival; Humans; Hydrogen-Ion Concentration; Lipid Metabolism; Lipid Peroxidation; Liposomes; Microscopy, Electron, Transmission; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Protein Multimerization; Recombinant Proteins; Synaptic Vesicles

Parkinson's disease (PD) is recognized as the second most common neurodegenerative disorder and has affected approximately one million people in the United States alone. A large body of evidence has suggested that deposition of aggregated alpha-synuclein (α-Syn), a brain protein abundant near presynaptic termini, in intracellular protein inclusions (Lewy bodies) results in neuronal cell damage and ultimately contributes to the progression of PD. However, the exact mechanism is still unclear. One hypothesis is that α-Syn aggregates disrupt the cell membrane's integrity, eventually leading to cell death. We used scanning ion conductance microscopy (SICM) to monitor the morphological changes of SH-SY5Y neuroblastoma cells and observed dramatic disruption of the cell membrane after adding α-Syn aggregates to the culturing media. This work demonstrates that SICM can be applied as a new approach to studying the cytotoxicity of α-Syn aggregates.

Topics: alpha-Synuclein; Cell Death; Cell Line, Tumor; Cell Membrane; Humans; Microscopy, Electrochemical, Scanning; Neuroblastoma; Neurons; Parkinson Disease; Protein Aggregation, Pathological

Topics: alpha-Synuclein; Animals; Blood-Brain Barrier; Cells, Cultured; Graphite; Humans; Lewy Bodies; Mice; Parkinson Disease; Protein Aggregation, Pathological; Quantum Dots; Synapses

The presynaptic protein, α-synuclein (α-syn), has been shown to play a crucial role in multiple neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), and dementia with Lewy bodies (DLB). The three major domains of α-syn protein were shown to govern its membrane interaction, protein fibrillation, and chaperone activity. So far, four different alternatively spliced isoforms of α-syn, which lack either exon 3 (syn-126) or exon 5 (syn-112) or both (syn-98) resulting in altered function of the proteins, have been identified. In the present study, we have identified the smallest isoform of α-syn due to the skipping of exons 3 and 4 generating a 238 bp transcript. Due to the presence of a premature stop codon, the 238 bp transcript generated a 41 aa N-terminal peptide instead of the 78 aa protein, which is secreted into the extracellular medium when overexpressed in cells. The presence of 41-syn was initially noticed in the substantia nigra of PD autopsy tissues, as well as in cells undergoing oxidative stress. In vitro studies inferred that 41-syn neither aggregates nor alters the aggregation propensity of either WT or 112-syn. Overexpression of 41-syn or treatment of cells with 41-syn peptide did not affect cell viability. However, PC-12 cells treated with 41-syn exhibited a time and dose dependent enhancement in the cellular uptake of dopamine. Based on the physiological role of the N-terminal region of α-syn in modulating membrane trafficking events, we believe that the identification of 41-syn may provide novel impetus in unraveling the physiological basis of alternative splicing events in governing PD pathophysiology.

Topics: alpha-Synuclein; Alternative Splicing; Animals; Autopsy; Cell Line, Tumor; Cell Survival; Dopamine; Homeostasis; Humans; Neurons; Oxidative Stress; Parkinson Disease; Pars Compacta; PC12 Cells; Protein Aggregation, Pathological; Protein Isoforms; Rats; RNA Isoforms; RNA, Messenger; Synapses

Amyloid formation of α-synuclein (α-Syn) and its familial mutations are directly linked with Parkinson's disease (PD) pathogenesis. Recently, a new familial α-Syn mutation (A53E) was discovered, associated with an early onset aggressive form of PD, which delays α-Syn aggregation. When we overexpressed wild-type (WT) and A53E proteins in cells, showed neither toxicity nor aggregate formation, suggesting merely overexpression may not recapitulate the PD phenotype in cell models. We hypothesized that cells expressing the A53E mutant might possess enhanced susceptibility to PD-associated toxicants compared to that of the WT. When cells were treated with PD toxicants (dopamine and rotenone), cells expressing A53E showed more susceptibility to cell death along with compromised mitochondrial potential and an increased production of reactive oxygen species. The higher toxicity of A53E could be due to more oligomers being formed in cells as confirmed by a dot blot assay using amyloid specific OC and A11 antibody and using an  in vitro aggregation study. The cellular model presented here suggests that along with familial mutation, environmental and other cellular factors might play a crucial role in dictating PD pathogenesis.

Topics: alpha-Synuclein; Apoptosis; Cell Line, Tumor; Dopamine; Humans; Kinetics; Membrane Potential, Mitochondrial; Mitochondria; Mutation; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization; Reactive Oxygen Species; Rotenone

Increasing evidence suggests that α-synuclein (αS) aggregates in brains of individuals with Parkinson's disease and dementia with Lewy bodies can spread in a prion-like manner. Although the initial αS nuclei are pivotal in determining αS fibril polymorphs and resulting phenotypes, it is not clear how the initial fibril seeds are generated. Previous studies have shown that αS truncation might have an important role in αS aggregation. However, little is known about how this truncation influences αS's propagation properties. In the present study, we generated αS fibrils from a series of truncated human αS constructs, characterized their structures and conformational stabilities, and investigated their ability to convert the conformation of full-length αS

Topics: alpha-Synuclein; Animals; Humans; Lewy Bodies; Mice; Mutant Proteins; Parkinson Disease; Prions; Protein Aggregation, Pathological; Protein Conformation

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-β. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Aptamers, Peptide; Humans; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Several neurodegenerative diseases have a common pathophysiology where selective damage to neurons results from the accumulation of amyloid oligomer proteins formed via fibrilization. Considering that the formation of amyloid oligomers leads to cytotoxicity, the development of chemical compounds that are able to effectively cross the blood-brain barrier (BBB) and inhibit this conversion to oligomers and/or fibrils is essential for neurodegenerative disease therapy. We previously reported that pyrroloquinoline quinone (PQQ) prevented aggregation and fibrillation of α-synuclein, amyloid β

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Amyloidogenic Proteins; Animals; Blood-Brain Barrier; Cell Line, Tumor; Cell Survival; Esterification; Esters; Humans; Mice; Neurons; Peptide Fragments; Permeability; PQQ Cofactor; Prion Proteins; Protein Aggregation, Pathological

Quinolinic acid (QA), a downstream neurometabolite in the kynurenine pathway, the biosynthetic pathway of tryptophan, is associated with neurodegenerative diseases pathology. Mutations in genes encoding kynurenine pathway enzymes, which control the level of QA production, are linked with elevated risk of developing Parkinson's disease. Recent findings have revealed the accumulation and deposition of QA in post-mortem samples, as well as in cellular models of Alzheimer's disease and related disorders. Furthermore, intrastriatal inoculation of mice with QA results in increased levels of phosphorylated α-synuclein and neurodegenerative pathological and behavioral characteristics. However, the cellular and molecular mechanisms underlying the involvement of QA accumulation in protein aggregation and neurodegeneration remain elusive. We recently established that self-assembled ordered structures are formed by various metabolites and hypothesized that these "metabolite amyloids" may seed amyloidogenic proteins. Here we demonstrate the formation of QA amyloid-like fibrillar assemblies and seeding of α-synuclein aggregation by these nanostructures both in vitro and in cell culture. Notably, α-synuclein aggregation kinetics was accelerated by an order of magnitude. Additional amyloid-like properties of QA assemblies were demonstrated using thioflavin T assay, powder X-ray diffraction and cell apoptosis analysis. Moreover, fluorescently labeled QA assemblies were internalized by neuronal cells and co-localized with α-synuclein aggregates. In addition, we observed cell-to-cell propagation of fluorescently labeled QA assemblies in a co-culture of treated and untreated cells. Our findings suggest that excess QA levels, due to mutations in the kynurenine pathway, for example, may lead to the formation of metabolite assemblies that seed α-synuclein aggregation, resulting in neuronal toxicity and induction of Parkinson's disease.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Quinolinic Acid; Spectrum Analysis; Structure-Activity Relationship

The accumulation of specific phosphorylated protein aggregates in the brain is a hallmark of severe neurodegenerative disorders. Specifically, hyperphosphorylated tau (hp-tau) accumulates in Alzheimer disease, frontotemporal dementia with Parkinsonism linked to chromosome 17, and progressive supranuclear palsy; furthermore, phosphorylated α-synuclein (p-αSyn) accumulates in Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. Moreover, codeposition of different pathological protein aggregates is common in the brains of individuals with neurodegenerative diseases. In the present report, we describe the detection of p-αSyn aggregates in the brain of rTg4510 mice that overexpress human P301L mutant tau. Immunohistochemistry showed that hp-tau and p-αSyn aggregates were found within the same neuronal cells in rTg4510 mice and increased with age. Moreover, semiquantitative analysis revealed a significant regional correlation between hp-tau and p-αSyn accumulation. These results indicate that endogenous mouse αSyn protein is phosphorylated and accumulates with hp-tau aggregation in neurons and suggest that the overexpression of human P301L mutant tau may enhance endogenous αSyn phosphorylation and aggregation via a similar hyperphosphorylation mechanism in vivo. This synergic effect between tau and αSyn accumulation may exacerbate the pathology of several neurodegenerative disorders that show a cooccurrence of hp-tau and p-αSyn aggregation.

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphorylation; Protein Aggregation, Pathological; tau Proteins; Tauopathies

Intrinsically disordered proteins (IDPs) experience a diverse spectrum of motions that are difficult to characterize with a single experimental technique. Herein we combine high- and low-field nuclear spin relaxation, nanosecond fluorescence correlation spectroscopy (nsFCS), and long molecular dynamics simulations of alpha-synuclein, an IDP involved in Parkinson disease, to obtain a comprehensive picture of its conformational dynamics. The combined analysis shows that fast motions below 2 ns caused by local dihedral angle fluctuations and conformational sampling within and between Ramachandran substates decorrelate most of the backbone N-H orientational memory. However, slow motions with correlation times of up to ca. 13 ns from segmental dynamics are present throughout the alpha-synuclein chain, in particular in its C-terminal domain, and global chain reconfiguration occurs on a timescale of ca. 60 ns. Our study demonstrates a powerful strategy to determine residue-specific protein dynamics in IDPs at different time and length scales.

Topics: alpha-Synuclein; Humans; Intrinsically Disordered Proteins; Molecular Dynamics Simulation; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation; Protein Domains; Protein Folding; Spectrometry, Fluorescence

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wild-type α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD

Topics: alpha-Synuclein; Amyloid; Animals; Caenorhabditis elegans; Dopaminergic Neurons; High-Throughput Screening Assays; Humans; Neuroblastoma; Parkinson Disease; Protein Aggregation, Pathological; Small Molecule Libraries; Tumor Cells, Cultured

We aimed to characterize in vivo α-synuclein (α-syn) aggregates in skin nerves to ascertain: 1) the optimal marker to identify them; 2) possible differences between synucleinopathies that may justify the clinical variability. We studied multiple skin nerve α-syn deposits in 44 patients with synucleinopathy: 15 idiopathic Parkinson's disease (IPD), 12 dementia with Lewy Bodies (DLB), 5 pure autonomic failure (PAF) and 12 multiple system atrophy (MSA). Ten healthy subjects were used as controls. Antibodies against native α-syn, C-terminal α-syn epitopes such as phosphorylation at serine 129 (p-syn) and to conformation-specific for α-syn mature amyloid fibrils (syn-F1) were used. We found that p-syn showed the highest sensitivity and specificity in disclosing skin α-syn deposits. In MSA abnormal deposits were only found in somatic fibers mainly at distal sites differently from PAF, IPD and DLB displaying α-syn deposits in autonomic fibers mainly at proximal sites. PAF and DLB showed the highest p-syn load with a widespread involvement of autonomic skin nerve fibers.. 1) p-syn in skin nerves was the optimal marker for the in vivo diagnosis of synucleinopathies; 2) the localization and load differences of aggregates may help to identify specific diagnostic traits and support a different pathogenesis among synucleinopathies.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Amyloid; Brain; Female; Humans; Lewy Body Disease; Male; Multiple System Atrophy; Nerve Fibers; Parkinson Disease; Protein Aggregation, Pathological; Pure Autonomic Failure; Skin; Skin Diseases

Parkinson's disease (PD) is a common neurodegenerative disease which usually associates with neuroinflammation. The main pathological characteristics of PD are dopaminergic neurons death and the presence of Lewy bodies which are composed of aggregated α-synuclein (α-Syn). Truncated forms of α-Syn are found in the brain of PD patients, and account for 10-30% of total synuclein in Lewy bodies. Caspase-1, which plays an important role in neuroinflammation, cleaves full-length α-Syn (α-Syn FL) to generate a C-terminus 19-residues truncated α-Syn (α-Syn121). However, the role of truncated α-Syn in the onset and/or pathogenesis of PD is unclear. Here, we used α-Syn121 as a model to explore its aggregation, membrane disruption and cytotoxicity properties. Compared with α-Syn FL, α-Syn121 aggregated at an accelerated rate, and formed amorphous aggregates rich in random coil structures rather than β-sheet-rich linear fibrils formed by α-Syn FL. Importantly, higher cytotoxicity with lower membrane disruption capacity was found for α-Syn121 aggregates. Furthermore, α-Syn121 aggregates could activate the apoptosis signaling pathway and stimulate the caspase-1-mediated cleavage of α-Syn FL to generate α-Syn121, which as a result leading to increased levels of endogenous α-Syn121 and intracellular S129 phosphorylated α-Syn inclusions. Together, our data suggests a hidden vicious cycle in PD that α-Syn121 rapidly forms amorphous aggregates, which activate caspase-1 to cleave α-Syn FL and generate more α-Syn121, and this cycle may contribute to the onset and/or pathogenesis of PD.

Topics: alpha-Synuclein; Amyloid; Apoptosis; Caspase 1; Cell Line; Humans; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary; Proteolysis; Reactive Oxygen Species

α-Synuclein (αsyn) aggregates into toxic fibrils in multiple neurodegenerative diseases where these fibrils form characteristic pathological inclusions such as Lewy bodies (LBs). The mechanisms initiating αsyn aggregation into fibrils are unclear, but ubiquitous post-translational modifications of αsyn present in LBs may play a role. Specific C-terminally (C)-truncated forms of αsyn are present within human pathological inclusions and form under physiological conditions likely in lysosome-associated pathways, but the roles for these C-truncated forms of αsyn in inclusion formation and disease are not well understood. Herein, we characterized the

Topics: alpha-Synuclein; Amyloid; Animals; Antibodies, Monoclonal; HEK293 Cells; Humans; Mice, Inbred BALB C; Peptide Fragments; Protein Aggregation, Pathological; Protein Multimerization; Proteolysis

Leucine-rich repeat kinase 2 (LRRK2) is the most common causative gene for autosomal dominant Parkinson's disease (PD) and is also known to be a susceptibility gene for sporadic PD. Although clinical symptoms with LRRK2 mutations are similar to those in sporadic PD, their pathologies are heterogeneous and include nigral degeneration with abnormal inclusions containing alpha-synuclein, tau, TAR DNA-binding protein 43, and ubiquitin, or pure nigral degeneration with no protein aggregation pathologies. We discovered two families harboring heterozygous and homozygous c.4332 G > A; p.R1441H in LRRK2 with consanguinity, sharing a common founder. They lived in the city of Makurazaki, located in a rural area of the southern region, the Kagoshima prefecture, in Kyushu, Japan. All patients presented late-onset parkinsonism without apparent cognitive decline and demonstrated a good response to levodopa. We obtained three autopsied cases that all presented with isolated nigral degeneration with no alpha-synuclein or other protein inclusions. This is the first report of neuropathological findings in patients with LRRK2 p.R1441H mutations that includes both homozygous and heterozygous mutations. Our findings in this study suggest that isolated nigral degeneration is the primary pathology in patients with LRRK2 p.R1441H mutations, and that protein aggregation of alpha-synuclein or tau might be secondary changes.

Topics: Aged; alpha-Synuclein; Astrocytes; Autopsy; DNA-Binding Proteins; Female; Gene Expression Regulation; Histidine; Homozygote; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Male; Middle Aged; Mutation; Neurodegenerative Diseases; Parkinsonian Disorders; Proline; Protein Aggregation, Pathological; Substantia Nigra; tau Proteins

Amyloid-β (Aβ) peptide and α-synuclein (α-syn) are major components of senile plaques in Alzheimer's disease (AD) and Lewy bodies in Parkinson's disease (PD), respectively. Co-occurrence of Aβ and α-syn in the senile brains of AD and LB diseases suggests interactions between the two proteins. However, the significance of the overlapping deposition, especially the effects of α-syn on the Aβ aggregation, still remains to be clarified. In the present study, we investigated the effects of α-syn pre-formed fibrils (PFFs) injection on the cognitive behaviors and Aβ deposition in the brain of APP/PS1 transgenic AD mice by using Morris water maze (MWM) test, immunohistochemistry and western blot techniques. We found that APP/PS1 transgenic mice exhibited an obvious elevation in the α-syn load, as well as Aβ deposition in the brain compared with wild type of C57 BL littermates. 5 months after cerebral injection of exogenous α-syn, MWM tests showed an alleviation in cognitive impairments in APP/PS1 mice; western blot and immunohistochemistry experiments also exhibited a significant reduction in Aβ level in the brain of APP/PS1 mice injected with α-syn. These results suggest that α-syn aggregated in the brain of AD may act as a protective factor and defend the brain tissue from early Aβ deposition and cognitive deficits.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Brain; Cognition Disorders; Cognitive Dysfunction; Disease Models, Animal; Humans; Male; Maze Learning; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Transgenic; Plaque, Amyloid; Presenilin-1; Protein Aggregation, Pathological; Spatial Memory

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson's disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

Topics: alpha-Synuclein; Fluorescent Dyes; Humans; Hydrophobic and Hydrophilic Interactions; Optical Imaging; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization; Solubility

Accumulation of alpha-synuclein (ASYN) in neurons and other CNS cell types may contribute to the underlying pathology of synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Multiple Systems Atrophy (MSA). In support of this hypothesis for PD, ASYN immunopositive aggregates are a prominent pathological feature of PD, and mutations and gene multiplications of human wild type (WT) ASYN cause rare familial autosomal-dominant forms of PD. Targeted therapeutics that reduce the accumulation of ASYN could prevent or slow the neurodegenerative processes in PD and other synucleinopathies. NPT200-11 is a novel small molecule inhibitor of ASYN misfolding and aggregation. The effects of NPT200-11 on ASYN neuropathology were evaluated in animal models over expressing human alpha synuclein. Longitudinal studies using retinal imaging in mice expressing a hASYN::GFP fusion protein revealed that 2 months of once daily administration of NPT200-11 (5 mg/kg IP) resulted in a time-dependent and progressive reduction in retinal ASYN pathology. The effects of NPT200-11 on ASYN pathology in cerebral cortex and on other disease-relevant endpoints was evaluated in the Line 61 transgenic mouse model overexpressing human wild type ASYN. Results from these studies demonstrated that NPT200-11 reduced alpha-synuclein pathology in cortex, reduced associated neuroinflammation (astrogliosis), normalized striatal levels of the dopamine transporter (DAT) and improved motor function. To gain insight into the relationship between dose, exposure, and therapeutic benefit pharmacokinetic studies were also conducted in mice. These studies demonstrated that NPT200-11 is orally bioavailable and brain penetrating and established target plasma and brain exposures for future studies of potential therapeutic benefit.

Topics: alpha-Synuclein; Animals; Cerebral Cortex; Disease Models, Animal; Gene Expression Regulation; Humans; Inflammation; Lewy Body Disease; Mice; Mice, Transgenic; Multiple System Atrophy; Neurons; Parkinson Disease; Piperidines; Protein Aggregation, Pathological; Protein Folding; Pyrazines; Pyrimidines; Retina

α-synclein (αS) aggregation is a representative molecular feature of the pathogenesis of Parkinson's disease (PD). Epigallocatechin gallate (EGCG) can prevent αS aggregation

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Catechin; Cell Line; Disease Models, Animal; Dopaminergic Neurons; Drug Carriers; Humans; Membrane Proteins; Mice; Molecular Targeted Therapy; Nanoparticles; Neuroprotective Agents; Parkinson Disease; Protein Aggregation, Pathological; Treatment Outcome

Increasing evidence suggests the important role of sirtuin 2 (SIRT2) in the pathology of Parkinson's disease (PD). However, the association between potential functional polymorphisms in the SIRT2 gene and PD still needs to be identified. Exploring the molecular mechanism underlying this potential association could also provide novel insights into the pathogenesis of this disorder.. Bioinformatics analysis and screening were first performed to find potential microRNAs (miRNAs) that could target the SIRT2 gene, and molecular biology experiments were carried out to further identify the regulation between miRNA and SIRT2 and characterize the pivotal role of miRNA in PD models. Moreover, a clinical case-control study was performed with 304 PD patients and 312 healthy controls from the Chinese Han population to identify the possible association of single nucleotide polymorphisms (SNPs) within the miRNA binding sites of SIRT2 with the risk of PD.. Here, we demonstrate that miR-486-3p binds to the 3' UTR of SIRT2 and influences the translation of SIRT2. MiR-486-3p mimics can decrease the level of SIRT2 and reduce a-synuclein (α-syn)-induced aggregation and toxicity, which may contribute to the progression of PD. Interestingly, we find that a SNP, rs2241703, may disrupt miR-486-3p binding sites in the 3' UTR of SIRT2, subsequently influencing the translation of SIRT2. Through the clinical case-control study, we further verify that rs2241703 is associated with PD risk in the Chinese Han population.. The present study confirms that the rs2241703 polymorphism in the SIRT2 gene is associated with PD in the Chinese Han population, provides the potential mechanism of the susceptibility locus in determining PD risk and reveals a potential target of miRNA for the treatment and prevention of PD.

Topics: 3' Untranslated Regions; Aged; alpha-Synuclein; Asian People; Case-Control Studies; Cell Line; Female; Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Male; MicroRNAs; Middle Aged; Parkinson Disease; Polymorphism, Single Nucleotide; Protein Aggregation, Pathological; Protein Biosynthesis; Sirtuin 2

The hallmark of Parkinson's disease (PD) is the intracellular protein aggregation forming Lewy Bodies (LB) and Lewy neuritis which comprise mostly of a protein, alpha synuclein (α-syn). Molecular dynamics (MD) simulation methods can augment experimental techniques to understand misfolding and aggregation pathways with atomistic resolution. The quality of MD simulations for proteins and peptides depends greatly on the accuracy of empirical force fields. The aim of this work is to investigate the effects of different force fields on the structural character of β hairpin fragment of α-syn (residues 35-56) peptide in aqueous solution. Six independent MD simulations are done in explicit solvent using, AMBER03, AMBER99SB, GROMOS96 43A1, GROMOS96 53A6, OPLS-AA, and CHARMM27 force fields with CMAP corrections. The performance of each force field is assessed from several structural parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), formation of β-turn, the stability of folded β-hairpin structure, and the favourable conformations obtained for different force fields. In this study, CMAP correction of CHARMM27 force field is found to overestimate the helical conformation, while GROMOS96 53A6 is found to most successfully capture the conformational dynamics of α-syn β-hairpin fragment as elicited from NMR.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Humans; Hydrogen Bonding; Lewy Bodies; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Parkinson Disease; Protein Aggregation, Pathological; Protein Structure, Secondary; Solvents; Thermodynamics; Water

See Stayte and Vissel (doi:10.1093/awx064) for a scientific commentary on this article. Multiple system atrophy is a fatal sporadic adult-onset neurodegenerative disorder with no symptomatic or disease-modifying treatment available. The cytopathological hallmark of multiple system atrophy is the accumulation of α-synuclein aggregates in oligodendrocytes, forming glial cytoplasmic inclusions. Impaired insulin/insulin-like growth factor-1 signalling (IGF-1) and insulin resistance (i.e. decreased insulin/IGF-1) have been reported in other neurodegenerative disorders such as Alzheimer's disease. Increasing evidence also suggests impaired insulin/IGF-1 signalling in multiple system atrophy, as corroborated by increased insulin and IGF-1 plasma concentrations in multiple system atrophy patients and reduced IGF-1 brain levels in a transgenic mouse model of multiple system atrophy. We here tested the hypothesis that multiple system atrophy is associated with brain insulin resistance and showed increased expression of the key downstream messenger insulin receptor substrate-1 phosphorylated at serine residue 312 in neurons and oligodendrocytes in the putamen of patients with multiple system atrophy. Furthermore, the expression of insulin receptor substrate 1 (IRS-1) phosphorylated at serine residue 312 was more apparent in inclusion bearing oligodendrocytes in the putamen. By contrast, it was not different between both groups in the temporal cortex, a less vulnerable structure compared to the putamen. These findings suggest that insulin resistance may occur in multiple system atrophy in regions where the neurodegenerative process is most severe and point to a possible relation between α-synuclein aggregates and insulin resistance. We also observed insulin resistance in the striatum of transgenic multiple system atrophy mice and further demonstrate that the glucagon-like peptide-1 analogue exendin-4, a well-tolerated and Federal Drug Agency-approved antidiabetic drug, has positive effects on insulin resistance and monomeric α-synuclein load in the striatum, as well as survival of nigral dopamine neurons. Additionally, plasma levels of exosomal neural-derived IRS-1 phosphorylated at serine residue 307 (corresponding to serine residue 312 in humans) negatively correlated with survival of nigral dopamine neurons in multiple system atrophy mice treated with exendin-4. This finding suggests the potential for developing this peripheral biomarker candidate as an objective

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Animals; Cell Survival; Corpus Striatum; Dopaminergic Neurons; Exenatide; Female; Humans; Insulin Receptor Substrate Proteins; Insulin Resistance; Male; Mice; Mice, Transgenic; Middle Aged; Multiple System Atrophy; Neurons; Oligodendroglia; Peptides; Phosphorylation; Protein Aggregation, Pathological; Putamen; Substantia Nigra; Temporal Lobe; Venoms

Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.

Topics: alpha-Synuclein; Animals; Autophagy; Brain; Cell Line; Glycogen Synthase Kinase 3 beta; HeLa Cells; Humans; Indoles; Male; Mice; Mice, Inbred C57BL; MPTP Poisoning; Neuroprotective Agents; Oximes; Protein Aggregation, Pathological; Saccharomyces cerevisiae

Although misfolded and aggregated α-synuclein (α-syn) is recognized in the disease progression of synucleinopathies, its role in the impairment of cortical circuitries and synaptic plasticity remains incompletely understood. We investigated how α-synuclein accumulation affects synaptic plasticity in the mouse somatosensory cortex using two distinct approaches. Long-term

Topics: Aging; alpha-Synuclein; Animals; Dendritic Spines; Female; Humans; Male; Mice, Inbred C57BL; Mice, Transgenic; Neocortex; Protein Aggregation, Pathological; Pyramidal Cells; Up-Regulation

A limitation of the amyloid hypothesis in explaining the development of neurodegenerative diseases is that the level of amyloidogenic polypeptide in vivo is below the critical concentration required to form the aggregates observed in post-mortem brains. We discovered a novel, on-surface aggregation pathway of amyloidogenic polypeptide that eliminates this long-standing controversy. We applied atomic force microscope (AFM) to demonstrate directly that on-surface aggregation takes place at a concentration at which no aggregation in solution is observed. The experiments were performed with the full-size Aβ protein (Aβ42), a decapeptide Aβ(14-23) and α-synuclein; all three systems demonstrate a dramatic preference of the on-surface aggregation pathway compared to the aggregation in the bulk solution. Time-lapse AFM imaging, in solution, show that over time, oligomers increase in size and number and release in solution, suggesting that assembled aggregates can serve as nuclei for aggregation in bulk solution. Computational modeling performed with the all-atom MD simulations for Aβ(14-23) peptide shows that surface interactions induce conformational transitions of the monomer, which facilitate interactions with another monomer that undergoes conformational changes stabilizing the dimer assembly. Our findings suggest that interactions of amyloidogenic polypeptides with cellular surfaces play a major role in determining disease onset.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Microscopy, Atomic Force; Molecular Dynamics Simulation; Protein Aggregation, Pathological; Signal Transduction; Surface Properties

The accumulation of α-synuclein (α-syn) fibrils in neuronal inclusions is the defining pathological process in Parkinson's disease (PD). A pathogenic role for α-syn fibril accumulation is supported by the identification of dominantly inherited α-syn (

Topics: alpha-Synuclein; Amino Acid Substitution; Amyloid; Humans; Mutation, Missense; Parkinson Disease; Protein Aggregation, Pathological

Amyloid fibril formation has been implicated in the pathogenesis of neurodegenerative diseases. Fibrillation generates numerous conformers. Presumably, the conformers may possess specific biological properties, thus providing a biochemical framework for strains of prions. However, the precise relationship between various fibril conformers and their pathogenic functions has not been determined because of limited accessibility to adequate amounts of fibrils from tissue samples. α-Synuclein is one such protein, and it has been implicated in Parkinson disease. Using a technique known as protein misfolding cyclic amplification, originally developed for amplifying prions, we established a procedure through which the amplification of α-synuclein fibrils is possible. With a trace amount of seeds, we succeeded in amplifying α-synuclein fibrils. The replication of the seeds was faithful in terms of conformation even after multiple rounds of cyclic amplification. Moreover, two transgenic mouse strains each representing a distinct synucleinopathy were used to investigate different conformers by using this technique. The amplified α-synuclein fibrils derived from the tissue extracts of these two strains led to the production of two different fibril conformers with distinct proteinase K digestion profiles. Together, our results demonstrated that a trace amount of α-synuclein fibrils in tissue extracts could be amplified with their conformations conserved. This procedure should be useful in amplifying α-synuclein fibrils from the brains and body fluids of patients afflicted with synucleinopathies and may serve as a potential diagnostic tool for Parkinson disease and other synucleinopathies.

Topics: alpha-Synuclein; Amyloid; Animals; Brain; Humans; Mice; Protein Aggregation, Pathological; Protein Conformation; Protein Folding

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.

Topics: Aging; alpha-Synuclein; Animals; Cell Differentiation; Cell Survival; Cells, Cultured; Disease Models, Animal; Drosophila; Enzyme Inhibitors; Female; Glycosylation; Hippocampus; Humans; Induced Pluripotent Stem Cells; Male; Mice; Mice, Transgenic; Neurodegenerative Diseases; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Pyruvaldehyde; Rats; Yeasts

Clinical studies report significant increases in acrolein (an α,β-unsaturated aldehyde) in the substantia nigra (SN) of patients with Parkinson's disease (PD). In the present study, acrolein-induced neurotoxicity in the nigrostriatal dopaminergic system was investigated by local infusion of acrolein (15, 50, 150 nmoles/0.5 μl) in the SN of Sprague-Dawley rats. Acrolein-induced neurodegeneration of nigrostriatal dopaminergic system was delineated by reductions in tyrosine hydroxylase (TH) levels, dopamine transporter levels and TH-positive neurons in the infused SN as well as in striatal dopamine content. At the same time, apomorphine-induced turning behavior was evident in rats subjected to a unilateral infusion of acrolein in SN. Acrolein was pro-oxidative by increasing 4-hydroxy-2-nonenal and heme oxygenase-1 levels. Furthermore, acrolein conjugated with proteins at lysine residue and induced α-synuclein aggregation in the infused SN. Acrolein was pro-inflammatory by activating astrocytes and microglia. In addition, acrolein activated caspase 1 in the infused SN, suggesting acrolein-induced inflammasome formation. The neurotoxic mechanisms underlying acrolein-induced neurotoxicity involved programmed cell death, including apoptosis and necroptosis. Compared with well-known Parkinsonian neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone which do not exist in the SN of PD patients, our in vivo study shows that acrolein acts as a Parkinsonian neurotoxin in the nigrostriatal dopaminergic system of rat brain.

Topics: Acrolein; alpha-Synuclein; Animals; Cell Death; Disease Models, Animal; Dopaminergic Neurons; Encephalitis; Male; Oxidative Stress; Parkinsonian Disorders; Protein Aggregation, Pathological; Rats, Sprague-Dawley; Substantia Nigra

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aβ

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Catechin; Cell Survival; Cells, Cultured; Chickens; Dopaminergic Neurons; HEK293 Cells; Humans; Lysine; Methionine; Mice; Micrococcus luteus; Microtubule-Associated Proteins; Muramidase; Neuroprotective Agents; Oxidation-Reduction; Peptide Fragments; Protein Aggregation, Pathological; Quinones; Tyrosine 3-Monooxygenase

Parkinson's disease (PD) is a common neurodegenerative disorder that affects ~2% of the human population aged >65. α‑synuclein serves a role in the pathogenesis of PD as it is a primary component of Lewy bodies, a pathological feature of PD. Endosomal‑lysosomal dysfunction may be a key factor involved in the pathophysiology of PD, and may cause PD‑associated neurodegeneration via α‑synuclein‑dependent and ‑independent mechanisms. The D620N mutation in the endosomal‑lysosomal gene, vacuolar protein sorting‑associated protein 35 (VPS35), has been linked to PD. To clarify the underlying cellular mechanism of the VPS35 D620N mutation in PD, cell growth and endosomal‑lysosomal functions were investigated in Saccharomyces cerevisiae (sc) yeast cells that exhibited various expression levels of scVPS35, in the presence or absence of non‑toxic expression levels of α‑synuclein. Overexpression of the scVPS35 D686N mutation (the yeast equivalent of D620N) did not lead to toxicity in yeast. However, the co‑expression of high copy numbers of scVPS35 D686N and low copy numbers of α‑synuclein caused toxicity, whereas the co‑expression of scVPS35 wild‑type and α‑synuclein did not. In addition, the scVPS35 D686N mutant enhanced α‑synuclein aggregation. Fragmentation of vacuoles and subsequent inhibition of lysosome function was evident in yeast cells bearing the scVPS35 mutant. The results of the present study suggested that α‑synuclein and scVPS35 were interlinked via the endosomal‑lysosome pathway, which is important for the pathogenesis of PD.

Topics: alpha-Synuclein; Humans; Lysosomes; Microbial Viability; Mutation; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Saccharomyces cerevisiae; Vesicular Transport Proteins; Yeasts

Intracellular accumulation of α-synuclein (α-syn) are hallmarks of synucleinopathies, including Parkinson's disease (PD). Exogenous addition of preformed α-syn fibrils (PFFs) into primary hippocampal neurons induced α-syn aggregation and accumulation. Likewise, intrastriatal inoculation of PFFs into mice and non-human primates generates Lewy bodies and Lewy neurites associated with PD-like neurodegeneration. Herein, we investigate the putative effects of synthetic human PFFs on cultured rat ventral midbrain dopamine (DA) neurons. A time- and dose-dependent accumulation of α-syn was observed following PFFs exposure that also underwent phosphorylation at serine 129. PFFs treatment decreased the expression levels of synaptic proteins, caused alterations in axonal transport-related proteins, and increased H2AX Ser139 phosphorylation. Mitochondrial impairment (including modulation of mitochondrial dynamics-associated protein content), enhanced oxidative stress, and an inflammatory response were also detected in our experimental paradigm. In attempt to unravel a potential molecular mechanism of PFFs neurotoxicity, the expression of inducible nitric oxide synthase was blocked; a significant decline in protein nitration levels and protection against PFFs-induced DA neuron death were observed. Combined exposure to PFFs and rotenone resulted in an additive toxicity. Strikingly, many of the harmful effects found were more prominent in DA rather than non-DA neurons, suggestive of higher susceptibility to degenerate. These findings provide new insights into the role of α-syn in the pathogenesis of PD and could represent a novel and valuable model to study DA-related neurodegeneration.

Topics: alpha-Synuclein; Animals; Cell Survival; Cells, Cultured; Dopaminergic Neurons; Humans; Inflammation; Mesencephalon; Mitochondria; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Rats, Sprague-Dawley

Proteins implicated in neurodegenerative conditions such as Alzheimer's disease (AD) and Dementia with Lewy Bodies (DLB) have been identified in bodily fluids encased in extracellular vesicles called exosomes. Whether exosomes found in DLB patients can transmit pathology is not clear. In this study, exosomes were successfully harvested through ultracentrifugation from brain tissue from DLB and AD patients as well as non-diseased brain tissue. Exosomes extracted from brains diagnosed with either AD or DLB contained aggregate-prone proteins. Furthermore, injection of brain-derived exosomes from DLB patients into the brains of wild type mice induced α-synuclein (α-syn) aggregation. As assessed through immunofluorescent double labeling, α-syn aggregation was observed in MAP2

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Alzheimer Disease; Animals; Astrocytes; Brain; Cell Line, Tumor; Endocytosis; Exosomes; Humans; Lewy Body Disease; Male; Mice, Inbred C57BL; Mice, Inbred DBA; Neurons; Protein Aggregation, Pathological; Rats

Direct cell-to-cell transmission of proteopathic α-synuclein (α-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of α-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied α-syn seed species. Herein, we employed fluorescently labeled α-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized α-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous α-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous α-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of α-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.

Topics: alpha-Synuclein; Amino Acid Substitution; Animals; Cells, Cultured; Coloring Agents; Embryo, Mammalian; Endocytosis; Endosomes; Fluorescent Dyes; Green Fluorescent Proteins; Hippocampus; Humans; Hydrogen-Ion Concentration; Lysosomes; Mice; Microscopy, Electron, Transmission; Mutation; Neurons; Porphobilinogen; Protein Aggregation, Pathological; Recombinant Fusion Proteins; Rhodamines; Trypan Blue

Parkinson's disease (PD) is characterized neuropathologically by intracellular aggregates of fibrillar α-synuclein, termed Lewy bodies (LBs). Approximately 90% of α-synuclein deposited as LBs is phosphorylated at Ser129 in brains with PD. In contrast, only 4% of total α-synuclein is phosphorylated at Ser129 in brains with normal individuals. It is unclear why extensive phosphorylation occurs in the pathological process of PD. To address this issue, we investigated a mechanism and role of Ser129-phosphorylation in regulating accumulation of α-synuclein. In CHO cells, the levels of Ser129-phosphorylated soluble α-synuclein were maintained constantly to those of total α-synuclein in intracellular and extracellular spaces. In SH-SY5Y cells and rat primary cortical neurons, mitochondrial impairment by rotenone or MPP

Topics: alpha-Synuclein; Animals; Calcium; Cations, Divalent; Cell Line, Tumor; Cerebral Cortex; CHO Cells; Cricetulus; Extracellular Space; Humans; Mutation; Neurons; Parkinsonian Disorders; Phosphorylation; Proteasome Endopeptidase Complex; Protein Aggregation, Pathological; Rats, Sprague-Dawley; Stress, Physiological

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and its causes remain unknown. A major hallmark of the disease is the increasing presence of aggregated alpha-synuclein (aSyn). Furthermore, there is a solid consensus on iron (Fe) accumulation in several regions of PD brains during disease progression. In our study, we focused on the interaction of Fe and aggregating aSyn in vivo in a transgenic mouse model overexpressing human aSyn bearing the A53T mutation (prnp.aSyn.A53T). We utilized a neonatal iron-feeding model to exacerbate the motor phenotype of the transgenic mouse model. Beginning from day 100, mice were treated with deferiprone (DFP), a ferric chelator that is able to cross the blood-brain barrier and is currently used in clinics as treatment for hemosiderosis. Our paradigm resulted in an impairment of the learning abilities in the rotarod task and the novel object recognition test. DFP treatment significantly improved the performance in both tasks. Although this was not accompanied by alterations in aSyn aggregation, our results support DFP as possible therapeutic option in PD.

Topics: alpha-Synuclein; Animals; Cell Count; Deferiprone; Drug Evaluation, Preclinical; Female; Gait Disorders, Neurologic; Humans; Iron; Iron Chelating Agents; Learning Disabilities; Male; Mice; Mice, Transgenic; Neurons; Parkinsonian Disorders; Protein Aggregation, Pathological; Pyridones; Recognition, Psychology; Rotarod Performance Test

Neurodegenerative diseases such as Parkinson's and Alzheimer's disease share the pathological hallmark of fibrillar protein aggregates. The specific detection of these protein aggregates by positron emission tomography (PET) in the patient brain can yield valuable information for diagnosis and disease progression. However, the identification of novel small compounds that bind fibrillar protein aggregates has been a challenge. In this study, microscale thermophoresis (MST) was applied to assess the binding affinity of known small molecule ligands of α-synuclein fibrils, which were also tested in parallel in a thioflavin T fluorescence competition assay for further validation. In addition, a MST assay was also developed for the detection of the interaction between a variety of small molecules and tau fibrils. The results of this study demonstrate that MST is a powerful and practical methodology to quantify interactions between small molecules and protein fibrillar aggregates, which suggests that it can be applied for the identification and development of PET radioligands and potentially of therapeutic candidates for protein misfolding diseases.

Topics: alpha-Synuclein; Benzothiazoles; Binding, Competitive; Dose-Response Relationship, Drug; Drug Discovery; Humans; Indoles; Microscopy, Atomic Force; Optical Imaging; Protective Agents; Protein Aggregation, Pathological; Protein Binding; tau Proteins; Thermal Diffusion; Thiazoles

The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Animals; Blotting, Western; Brain; Cognition Disorders; Cyclophilins; Cyclosporine; Disease Models, Animal; Female; HEK293 Cells; Humans; Male; Mice, Transgenic; Microscopy, Electron, Transmission; Neurodegenerative Diseases; Peptidyl-Prolyl Isomerase F; Protein Aggregates; Protein Aggregation, Pathological; tau Proteins; Tauopathies

Aggregated forms of α-synuclein play a crucial role in the pathogenesis of synucleinopathies such as Parkinson's disease (PD). However, the molecular mechanisms underlying the pathogenic effects of α-synuclein are not completely understood. Here we show that asparagine endopeptidase (AEP) cleaves human α-synuclein, triggers its aggregation and escalates its neurotoxicity, thus leading to dopaminergic neuronal loss and motor impairments in a mouse model. AEP is activated and cleaves human α-synuclein at N103 in an age-dependent manner. AEP is highly activated in human brains with PD, and it fragments α-synuclein, which is found aggregated in Lewy bodies. Overexpression of the AEP-cleaved α-synuclein

Topics: alpha-Synuclein; Animals; Asparagine; Cysteine Endopeptidases; Disease Models, Animal; Humans; Mice; Parkinson Disease; Protein Aggregation, Pathological; Proteins; Proteolysis

α-Synuclein (αS) is the primary protein associated with Parkinson's disease, and it undergoes aggregation from its intrinsically disordered monomeric form to a cross-β fibrillar form. The closely related homolog β-synuclein (βS) is essentially fibril-resistant under cytoplasmic physiological conditions. Toxic gain-of-function by βS has been linked to dysfunction, but the aggregation behavior of βS under altered pH is not well-understood. In this work, we compare fibril formation of αS and βS at pH 7.3 and mildly acidic pH 5.8, and we demonstrate that pH serves as an on/off switch for βS fibrillation. Using αS/βS domain-swapped chimera constructs and single residue substitutions in βS, we localized the switch to acidic residues in the N-terminal and non-amyloid component domains of βS. Computational models of βS fibril structures indicate that key glutamate residues (Glu-31 and Glu-61) in these domains may be sites of pH-sensitive interactions, and variants E31A and E61A show dramatically altered pH sensitivity for fibril formation supporting the importance of these charged side chains in fibril formation of βS. Our results demonstrate that relatively small changes in pH, which occur frequently in the cytoplasm and in secretory pathways, may induce the formation of βS fibrils and suggest a complex role for βS in synuclein cellular homeostasis and Parkinson's disease.

Topics: alpha-Synuclein; Amino Acid Substitution; beta-Synuclein; Glutamic Acid; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Microfibrils; Models, Molecular; Mutagenesis, Site-Directed; Parkinson Disease; Peptide Fragments; Point Mutation; Protein Aggregation, Pathological; Protein Interaction Domains and Motifs; Recombinant Fusion Proteins

The pathology of Parkinson's disease and other synucleinopathies is characterized by the formation of intracellular inclusions comprised primarily of misfolded, fibrillar α-synuclein (α-syn). One strategy to slow disease progression is to prevent the misfolding and aggregation of its native monomeric form. Here we present findings that support the contention that the tricyclic antidepressant compound nortriptyline (NOR) has disease-modifying potential for synucleinopathies. Findings from in vitro aggregation and kinetics assays support the view that NOR inhibits aggregation of α-syn by directly binding to the soluble, monomeric form, and by enhancing reconfiguration of the monomer, inhibits formation of toxic conformations of the protein. We go on to demonstrate that NOR inhibits the accumulation, aggregation and neurotoxicity of α-syn in multiple cell and animal models. These findings suggest that NOR, a compound with established safety and efficacy for treatment of depression, may slow progression of α-syn pathology by directly binding to soluble, native, α-syn, thereby inhibiting pathological aggregation and preserving its normal functions.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Antidepressive Agents, Tricyclic; Brain; Cell Line, Tumor; Drosophila; Escherichia coli; Humans; Male; Mice; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Nortriptyline; Protein Aggregation, Pathological; Protein Unfolding; Random Allocation; Rats, Sprague-Dawley; Recombinant Proteins

The transition of intrinsically disordered, monomeric α-synuclein into β-sheet-rich oligomers and fibrils is associated with multiple neurodegenerative diseases. Fibrillar aggregates possessing distinct structures that differ in toxicity have been observed in different pathological phenotypes. Understanding the mechanism of the formation of various fibril polymorphs with differing cytotoxic effects is essential for determining how the aggregation reaction could be modulated to favor nontoxic fibrils over toxic fibrils. In this study, two morphologically different α-synuclein fibrils, one helical and the other ribbon-like, are shown to form together. Surprisingly, a widely used small molecule for probing aggregation reactions, thioflavin T (ThT), was found to tune the structural heterogeneity found in the fibrils. The ribbon-like fibrils formed in the presence of ThT were found to have a longer structural core than the helical fibrils formed in the absence of ThT. The ribbon-like fibrils are also more toxic to cells. By facilitating the formation of ribbon-like fibrils over helical fibrils, ThT reduced the extent of fibril polymorphism. This study highlights the role of a small molecule such as ThT in selectively favoring the formation of a specific type of fibril by binding to aggregates formed early on one of multiple pathways, thereby altering the structural core and external morphology of the fibrils formed.

Topics: alpha-Synuclein; Benzothiazoles; Humans; Multiprotein Complexes; Protein Aggregation, Pathological; Protein Multimerization; Thiazoles

In neurodegenerative diseases, seeding is a process initiated by the internalization of exogenous protein aggregates. Multiple pathways for internalization of aggregates have been proposed, including direct membrane penetration and endocytosis. To decipher the seeding mechanisms of alpha-synuclein (αS) aggregates in human cells, we visualized αS aggregation, endo-lysosome distribution, and endo-lysosome rupture in real-time. Our data suggest that exogenous αS can seed endogenous cytoplasmic αS by either directly penetrating the plasma membrane or via endocytosis-mediated endo-lysosome rupture, leading to formation of endo-lysosome-free or endo-lysosome-associated αS aggregates, respectively. Further, we demonstrate that αS aggregates isolated from postmortem human brains with diffuse Lewy body disease (DLBD) preferentially show endocytosis-mediated seeding associated with endo-lysosome rupture and have significantly reduced seeding activity compared to recombinant αS aggregates. Colocalization of αS pathology with galectin-3 (a marker of endo-lysosomal membrane rupture) in the basal forebrain of DLBD, but not in age-matched controls, suggests endo-lysosome rupture is involved in the formation of αS pathology in humans. Interestingly, cells with endo-lysosomal membrane permeabilization (LMP) are more vulnerable to the seeding effects of αS aggregates. This study suggests that endo-lysosomal impairment in neurons might play an important role in PD progression.

Topics: alpha-Synuclein; Brain; Endocytosis; Endosomes; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Intracellular Membranes; Lewy Bodies; Lysosomes; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological

Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (SNCA-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.

Topics: alpha-Synuclein; Animals; Antibody Affinity; Bacteriophages; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Parkinson Disease; Peptides; Protein Aggregation, Pathological; RNA, Viral; Vaccines; Virion

Cell-to-cell transmission of intracellular protein aggregates is considered a central event in many neurodegenerative diseases, but little is known about the underlying molecular mechanisms. A new study employs fluorescence quenching to examine the fate of α-synuclein, a key molecule in the pathology of Parkinson's disease and related disorders, in primary cultured neurons, finding that endocytosis and lysosomal processing of exogenous fibrils may explain the transmission of α-synuclein pathology.

Topics: alpha-Synuclein; Animals; Endocytosis; Humans; Lysosomes; Models, Biological; Neurons; Protein Aggregation, Pathological

α-Synuclein (aSyn) is the main driver of neurodegenerative diseases known as "synucleinopathies," but the mechanisms underlying this toxicity remain poorly understood. To investigate aSyn toxic mechanisms, we have developed a primary neuronal model in which a longitudinal survival analysis can be performed by following the overexpression of fluorescently tagged WT or pathologically mutant aSyn constructs. Most aSyn mutations linked to neurodegenerative disease hindered neuronal survival in this model; of these mutations, the E46K mutation proved to be the most toxic. While E46K induced robust PLK2-dependent aSyn phosphorylation at serine 129, inhibiting this phosphorylation did not alleviate aSyn toxicity, strongly suggesting that this pathological hallmark of synucleinopathies is an epiphenomenon. Optical pulse-chase experiments with Dendra2-tagged aSyn versions indicated that the E46K mutation does not alter aSyn protein turnover. Moreover, since the mutation did not promote overt aSyn aggregation, we conclude that E46K toxicity was driven by soluble species. Finally, we developed an assay to assess whether neurons expressing E46K aSyn affect the survival of neighboring control neurons. Although we identified a minor non-cell-autonomous component spatially restricted to proximal neurons, most E46K aSyn toxicity was cell autonomous. Thus, we have been able to recapitulate the toxicity of soluble aSyn species at a stage preceding aggregation, detecting non-cell-autonomous toxicity and evaluating how some of the main aSyn hallmarks are related to neuronal survival.

Topics: alpha-Synuclein; Amino Acid Substitution; Animals; Mutation, Missense; Neurodegenerative Diseases; Neurons; Phosphorylation; Protein Aggregation, Pathological; Rats; Rats, Sprague-Dawley

Alpha synuclein (α-syn) is a central player in neurodegeneration, but the mechanisms triggering its pathology are not fully understood. Here we found that α-syn is a highly efficient substrate for arginyltransferase ATE1 and is arginylated in vivo by a novel mid-chain mechanism that targets the acidic side chains of E46 and E83. Lack of arginylation leads to increased α-syn aggregation and causes the formation of larger pathological aggregates in neurons, accompanied by impairments in its ability to be cleared via normal degradation pathways. In the mouse brain, lack of arginylation leads to an increase in α-syn's insoluble fraction, accompanied by behavioral changes characteristic for neurodegenerative pathology. Our data show that lack of arginylation in the brain leads to neurodegeneration, and suggests that α-syn arginylation can be a previously unknown factor that facilitates normal α-syn folding and function in vivo.

Topics: alpha-Synuclein; Amino Acid Sequence; Aminoacyltransferases; Animals; Arginine; Brain; Cells, Cultured; Disease Models, Animal; Humans; Mass Spectrometry; Mice; Mice, Knockout; Models, Biological; Neurodegenerative Diseases; Neurons; Peptides; Protein Aggregates; Protein Aggregation, Pathological; Protein Processing, Post-Translational; Proteolysis; Recombinant Proteins; Substrate Specificity

α-synuclein-induced neurotoxicity is a core pathogenic event in neurodegenerative synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy. There is currently no disease-modifying therapy available for these diseases. We screened 1,600 FDA-approved drugs for their efficacy to protect LUHMES cells from degeneration induced by wild-type α-synuclein and identified dipyridamole, a non-selective phosphodiesterase inhibitor, as top hit. Systematic analysis of other phosphodiesterase inhibitors identified a specific phosphodiesterase 1 inhibitor as most potent to rescue from α-synuclein toxicity. Protection was mediated by an increase of cGMP and associated with the reduction of a specific α-synuclein oligomeric species. RNA interference experiments confirmed PDE1A and to a smaller extent PDE1C as molecular targets accounting for the protective efficacy. PDE1 inhibition also rescued dopaminergic neurons from wild-type α-synuclein induced degeneration in the substantia nigra of mice. In conclusion, this work identifies inhibition of PDE1A in particular as promising target for neuroprotective treatment of synucleinopathies.

Topics: alpha-Synuclein; Animals; Cell Line; Dipyridamole; Drug Discovery; Drug Evaluation, Preclinical; Enzyme Inhibitors; High-Throughput Screening Assays; Humans; Mice; Neurons; Neuroprotective Agents; Phosphodiesterase I; Protein Aggregation, Pathological; Vinca Alkaloids

Parkinson's disease is the second most common neurodegenerative disease. It is characterized by aggregation of the protein α-synuclein (α-syn) in Lewy bodies, mitochondrial dysfunction, and increased oxidative stress in the substantia nigra. Oxidative stress leads to several modifications of biomolecules including dityrosine (DiY) crosslinking in proteins, which has recently been detected in α-syn in Lewy bodies from Parkinson's disease patients. Here we report that α-syn is highly susceptible to ultraviolet-induced DiY formation. We investigated DiY formation of α-syn and nine tyrosine-to-alanine mutants and monitored its effect on α-syn fibril formation in vitro. Ultraviolet irradiation of intrinsically disordered α-syn generates DiY-modified monomers and dimers, which inhibit fibril formation of unmodified α-syn by interfering with fibril elongation. The inhibition depends on both the DiY group and its integration into α-syn. When preformed α-syn fibrils are crosslinked by DiY formation, they gain increased resistance to denaturation. DiY-stabilized α-syn fibrils retain their high seeding efficiency even after being exposed to denaturant concentrations that completely depolymerize non-crosslinked seeds. Oxidative stress-associated DiY crosslinking of α-syn therefore entails two opposing effects: (i) inhibition of aggregation by DiY-modified monomers and dimers, and (ii) stabilization of fibrillar aggregates against potential degradation mechanisms, which can lead to promotion of aggregation, especially in the presence of secondary nucleation.

Topics: alpha-Synuclein; Humans; Oxidative Stress; Protein Aggregation, Pathological; Protein Multimerization; Tyrosine; Ultraviolet Rays

Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.

Topics: alpha-Synuclein; Animals; Autophagosomes; Autophagy; Cell Survival; Disaccharides; Humans; Lysosomes; Microtubule-Associated Proteins; Neurons; Parkinson Disease; Primary Cell Culture; Protein Aggregation, Pathological; Rats; RNA-Binding Proteins; Trehalose

Approximately 90% of alpha-synuclein (α-Synuclein) deposited in Lewy bodies is phosphorylated at serine 129 suggesting that the accumulation of phosphorylated α-Synuclein is critical in the pathogenesis of Parkinson's disease. However, in vivo experiments addressing the role of phosphorylated α-Synuclein in the progression of Parkinson's disease have produced equivocal data. To clarify a role of Ser129 phosphorylation of α-Synuclein in pathology progression we performed stereotaxic injections targeting the mouse striatum with three fibrilar α-Synuclein types: wt-fibrils, phosphorylated S129 fibrils and, phosphorylation incompetent, S129A fibrils. Brain inoculation of all three fibrilar types caused seeding of the endogenous α-Synuclein. However, phosphorylated fibrils triggered the formation of more α-Synuclein inclusions in the Substantia Nigra pars compacta (SNpc), exacerbated pathology in the cortex and caused dopaminergic neuronal loss and fine motor impairment as early as 60 days post injection. Phosphorylated fibril injections also induced early changes in the innate immune response including alterations in macrophage recruitment and IL-10 release. Our study further shows that S129 phosphorylation facilitated α-Synuclein fibril uptake by neurons. Our results highlight the role of phosphorylated fibrilar α-Synuclein in pathology progression in vivo and suggest that targeting phosphorylated α-Synuclein assemblies might be important for delaying inclusion formation.

Topics: alpha-Synuclein; Amyloid; Animals; Cerebral Cortex; Female; Fluorescent Antibody Technique; Humans; Immunity, Innate; Lewy Bodies; Male; Mice; Motor Activity; Neurons; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Proteins

Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons.

Topics: alpha-Synuclein; Cell Line, Tumor; Cell Membrane; Cerebral Cortex; Humans; Lipid Bilayers; Mutation; Neurons; Nuclear Magnetic Resonance, Biomolecular; Parkinson Disease; Protein Aggregation, Pathological

The aggregation and deposition of α-synuclein (αSyn) in neuronal cells is correlated to pathogenesis of Parkinson's disease. Although the mechanism of αSyn aggregation and fibril formation has been studied extensively, the structural hallmarks that are directly responsible for toxicity toward cells are still under debate. Here, we have compared the structural characteristics of the toxic intermediate molecular species of αSyn and similar toxic species of another protein, GroES, using coherent X-ray diffraction analysis. Using coherent X-ray free electron laser pulses of SACLA, we analysed αSyn and GroES fibril intermediate species and characterized various aggregate structures. Unlike previous studies where an annular oligomeric form of αSyn was identified, particle reconstruction from scattering traces suggested that the specific forms of the toxic particles were varied, with the sizes of the particles falling within a specific range. We did however discover a common structural feature in both αSyn and GroES samples; the edges of the detected particles were nearly parallel and produced a characteristic diffraction pattern in the diffraction experiments. The presence of parallel-edged particles in toxic intermediates of αSyn and GroES fibrillogenesis pointed towards a plausible common molecular interface that leads to the formation of mature fibrils.

Topics: alpha-Synuclein; Animals; Cell Line, Tumor; Chaperonin 10; Humans; Mice; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; X-Ray Diffraction

Misfolded α-synuclein (αS) is hypothesized to spread throughout the central nervous system (CNS) by neuronal connectivity leading to widespread pathology. Increasing evidence indicates that it also has the potential to invade the CNS via peripheral nerves in a prion-like manner. On the basis of the effectiveness following peripheral routes of prion administration, we extend our previous studies of CNS neuroinvasion in M83 αS transgenic mice following hind limb muscle (intramuscular [i.m.]) injection of αS fibrils by comparing various peripheral sites of inoculations with different αS protein preparations. Following intravenous injection in the tail veins of homozygous M83 transgenic (M83. The misfolding and accumulation of α-synuclein (αS) inclusions are found in a number of neurodegenerative disorders and is a hallmark feature of Parkinson's disease (PD) and PD-related diseases. Similar characteristics have been observed between the infectious prion protein and αS, including its ability to spread from the peripheral nervous system and along neuroanatomical tracts within the central nervous system. In this study, we extend our previous results and investigate the efficiency of intravenous (i.v.), intraperitoneal (i.p.), and intramuscular (i.m.) routes of injection of αS fibrils and other protein controls. Our data reveal that injection of αS fibrils via these peripheral routes in αS-overexpressing mice are capable of inducing a robust αS pathology and in some cases cause paralysis. Furthermore, soluble, nonaggregated αS also induced αS pathology, albeit with much less efficiency. These findings further support and extend the idea of αS neuroinvasion from peripheral exposures.

Topics: alpha-Synuclein; Animals; Brain; Central Nervous System Diseases; Disease Models, Animal; Inclusion Bodies; Mice; Mice, Transgenic; Neurodegenerative Diseases; Phenotype; Protein Aggregates; Protein Aggregation, Pathological; Spinal Cord

The 140-residue natively disordered protein α-synuclein (aSN) is a central component in the development of a family of neurodegenerative diseases termed synucleinopathies. This is attributed to its ability to form cytotoxic aggregates such as oligomers and amyloid fibrils. Consequently there have been intense efforts to avoid aggregation or reroute the aggregation pathway using pharmaceutical agents such as small molecules, chaperones and antibodies. aSN's lack of persistent structure in the monomeric state, as well as the multitude of different oligomeric and even different fibrillar states, makes it difficult to raise antibodies that would be efficacious in neutralizing all conformations of aSN. However, the C-terminal 20-40 residues of aSN are a promising epitope for antibody development. It is primarily disordered in both monomeric and aggregated forms, and an anti-C-terminal antibody will therefore be able to bind all forms. Furthermore, it might not interfere with the folding of aSN into membranes, which could be important for its physiological role. Here we report a screen of a series of monoclonal antibodies, which all target the C-terminal of aSN. According to dot blot analyses, different antibodies bound different forms of aSN with different preferences and showed reduced binding to monomeric compared to aggregated (oligomeric and fibrillary) aSN. Consequently they have different effects on aSN's ability to fibrillate and permeabilize membranes. Generally, the antibodies with strongest binding to aggregated aSN in dot blot, also inhibited fibrillation and membrane permeabilization the most, and promoted formation of amorphous aggregates surrounded by small and thin fibers. This suggests that the development of antibodies that targets the C-terminus, exposed in the aggregated forms of aSN, may be beneficial for improved immunotherapy against PD.

Topics: alpha-Synuclein; Amyloid; Animals; Antibodies, Monoclonal; Cell Membrane Permeability; Humans; Mice; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named 'ochronosis' and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis.. Gel electrophoresis, Western Blot, Congo Red-based assays and electron microscopy were used to investigate the effects of HGA on the aggregation and fibrillation propensity of amyloidogenic proteins and peptides [Aβ(1-42), transthyretin, atrial natriuretic peptide, α-synuclein and SAA]. LC/MS and in silico analyses were undertaken to identify possible binding sites for HGA (or its oxidative metabolite, a benzoquinone acetate or BQA) in SAA.. We found that HGA might act as an amyloid aggregation enhancer in vitro for all the tested proteins and peptides in a time- and dose- dependent fashion, and identified a small crevice at the interface between two HGD subunits as a candidate binding site for HGA/BQA.. HGA might be an important amyloid co- component playing significant roles in AKU amyloidosis.. Our results provide a possible explanation for the clinically verified onset of amyloidotic processes in AKU and might lay the basis to setup proper pharmacological approaches to alkaptonuric ochronosis, which are still lacking.

Topics: Alkaptonuria; alpha-Synuclein; Amyloid beta-Peptides; Amyloidogenic Proteins; Amyloidosis; Atrial Natriuretic Factor; Binding Sites; Connective Tissue; Homogentisate 1,2-Dioxygenase; Homogentisic Acid; Humans; Ochronosis; Oxidation-Reduction; Prealbumin; Protein Aggregation, Pathological; Serum Amyloid A Protein

Copper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson's Disease. Hence, residue specific interaction of Cu. We investigated the residue specific mapping of Cu. Copper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu. Cu. These findings will help in the better understanding of Cu

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Binding Sites; Cell Line; Copper; Humans; Kinetics; Parkinson Disease; Protein Aggregation, Pathological

Parkinson disease (PD) is a highly prevalent and incurable neurodegenerative disease associated with the accumulation of misfolded α-synuclein (αSyn) aggregates. An important problem in this disease is the lack of a sensitive, specific, and noninvasive biochemical diagnosis to help in clinical evaluation, monitoring of disease progression, and early differential diagnosis from related neurodegenerative diseases.. To develop a novel assay with high sensitivity and specificity to detect small quantities of αSyn aggregates circulating in cerebrospinal fluid (CSF) of patients affected by PD and related synucleinopathies.. The strategy evaluated in this proof-of-concept study uses the protein misfolding cyclic amplification (PMCA) technology that detects minute amounts of misfolded oligomers by taking advantage of their ability to nucleate further aggregation, enabling a very high amplification of the signal. The technology was first adapted with synthetic αSyn oligomers prepared in vitro and used to screen in 2 blinded cohorts of CSF samples from German and Japanese patients with PD (n = 76) and individuals serving as controls affected by other neurologic disorders (n = 65), neurodegenerative diseases (n = 18), and Alzheimer disease (n = 14). The kinetics of αSyn aggregation were measured by αSyn-PMCA in the presence of CSF samples from the participants to detect αSyn oligomeric seeds present in this biological fluid. The assays were conducted from November 15, 2013, to August 28, 2015.. Kinetic parameters correlated with disease severity at the time of sample collection, measured by the Hoehn and Yahr scale, with the lowest grade indicating unilateral involvement with minimal or no functional impairment, and the highest grade defining patients with complete confinement to wheelchair or bed.. Studies with synthetic αSyn aggregates showed that αSyn-PMCA enabled to detect as little as 0.1 pg/mL of αSyn oligomers. The αSyn-PMCA signal was directly proportional to the amount of αSyn oligomers added to the reaction. A blinded study of CSF samples correctly identified patients affected by PD with an overall sensitivity of 88.5% (95% CI, 79.2%-94.6%) and specificity of 96.9% (95% CI, 89.3%-99.6%). The αSyn-PMCA results for different patients correlated with the severity of the clinical symptoms of PD (Japanese cohort: rs = -0.54, P = .006; German cohort: rs = -0.36, P = .02).. The findings suggest that detection of αSyn oligomers by αSyn-PMCA in the CSF of patients affected by PD may offer a good opportunity for a sensitive and specific biochemical diagnosis of the disease. Further studies are needed to investigate the usefulness of αSyn-PMCA to monitor disease progression and for preclinical identification of patients who may develop PD.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Biochemical Phenomena; Diagnostic Tests, Routine; Female; Humans; In Vitro Techniques; Lewy Body Disease; Male; Multiple System Atrophy; Outcome Assessment, Health Care; Parkinson Disease; Peptide Fragments; Predictive Value of Tests; Protein Aggregation, Pathological; Proteostasis Deficiencies; Retrospective Studies; Sensitivity and Specificity; Severity of Illness Index; tau Proteins

α-synuclein gene mutations can cause α-synuclein protein aggregation in the midbrain of Parkinson's disease (PD) patients. MicroRNAs (miRNAs) play a key role in the metabolism of α-synuclein but the mechanism involved in synucleinopathy remains unclear. In this study, we investigated the miRNA profiles in A53T-α-synuclein transgenic mice and analyzed the candidate miRNAs in the cerebrospinal fluid (CSF) of PD patients. The 12-month A53T-transgenic mouse displayed hyperactive movement and anxiolytic-like behaviors with α-synuclein aggregation in midbrain. A total of 317,759 total and 289,207 unique small RNA sequences in the midbrain of mice were identified by high-throughput deep sequencing. We found 644 miRNAs were significantly changed in the transgenic mice. Based on the conserved characteristic of miRNAs, we selected 11 candidates from the 40 remarkably expressed miRNAs and explored their expression in 44 CSF samples collected from PD patients. The results revealed that 11 microRNAs were differently expressed in CSF, emphatically as miR-144-5p, miR-200a-3p and miR-542-3p, which were dramatically up-regulated in both A53T-transgenic mice and PD patients, and had a helpful accuracy for the PD prediction. The ordered logistic regression analysis showed that the severity of PD has strong correlation with an up-expression of miR-144-5p, miR-200a-3p and miR-542-3p in CSF. Taken together, our data suggested that miRNAs in CSF, such as miR-144-5p, miR-200a-3p and miR-542-3p, may be useful to the PD diagnosis as potential biomarkers.

Topics: Adult; Aged; Alleles; alpha-Synuclein; Animals; Biomarkers; Case-Control Studies; Disease Models, Animal; Dopaminergic Neurons; Female; Gene Expression Profiling; Gene Expression Regulation; High-Throughput Nucleotide Sequencing; Humans; Hyperkinesis; Male; Mice; Mice, Transgenic; MicroRNAs; Middle Aged; Mutation; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; RNA Interference; Transcriptome

Ample in vitro and in vivo experimental evidence supports the hypothesis that intercellular transmission of α-synuclein (αS) is a mechanism underlying the spread of αS pathology in Parkinson's disease and related disorders. What remains unexplained is where and how initial transmissible αS aggregates form. In a previous study, we demonstrated that αS aggregates rapidly form in neurons with impaired nuclear membrane integrity due to the interaction between nuclear proaggregant factor(s) and αS and that such aggregates may serve as a source for αS seeding. In the present study, we identify histones as a potential nuclear proaggregant factor for αS aggregation in both apoptotic neurons and brains with αS pathology. We further demonstrate that histone-induced aggregates contain a range of αS oligomers, including protofibrils and mature fibrils, and that these αS aggregates can seed additional aggregation. Importantly, we demonstrate transmissibility in mouse brains from stereotaxic injection. This study provides new clues to the mechanism underlying initial pathological aggregation of αS in PD and related disorders, and could lead to novel diagnostic and therapeutic approaches.

Topics: alpha-Synuclein; Animals; Apoptosis; Blotting, Western; Brain; Cell Line, Tumor; Cytoplasm; Fluorescent Antibody Technique; Histones; Humans; Immunoprecipitation; Lewy Body Disease; Mice, Inbred C57BL; Microscopy, Confocal; Microscopy, Electron; Microscopy, Fluorescence; Neurons; Protein Aggregation, Pathological; Recombinant Proteins

Parkinson's disease (PD) presents with neuropathological inclusions called Lewy bodies, which are primarily composed of fibrillar α-synuclein. Recently, we characterized sheep with Gaucher disease and since GBA1 mutations represent the highest genetic risk factor for PD, we have investigated α-synuclein fibrillation in the sheep. Here we demonstrate that differences in six amino acid residues between sheep and human α-synuclein significantly alter in vitro fibril formation. Circular dichroism of recombinant human and sheep α-synuclein show that both proteins adopt the same secondary structure. Fibrils from human and sheep α-synuclein formed at pH7.0 or 4.5 were analyzed by Transmission Electron Microscopy (TEM). Unexpectedly, sheep α-synuclein form fibrils much less readily than human α-synuclein and this difference was more pronounced at the lysosomal pH of 4.5. Aggregation-propensity and intrinsic-solubility analysis revealed that sheep α-synuclein had lower aggregation-propensity and higher solubility. As a result of these observations, TEM was used to analyze fibrils formed at pH4.5 of various "sheep-like" human or "human-like" sheep mutant α-synucleins, together with their wild-type forms. Thioflavin T was used to monitor in situ α-synuclein fibril formation at pH7.0 and 4.5. Results show that "sheep-like" human α-synuclein has substantially lower fibril aggregation, and "human-like" sheep α-synuclein aggregates faster than wild-type forms, respectively. Seeding with WT human α-synuclein showed that "sheep-like" human α-synuclein could not be seeded, providing further evidence that sheep sequence is resistant to fibrillation. These findings provide new avenues to prevent/reduce fibrillation in PD, which may aid in the development of therapies.

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloid; Animals; Benzothiazoles; Humans; Hydrogen-Ion Concentration; Kinetics; Lewy Bodies; Mutation; Parkinson Disease; Protein Aggregation, Pathological; Sequence Alignment; Sheep; Solubility; Thiazoles

Mutations in leucine-rich repeat kinase 2 (LRRK2) and α-synuclein lead to Parkinson's disease (PD). Disruption of protein homeostasis is an emerging theme in PD pathogenesis, making mechanisms to reduce the accumulation of misfolded proteins an attractive therapeutic strategy. We determined if activating nuclear factor erythroid 2-related factor (Nrf2), a potential therapeutic target for neurodegeneration, could reduce PD-associated neuron toxicity by modulating the protein homeostasis network. Using a longitudinal imaging platform, we visualized the metabolism and location of mutant LRRK2 and α-synuclein in living neurons at the single-cell level. Nrf2 reduced PD-associated protein toxicity by a cell-autonomous mechanism that was time-dependent. Furthermore, Nrf2 activated distinct mechanisms to handle different misfolded proteins. Nrf2 decreased steady-state levels of α-synuclein in part by increasing α-synuclein degradation. In contrast, Nrf2 sequestered misfolded diffuse LRRK2 into more insoluble and homogeneous inclusion bodies. By identifying the stress response strategies activated by Nrf2, we also highlight endogenous coping responses that might be therapeutically bolstered to treat PD.

Topics: alpha-Synuclein; Animals; Cerebral Cortex; Genes, Reporter; HEK293 Cells; Humans; Hydroquinones; Inclusion Bodies; Induced Pluripotent Stem Cells; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Nerve Tissue Proteins; Neurons; NF-E2-Related Factor 2; Parkinson Disease; Primary Cell Culture; Protein Aggregation, Pathological; Proteostasis; Rats; Recombinant Fusion Proteins; Single-Cell Analysis; Time Factors

Phosphorylated alpha-synuclein (p-α-syn) containing Lewy bodies (LBs) and Lewy neurites (LNs) are neuropathological hallmarks of Parkinson's disease (PD) in the central nervous system (CNS). Since they have been also demonstrated in the enteric nervous system (ENS) of PD patients, the aim of the study was to analyze enteric p-α-syn positive aggregates and intestinal gene expression. Submucosal rectal biopsies were obtained from patients with PD and controls and processed for dual-label-immunohistochemistry for p-α-syn and PGP 9.5. p-α-syn positive aggregates in nerve fibers and neuronal somata were subjected to a morphometric analysis. mRNA expression of α-syn and dopaminergic, serotonergic, VIP (vaso intestinal peptide) ergic, cholinergic, muscarinergic neurotransmitter systems were investigated using qPCR. Frequency of p-α-syn positive nerve fibers was comparable between PD and controls. Although neuronal p-α-syn positive aggregates were detectable in both groups, total number and area of p-α-syn positive aggregates were increased in PD patients as was the number of small and large sized aggregates. Increased expression of dopamine receptor D1, VIP and serotonin receptor 3A was observed in PD patients, while serotonin receptor 4 and muscarinic receptor 3 (M3R) were downregulated. M3R expression correlated negative with the number of small sized p-α-syn positive aggregates. The findings strengthen the hypothesis that the CNS pathology of increased p-α-syn in PD also applies to the ENS, if elaborated morphometry is applied and give further insights in altered intestinal gene expression in PD. Although the mere presence of p-α-syn positive aggregates in the ENS should not be regarded as a criterion for PD diagnosis, elaborated morphometric analysis of p-α-syn positive aggregates in gastrointestinal biopsies could serve as a suitable tool for in-vivo diagnosis of PD.

Topics: Adult; Aged; Aged, 80 and over; alpha-Synuclein; Colonoscopy; Enteric Nervous System; Ganglia, Autonomic; Gene Expression Profiling; Humans; Immunohistochemistry; Middle Aged; Neurons; Parkinson Disease; Phosphorylation; Protein Aggregation, Pathological; Real-Time Polymerase Chain Reaction; Rectum; RNA, Messenger; Transcriptome

Parkinson's disease is a debilitating neurodegenerative disorder that is pathologically characterized by intracellular inclusions comprised primarily of alpha-synuclein (αSyn) that can also be transmitted from neuron to neuron. Several lines of evidence suggest that these inclusions cause neurodegeneration. Thus exploring strategies to improve neuronal survival in neurons with αSyn aggregates is critical. Previously, exposure to αSyn pre-formed fibrils (PFFs) has been shown to induce aggregation of endogenous αSyn resulting in cell death that is exacerbated by either starvation or inhibition of mTOR by rapamycin, both of which are able to induce autophagy, an intracellular protein degradation pathway. Since mTOR inhibition may also inhibit protein synthesis and starvation itself can be detrimental to neuronal survival, we investigated the effects of autophagy induction on neurons with αSyn inclusions by a starvation and mTOR-independent autophagy induction mechanism. We exposed mouse primary cortical neurons to PFFs to induce inclusion formation in the presence and absence of the disaccharide trehalose, which has been proposed to induce autophagy and stimulate lysosomal biogenesis. As expected, we observed that on exposure to PFFs, there was increased abundance of pS129-αSyn aggregates and cell death. Trehalose alone increased LC3-II levels, consistent with increased autophagosome levels that remained elevated with PFF exposure. Interestingly, trehalose alone increased cell viability over a 14-d time course. Trehalose was also able to restore cell viability to control levels, but PFFs still exhibited toxic effects on the cells. These data provide essential information regarding effects of trehalose on αSyn accumulation and neuronal survival on exposure to PFF.

Topics: alpha-Synuclein; Animals; Apoptosis; Autophagy; Cell Survival; Humans; Mice; Neurons; Parkinson Disease; Protein Aggregation, Pathological; TOR Serine-Threonine Kinases; Trehalose

Neuropathological and genetic findings suggest that the presynaptic protein α-synuclein (aSyn) is involved in the pathogenesis of synucleinopathy disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. Evidence suggests that the self-assembly of aSyn conformers bound to phospholipid membranes in an aggregation-prone state plays a key role in aSyn neurotoxicity. Accordingly, we hypothesized that protein binding partners of lipid-associated aSyn could inhibit the formation of toxic aSyn oligomers at membrane surfaces. To address this hypothesis, we characterized the protein endosulfine-alpha (ENSA), previously shown to interact selectively with membrane-bound aSyn, in terms of its effects on the membrane-induced aggregation and neurotoxicity of two familial aSyn mutants, A30P and G51D. We found that wild-type ENSA, but not the non-aSyn-binding S109E variant, interfered with membrane-induced aSyn self-assembly, aSyn-mediated vesicle disruption and aSyn neurotoxicity. Immunoblotting analyses revealed that ENSA was down-regulated in the brains of synucleinopathy patients versus non-diseased individuals. Collectively, these results suggest that ENSA can alleviate neurotoxic effects of membrane-bound aSyn via an apparent chaperone-like activity at the membrane surface, and a decrease in ENSA expression may contribute to aSyn neuropathology in synucleinopathy disorders. More generally, our findings suggest that promoting interactions between lipid-bound, amyloidogenic proteins and their binding partners is a viable strategy to alleviate cytotoxicity in a range of protein misfolding disorders.

Topics: Adenoviridae; Aged; Aged, 80 and over; alpha-Synuclein; Animals; Brain; Cell Membrane; Cells, Cultured; Cohort Studies; Dopaminergic Neurons; Escherichia coli; Female; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Lewy Body Disease; Male; Middle Aged; Neuroprotective Agents; Peptides; Protein Aggregation, Pathological; Rats, Sprague-Dawley; Recombinant Proteins; Unilamellar Liposomes

The pathogenesis of Parkinson's disease is closely associated with the aggregation of the α-synuclein protein. Several familial mutants have been identified and shown to affect the aggregation kinetics of α-synuclein through distinct molecular mechanisms. Quantitative evaluation of the relative stabilities of the wild type and mutant fibrils is crucial for understanding the aggregation process and identifying the key component steps. In this work, we examined two topologically different α-synuclein fibril structures that are either determined by solid-state NMR method or modeled based on solid-state NMR data, and characterized their conformational properties and thermodynamic stabilities using molecular dynamics simulations. We show that the two fibril morphologies have comparable size, solvent exposure, secondary structures, and similar molecule/peptide binding modes; but different stabilities. Familial mutations do not significantly alter the overall fibril structures but shift their relative stabilities. Distinct mutations display altered fibril conformational behavior, suggesting different propagation preferences, reminiscent of cross-seeding among prion strains and tau deletion mutants. The simulations quantify the hydrophobic and electrostatic interactions, as well as N-terminal dynamics, that may contribute to the divergent aggregation kinetics that has been observed experimentally. Our results indicate that small molecule and peptide inhibitors may share the same binding region, providing molecular recognition that is independent of fibril conformation.

Topics: alpha-Synuclein; Humans; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Mutant Proteins; Mutation; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation

Parkinson's disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

Topics: 3,4-Dihydroxyphenylacetic Acid; alpha-Synuclein; Animals; Biological Transport; Cell Line; Cell Membrane; Humans; Magnetic Resonance Spectroscopy; Mice; Neurons; Permeability; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization; Synaptic Vesicles; Tandem Mass Spectrometry

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Topics: Algorithms; alpha-Synuclein; Amino Acid Sequence; Animals; Animals, Genetically Modified; Biological Products; Caenorhabditis elegans; Cell Line, Tumor; Cholestanols; Humans; Membrane Lipids; Molecular Structure; Neuroblastoma; Paresis; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization

The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson's disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP

Topics: 1-Methyl-4-phenylpyridinium; Adenylate Kinase; Aged; Aged, 80 and over; alpha-Synuclein; Animals; Cell Death; Dopaminergic Neurons; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; GTPase-Activating Proteins; Humans; Lewy Bodies; Mice; Mice, Inbred C57BL; Mice, Knockout; MPTP Poisoning; Nerve Tissue Proteins; Phosphorylation; Pleckstrin Homology Domains; Protein Aggregation, Pathological; Protein Binding; Protein Interaction Mapping; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-fyn

The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson's disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion. For fibrils aggregated in high-salt buffers (including those prepared in buffers with a physiological salt concentration) the measurements are consistent with αS molecules in a more tightly-packed, antiparallel intramolecular conformation, and suggest a structure characterized by two twisting stacks of approximately five hydrogen-bonded intermolecular β-sheets each. We find evidence that the high-frequency peak in the amide-I spectrum of αS fibrils involves a normal mode that differs fundamentally from the canonical high-frequency antiparallel β-sheet mode. The high sensitivity of the fibril structure to the ionic strength might form the basis of differences in αS-related pathologies.

Topics: alpha-Synuclein; Amyloid; Humans; Hydrogen Bonding; Microscopy, Atomic Force; Osmolar Concentration; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation, beta-Strand; Spectrophotometry, Infrared

α-Synuclein is a defining, key component of Lewy bodies and Lewy neurites in Parkinson's disease (PD) and dementia with Lewy bodies (DLB), as well as glial cytoplasmic inclusions in multiple system atrophy (MSA). The distribution and spreading of these pathologies are closely correlated with disease progression. Recent studies have revealed that intracerebral injection of synthetic α-synuclein fibrils or pathological α-synuclein prepared from DLB or MSA brains into wild-type or transgenic animal brains induced prion-like propagation of phosphorylated α-synuclein pathology. The common marmoset is a very small primate that is expected to be a useful model of human diseases. Here, we show that intracerebral injection of synthetic α-synuclein fibrils into adult wild-type marmoset brains (caudate nucleus and/or putamen) resulted in spreading of abundant α-synuclein pathologies, which were positive for various antibodies to α-synuclein, including phospho Ser129-specific antibody, anti-ubiquitin and anti-p62 antibodies, at three months after injection. Remarkably, robust Lewy body-like inclusions were formed in tyrosine hydroxylase (TH)-positive neurons in these marmosets, strongly suggesting the retrograde spreading of abnormal α-synuclein from striatum to substantia nigra. Moreover, a significant decrease in the numbers of TH-positive neurons was observed in the injection-side of the brain, where α-synuclein inclusions were deposited. Furthermore, most of the α-synuclein inclusions were positive for 1-fluoro-2,5-bis (3-carboxy-4-hydroxystyryl) benzene (FSB) and thioflavin-S, which are dyes widely used to visualize the presence of amyloid. Thus, injection of synthetic α-synuclein fibrils into brains of non-transgenic primates induced PD-like α-synuclein pathologies within only 3 months after injection. Finally, we provide evidence indicating that neurons with abnormal α-synuclein inclusions may be cleared by microglial cells. This is the first marmoset model for α-synuclein propagation. It should be helpful in studies to elucidate mechanisms of disease progression and in development and evaluation of disease-modifying drugs for α-synucleinopathies.

Topics: alpha-Synuclein; Animals; Benzothiazoles; Brain; Callithrix; Female; Immunohistochemistry; Lewy Bodies; Microglia; Nerve Degeneration; Neurons; Parkinsonian Disorders; Protein Aggregation, Pathological; Recombinant Proteins; Sequence Homology, Amino Acid; Thiazoles; Tyrosine 3-Monooxygenase

Synucleinophaties are progressive neurodegenerative disorders with no cure to date. An attractive strategy to tackle this problem is repurposing already tested safe drugs against novel targets. In this way, doxycycline prevents neurodegeneration in Parkinson models by modulating neuroinflammation. However, anti-inflammatory therapy per se is insufficient to account for neuroprotection. Herein we characterise novel targets of doxycycline describing the structural background supporting its effectiveness as a neuroprotector at subantibiotic doses. Our results show that doxycycline reshapes α-synuclein oligomers into off-pathway, high-molecular-weight species that do not evolve into fibrils. Off-pathway species present less hydrophobic surface than on-pathway oligomers and display different β-sheet structural arrangement. These structural changes affect the α-synuclein ability to destabilize biological membranes, cell viability, and formation of additional toxic species. Altogether, these mechanisms could act synergically giving novel targets for repurposing this drug.

Topics: alpha-Synuclein; Cell Line, Tumor; Cell Survival; Doxycycline; Drug Repositioning; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation, beta-Strand; Protein Multimerization; Spectroscopy, Fourier Transform Infrared

Mutations in the glucocerebrosidase gene (

Topics: alpha-Synuclein; Animals; Carbamates; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Glucosyltransferases; Humans; Mice; Mutation; Parkinson Disease; Protein Aggregation, Pathological; Quinuclidines; tau Proteins; Ubiquitin

Parkinson's disease is characterized by the presence of insoluble and neurotoxic aggregates (amyloid fibrils) of an intrinsically disordered protein α-synuclein. In this study we have examined the effects of four naturally occurring polyphenols in combination with β-cyclodextrin (β-CD) on the aggregation of α-synuclein in the presence of macromolecular crowding agents. Our results reveal that even at sub-stoichiometric concentrations of the individual components, the polyphenol-β-CD combination(s) not only inhibited the aggregation of the proteins but was also effective in disaggregating preformed fibrils. Curcumin was found to be the most efficient, followed by baicalein with (-)-epigallocatechin gallate and resveratrol coming in next, the latter two exhibiting very similar effects. Our results suggest that the efficiency of curcumin results from a balanced composition of the phenolic OH groups, benzene rings and flexibility. The latter ensures proper positioning of the functional groups to maximize the underlying interactions with both the monomeric form of α-synuclein and its aggregates. The uniqueness of β-CD was reinforced by the observation that none of the other cyclodextrin variants [α-CD and HP-β-CD] used was as effective, in spite of these possessing better water solubility. Moreover, the fact that the combinations remained effective under conditions of macromolecular crowding suggests that these have the potential to be developed into viable drug compositions in the near future. MTT assays on cell viability independently confirmed this hypothesis wherein these combinations (and the polyphenols alone too) appreciably impeded the toxicity of the prefibrillar α-synuclein aggregates on the mouse neuroblastoma cell lines (N2a cells).

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Animals; beta-Cyclodextrins; Catechin; Cell Line; Cell Survival; Circular Dichroism; Curcumin; Humans; Mice; Parkinson Disease; Polyphenols; Protein Aggregation, Pathological

Amyloid polymorphs have become one of the focal points of molecular studies of neurodegenerative diseases like Parkinson's disease. Due to their distinct biochemical properties and prion-like characteristics, insights into the molecular origin and stability of amyloid polymorphs over time are crucial for understanding the potential role of amyloid polymorphism in these diseases. Here, we systematically study the fibrillization of recombinantly produced human α-synuclein (αSyn) over an extended period of time to unravel the origin and temporal evolution of polymorphism. We follow morphological changes in the same fibril sample with atomic force microscopy over a period of 1 year. We show that wild-type (wt) αSyn fibrils undergo a slow maturation over time after reaching the plateau phase of aggregation (as detected in a Thioflavin-T fluorescence assay). This maturation, visualized by changes in the fibril periodicity over time, is absent in the disease mutant fibrils. The β-sheet content of the plateau phase and matured fibrils, obtained using Fourier transform infrared spectroscopy, is however similar for the αSyn protein sequences, suggesting that the morphological changes in wt αSyn fibrils are tertiary or quaternary in origin. Furthermore, results from a reversibility assay show that the plateau phase fibrils do not disassemble over time. Together, the observed changes in the periodicity distributions and stability of the fibrillar core over time point toward two distinct mechanisms that determine the morphology of wt αSyn fibrils: competitive growth between different polymorphs during the fibrillization phase followed by a process wherein fibrils undergo slow maturation or annealing.

Topics: alpha-Synuclein; Benzothiazoles; Circular Dichroism; Humans; Microscopy, Atomic Force; Multiprotein Complexes; Mutation; Protein Aggregation, Pathological; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thiazoles; Time Factors

Ubiquitin C-terminal Hydrolase-1 (UCHL1) is a deubiquitinating enzyme, which plays a key role in Parkinson's disease (PD). It is one of the most important proteins, which constitute Lewy body in PD patient. However, how this well folded highly soluble protein presents in this proteinaceous aggregate is still unclear. We report here that UCHL1 undergoes S-nitrosylation in vitro and rotenone induced PD mouse model. The preferential nitrosylation in the Cys 90, Cys 152 and Cys 220 has been observed which alters the catalytic activity and structural stability. We show here that nitrosylation induces structural instability and produces amorphous aggregate, which provides a nucleation to the native α-synuclein for faster aggregation. Our findings provide a new link between UCHL1-nitrosylation and PD pathology.

Topics: alpha-Synuclein; Animals; Humans; Mice; Parkinson Disease, Secondary; Protein Aggregation, Pathological; Rotenone; Ubiquitin Thiolesterase

Protein aggregation is involved in a variety of diseases. Alteration of the aggregation pathway, either to produce less toxic structures or to increase aggregate clearance, is a promising therapeutic route. Both active and passive immunization has been used for this purpose. However, the mechanism of action of antibodies on protein aggregates is not completely clear especially given poor ability of antibodies to cross blood-brain barrier. Here, we have shown that antibodies can interfere with protein aggregation at substoichiometric concentrations (as low as 1:1000 antibody to protein ratio). This is an indication that antibodies interact with aggregation intermediates in chaperone-like manner altering the aggregation pathways at very low antibody levels. This observation supports earlier suggestions that antibodies can inhibit aggregation by interaction with low abundance aggregation intermediates.

Topics: alpha-Synuclein; Antibodies; Benzothiazoles; Biocatalysis; Biological Assay; Circular Dichroism; Fluorescence; Kinetics; Protein Aggregates; Protein Aggregation, Pathological; Thiazoles

We previously reported a loss of cholinergic neurons within the pedunculopontine tegmental nucleus (PPTg) in rats that had been intra-nigrally lesioned with the proteasomal inhibitor lactacystin, with levels of neuronal loss corresponding to that seen in the post-mortem pedunculopontine nucleus (PPN) of advanced Parkinson's disease (PD) patients. Here we reveal lower expression values of the acetylcholine synthesising enzyme, choline acetyltransferase, within the remaining PPTg cholinergic neurons of lesioned rats compared to sham controls. We further characterise this animal model entailing dopaminergic- and non-dopaminergic neurodegeneration by reporting on stereological counts of non-cholinergic neurons, to determine whether the toxin is neuro-type specific. Cell counts between lesioned and sham-lesioned rats were analysed in terms of the topological distribution pattern across the rostro-caudal extent of the PPTg. The study also reports somatic hypotrophy in the remaining non-cholinergic neurons, particularly on the side closest to the nigral lesion. The cytotoxicity affecting the PPTg in this rat model of PD involves overexpression and accumulation of alpha-synuclein (αSYN), affecting cholinergic and non-cholinergic neurons as well as microglia on the lesioned hemispheric side. We ascertained that microglia within the PPTg become fully activated due to the extensive neuronal damage and neuronal death resulting from a lactacystin nigral lesion, displaying a distinct rostro-caudal distribution profile which correlates with PPTg neuronal loss, with the added implication that lactacystin-induced αSYN aggregation might trigger neuronophagia for promoting PPTg cell loss. The data provide critical insights into the mechanisms underlying the lactacystin rat model of PD, for studying the PPTg in health and when modelling neurodegenerative disease.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Count; Choline O-Acetyltransferase; Cholinergic Neurons; Disease Models, Animal; Dopaminergic Neurons; Male; Microglia; Neurons; Parkinson Disease; Parkinsonian Disorders; Pars Compacta; Pedunculopontine Tegmental Nucleus; Protein Aggregation, Pathological; Rats; Rats, Sprague-Dawley; Tyrosine 3-Monooxygenase

Amyloid formation and aberrant protein aggregation are hallmarks of more than 30 different human diseases. The proteins that form amyloid can be divided into two structural classes: those that form compact, well-ordered, globular structures in their unaggregated state and those that are intrinsically disordered in their unaggregated states. The latter include the Aβ peptide of Alzheimer's disease, islet amyloid polypeptide (IAPP, amylin) implicated in type 2 diabetes and α-synuclein, which is linked to Parkinson's disease. Work in the last 10 years has highlighted the potential role of pre-amyloid intermediates in cytotoxicity and has focused attention on their properties. A number of intrinsically disordered proteins appear to form helical intermediates during amyloid formation. We discuss the spectroscopic methods employed to detect and characterize helical intermediates in homogenous solution and in membrane-catalyzed amyloid formation, with the emphasis on the application of circular dichroism (CD). IAPP is used as an example, but the methods are generally applicable.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloidogenic Proteins; Humans; Intrinsically Disordered Proteins; Molecular Biology; Protein Aggregation, Pathological

The cell-to-cell transmission of protein aggregates has been implicated in the progression of pathological phenotypes in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In recent years, several experimental model systems have been developed to study the mechanisms of cell-to-cell transmission. Herein, we describe cell culture models with which cell-to-cell transmission of α-synuclein can be quantitatively analyzed. The principle underlying these models could be applied to developing model systems for transmission of other protein aggregates, such as tau and TDP-43.

Topics: alpha-Synuclein; Alzheimer Disease; Amyotrophic Lateral Sclerosis; Cell Culture Techniques; Humans; Molecular Biology; Parkinson Disease; Protein Aggregation, Pathological

The abnormal accumulation of alpha-synuclein (α-syn) has been linked to a number of neurodegenerative disorders, the most noteworthy of which is Parkinson's disease. Alpha-synuclein itself is not toxic and fulfills various physiological roles in the central nervous system. However, specific types of aggregates have been shown to be toxic, and metals have been linked to the assembly of these toxic aggregates. In this paper, we have characterized a transgenic mouse that overexpresses the A53T mutation of human α-syn, specifically assessing cognition, motor performance, and subtle anatomical markers that have all been observed in synucleinopathies in humans. We hypothesized that treatment with the moderate-affinity metal chelator, clioquinol (CQ), would reduce the interaction between metals and α-syn to subsequently improve the phenotype of the A53T animal model. We showed that CQ prevents an iron-synuclein interaction, the formation of urea-soluble α-syn aggregates, α-syn-related substantia nigra pars compacta cell loss, reduction in dendritic spine density of hippocampal and caudate putamen medium spiny neurons, and the decline in motor and cognitive function. In conclusion, our data suggests that CQ is capable of mitigating the pathological metal/α-syn interactions, suggesting that the modulation of metal ions warrants further study as a therapeutic approach for the synucleinopathies.

Topics: alpha-Synuclein; Animals; Brain; Clioquinol; Cognition Disorders; Disease Models, Animal; Exploratory Behavior; Humans; Maze Learning; Mice; Mice, Transgenic; Movement Disorders; Mutation; Protein Aggregation, Pathological; Recognition, Psychology; Silver Staining; Spatial Learning

Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity.. We first demonstrate excessive free radical production in a human induced pluripotent stem-derived α-S triplication model at basal levels and on application of picomolar doses of β-sheet-rich α-S oligomers. We probed the effects of different structural species of α-S in wild-type rat neuronal cultures and show that both oligomeric and fibrillar forms of α-S are capable of generating free radical production, but that only the oligomeric form results in reduction of endogenous glutathione and subsequent neuronal toxicity. We dissected the mechanism of oligomer-induced free radical production and found that it was interestingly independent of several known cellular enzymatic sources.. The oligomer-induced reactive oxygen species (ROS) production was entirely dependent on the presence of free metal ions as addition of metal chelators was able to block oligomer-induced ROS production and prevent oligomer-induced neuronal death.. Our findings further support the causative role of soluble amyloid oligomers in triggering neurodegeneration and shed light into the mechanisms by which these species cause neuronal damage, which, we show here, can be amenable to modulation through the use of metal chelation.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Apoptosis; Caspase 3; Caspase 7; Cell Differentiation; Enzyme Activation; Gene Duplication; Glutathione; Humans; Induced Pluripotent Stem Cells; Ions; Metals; Neurons; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Protein Conformation; Protein Multimerization; Rats; Reactive Oxygen Species; Structure-Activity Relationship

Alpha-synuclein (α-SYN) aggregates represent a key feature of Parkinson's disease, but the exact relationship between α-SYN aggregation and neurodegeneration remains incompletely understood. Therefore, the availability of a cellular assay that allows medium-throughput analysis of α-SYN-linked pathology will be of great value for studying the aggregation process and for advancing α-SYN-based therapies.. Here we describe a high-content neuronal cell assay that simultaneously measures oxidative stress-induced α-SYN aggregation and apoptosis.. We optimized an automated and reproducible assay to quantify both α-SYN aggregation and cell death in human SH-SY5Y neuroblastoma cells.. Quantification of α-SYN aggregates in cells has typically relied on manual imaging and counting or cell-free assays, which are time consuming and do not allow a concurrent analysis of cell viability. Our high-content analysis method for quantification of α-SYN aggregation allows simultaneous measurements of multiple cell parameters at a single-cell level in a fast, objective and automated manner.. The presented analysis approach offers a rapid, objective and multiparametric approach for the screening of compounds and genes that might alter α-SYN aggregation and/or toxicity.

Topics: alpha-Synuclein; Apoptosis; Benzothiazoles; Blotting, Western; Cell Count; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Genetic Vectors; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Indoles; Lentivirus; Microscopy, Fluorescence; Oxidative Stress; Parkinsonian Disorders; Protein Aggregation, Pathological; Protein Multimerization; Software; Thiazoles

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.

Topics: alpha-Synuclein; Amino Acid Substitution; Animals; Animals, Newborn; Cell Line; Cells, Cultured; Dopaminergic Neurons; Endocytosis; Humans; Mesencephalon; Mice; Mutation; Parkinson Disease; Phosphorylation; Protein Aggregation, Pathological; Protein Folding; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Recombinant Proteins; Serine; Synaptosomes

Alpha-synuclein (α-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, α-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated α-syn is a neuropathological feature of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble α-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of α-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated α-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal α-syn biology in transgenic animal models harbouring different α-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Topics: alpha-Synuclein; Brain; Brain Chemistry; Humans; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Ultracentrifugation

Acupuncture has historically been practiced to treat medical disorders by mechanically stimulating specific acupoints with fine needles. Despite its well-documented efficacy, its biological basis remains largely elusive. In this study, we found that mechanical stimulation at the acupoint of Yanglingquan (GB34) promoted the autophagic clearance of α-synuclein (α-syn), a well known aggregation-prone protein closely related to Parkinson's disease (PD), in the substantia nigra par compacta (SNpc) of the brain in a PD mouse model. We found the protein clearance arose from the activation of the autophagy-lysosome pathway (ALP) in a mammalian target of rapamycin (mTOR)-independent approach. Further, we observed the recovery in the activity of dopaminergic neurons in SNpc, and improvement in the motor function at the behavior level of PD mice. Whereas acupuncture and rapamycin, a chemical mTOR inhibitor, show comparable α-syn clearance and therapeutic effects in the PD mouse model, the latter adopts a distinctly different, mTOR-dependent, autophagy induction process. Due to this fundamental difference, acupuncture may circumvent adverse effects of the rapamycin treatment. The newly discovered connection between acupuncture and autophagy not only provides a new route to understanding the molecular mechanism of acupuncture but also sheds new light on cost-effective and safe therapy of neurodegenerative diseases.

Topics: Acupuncture; Acupuncture Therapy; alpha-Synuclein; Animals; Autophagy; Brain; Disease Models, Animal; Dopaminergic Neurons; Lysosomes; Male; Mice; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Signal Transduction; Substantia Nigra; TOR Serine-Threonine Kinases

Protein misfolding results in the accumulation of aggregated β-sheet-rich structures in Parkinson's disease (PD) and Alzheimer's disease. The toxic oligomer hypothesis stipulates that prefibrillar assemblies, such as soluble oligomers or protofibrils, are responsible for the poor prognosis of these diseases. Previous studies demonstrated that a small molecule related to the natural compound orcein, O4, directly binds to amyloid-β fibrils and stabilizes them, accelerating the formation of end-stage mature fibrils. Here we demonstrate a similar phenomenon during O4 treatment of α-synuclein (αsyn) aggregates, the protein responsible for PD pathology. While the drug did not change the kinetics of aggregate formation as measured by the amyloidophilic dye thioflavin T, O4 depleted αsyn oligomers and promoted the formation of sodium dodecyl sulfate and proteinase K resistant aggregates consisting of large fibril clusters. These fibril clusters exhibited reduced toxicity to human neuronal model cells and reduced seeding activity in vitro. The effectiveness of O4 decreased when it was added at later points in the αsyn aggregation pathway, which suggests that the incorporation of O4 into fibril assemblies stabilizes them against chemical, enzymatic, and mechanic degradation. These findings suggest that small molecules, which stabilize amyloid fibrils, can prevent fibril fragmentation and seeding and consequently prevent prion-like replication of misfolded αsyn. Inhibiting prion replication by fibril stabilization could thus be a therapeutic strategy for PD.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Humans; Parkinson Disease; Protein Aggregation, Pathological; Protein Folding; Protein Stability

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel β-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel β-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel β-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.

Topics: Acetylation; alpha-Synuclein; Amyloid; Biophysical Phenomena; Humans; Parkinson Disease; Protein Aggregation, Pathological; Protein Folding; Protein Structure, Secondary; Spectroscopy, Fourier Transform Infrared

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Apoptosis; Cells, Cultured; Humans; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Protein Transport; Proteostasis Deficiencies

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.

Topics: alpha-Synuclein; Animals; Dopaminergic Neurons; Drosophila melanogaster; Gaucher Disease; Glucosylceramidase; Humans; Lysosomes; Nerve Degeneration; Parkinson Disease; Phenotype; Protein Aggregation, Pathological

GFAP is the major intermediate filament protein in mature astrocytes. Its increased expression and aggregation was firstly associated to Alexander's disease, and successively in different neurological diseases including scrapie, Alzheimer's and Creutzfeld-Jacob diseases. Recently, ceftriaxone a multi-potent β-lactam antibiotic able to overcome the blood-brain barrier, successfully eliminated the cellular toxic effects of misfolded mutated GFAP, similarly to phenytoin sodium, in a cellular model of Alexander's disease and inhibited α-synuclein aggregation protecting PC12 cells from the exposure to 6-hydroxydopamine.. In this study, synchrotron radiation circular dichroism spectroscopy has been used to obtain structural information about the GFAP-ceftriaxone (phenytoin) interactions, while computational methods allowed the identification of the relevant putative binding site of either ceftriaxone or phenytoin on the dimer structure of GFAP, permitting to rationalize the spectroscopic experimental results.. We found that GFAP exhibited enhanced stability upon the addition of two equivalents of each ligands with ceftriaxone imparting a more spontaneous interactions and a more ordered complex system than phenytoin.. SRCD data and MD models indicate a stronger protective effect of ceftriaxone in neurological disorders characterized by an increased production and polymerization of GFAP.. This result, in addition to our previous works in which we documented that ceftriaxone interacts with α-synuclein inhibiting its pathological aggregation and that a cyclical treatment with this molecule in a patient with adult-onset Alexander's disease halted, and partly reversed, the progression of neurodegeneration, suggests the possibility of a chaperone-like effect of ceftriaxone on protein involved in specific neurodegenerative diseases.

Topics: Alexander Disease; alpha-Synuclein; Animals; Astrocytes; Binding Sites; Ceftriaxone; Glial Fibrillary Acidic Protein; Humans; Intermediate Filament Proteins; Molecular Docking Simulation; Molecular Dynamics Simulation; Nerve Degeneration; PC12 Cells; Phenytoin; Protein Aggregation, Pathological; Rats

α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells.

Topics: alpha-Synuclein; Cell Line; Humans; In Vitro Techniques; Lewy Bodies; Microtubules; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Recombinant Proteins; Sequence Deletion; tau Proteins

Aberrant protein-protein interactions are a common pathological hallmark among neurodegenerative diseases, including Parkinson's disease (PD). Thus far, mutations in more than 20 genes have been associated with PD. These genes encode for proteins involved in distinct intracellular pathways, complicating our understanding of the precise molecular mechanisms underlying the disease. Recent reports suggested that the endolysosomal protein ATP13A2 can determine the fate of alpha-synuclein (α-Syn), although no consensus has yet been reached on the mechanisms underlying this effect. Here, we describe, for the first time, the deleterious effect arising from the interaction between the ATP13A2 familial mutant Dup22 with α-Syn. We show that this ATP13A2 mutant can enhance α-Syn oligomerization and aggregation in cell culture. Additionally, we report the accumulation of both proteins in abnormal endoplasmic reticulum membranous structures and the activation of the protein kinase RNA-like endoplasmic reticulum kinase pathway. Ultimately, our data bring new insight into the molecular mechanisms underlying the interplay of these two proteins, opening novel perspectives for therapeutic intervention.

Topics: alpha-Synuclein; Brain; Cell Death; Cell Line; Endoplasmic Reticulum; Endosomes; Gene Expression Regulation; Humans; Lysosomes; Mutation; Parkinson Disease; Protein Aggregation, Pathological; Proton-Translocating ATPases

Intracellular α-synuclein deposits, known as Lewy bodies, have been linked to a range of neurodegenerative disorders, including Parkinson's disease. α-Synuclein binds to synthetic and biological lipids, and this interaction has been shown to play a crucial role for both α-synuclein's native function, including synaptic plasticity, and the initiation of its aggregation. Here, we describe the interplay between the lipid properties and the lipid binding and aggregation propensity of α-synuclein. In particular, we have observed that the binding of α-synuclein to model membranes is much stronger when the latter is in the fluid rather than the gel phase, and that this binding induces a segregation of the lipids into protein-poor and protein-rich populations. In addition, α-synuclein was found to aggregate at detectable rates only when interacting with membranes composed of the most soluble lipids investigated here. Overall, our results show that the chemical properties of lipids determine whether or not the lipids can trigger the aggregation of α-synuclein, thus affecting the balance between functional and aberrant behavior of the protein.

Topics: alpha-Synuclein; Cell Membrane; Humans; Kinetics; Lipid Bilayers; Parkinson Disease; Protein Aggregation, Pathological

The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, we assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 μg/μl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 μg/μl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α-synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamines, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles.

Topics: alpha-Synuclein; Animals; Dopaminergic Neurons; HEK293 Cells; Humans; Inclusion Bodies; Magnetite Nanoparticles; Primary Cell Culture; Proteasome Endopeptidase Complex; Protein Aggregation, Pathological; Rats; Rhodamines; Silicon Dioxide

Aggregates of abnormal proteins are widely observed in neuronal and glial cells of patients with various neurodegenerative diseases, and it has been proposed that prion-like behavior of these proteins can account for not only the onset but also the progression of these diseases. However, it is not yet clear which abnormal protein structures function most efficiently as seeds for prion-like propagation. In this study, we aimed to identify the most pathogenic species of α-synuclein (α-syn), the main component of the Lewy bodies and Lewy neurites that are observed in α-synucleinopathies. We prepared various forms of α-syn protein and examined their seeding properties in vitro in cells and in mouse experimental models. We also characterized these α-syn species by means of electron microscopy and thioflavin fluorescence assays and found that fragmented β sheet-rich fibrous structures of α-syn with a length of 50 nm or less are the most efficient promoters of accumulation of phosphorylated α-syn, which is the hallmark of α-synucleinopathies. These results indicate that fragmented amyloid-like aggregates of short α-syn fibrils are the key pathogenic seeds that trigger prion-like conversion.

Topics: alpha-Synuclein; Amyloid; Animals; Cell Line, Tumor; Humans; Lewy Bodies; Mice; Neurites; Parkinson Disease; Phosphorylation; Prions; Protein Aggregation, Pathological

Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease.

Topics: alpha-Synuclein; Amino Acid Substitution; Endosomes; HeLa Cells; Humans; Mutation, Missense; Parkinson Disease; Protein Aggregation, Pathological; Vesicular Transport Proteins

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.

Topics: alpha-Synuclein; Animals; Cell Survival; DNA Damage; Dopaminergic Neurons; Gene Expression Regulation, Developmental; HEK293 Cells; Humans; LIM-Homeodomain Proteins; Mesencephalon; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mitochondria; Oxidative Stress; Protein Aggregation, Pathological; Transcription Factors

Neurodegenerative diseases such as Parkinson's and Alzheimer's diseases are multifactorial disorders related to protein aggregation, metal dyshomeostasis, and oxidative stress. To advance understanding in this area and to contribute to therapeutic development, many efforts have been directed at devising suitable agents that can target metal ions associated with relevant biomolecules such as α-synuclein. This paper presents a new cyclodextrin-8-hydroxyquinoline conjugate and discusses the properties of four cyclodextrins 3-functionalized with 8-hydroxyquinoline as copper(II) chelators and inhibitors of copper-induced synuclein aggregation. The encouraging results establish the potential of cyclodextrin-8-hydroxyquinoline conjugates as chelators for the control of copper toxicity.

Topics: alpha-Synuclein; Antitoxins; Binding Sites; Copper; Cyclodextrins; Hydroxyquinolines; Protein Aggregation, Pathological

The misfolding and aggregation of a small, natively unfolded protein α-synuclein (α-syn) is presumably an important factor in the development of Parkinson's disease. However, the mechanism of α-syn aggregation into amyloid fibrils and their morphology are not well understood. To elucidate the aggregation kinetics and the morphology of aggregates by the use of fluorescent techniques the protein needs to be suitably labeled. In this study, using atomic force microscopy, we demonstrate a significant effect of fluorescent labels on the α-syn fibrillization process. We studied in detail the morphology of α-syn aggregates as a function of the composition of mixtures of labeled and wild type (WT) α-syn in solution using different types of fluorescent dyes. Although the overall charge of the fluorophores we used and their chemical structure varied significantly, the morphology of α-syn fibrils changed in a similar way in all cases. The increase in the fraction of labeled α-syn in solution led to shortening of the fibrils as compared to those from WT-only α-syn, whereas the height of the fibrils remained mainly unaffected. The twisted fibril morphology observed in the WT and A140C α-syn mutant completely disappeared when the A140C α-syn mutant was 100% fluorescently labeled.

Topics: alpha-Synuclein; Amyloid; Escherichia coli; Fluorescent Dyes; Kinetics; Microscopy, Atomic Force; Parkinson Disease; Protein Aggregation, Pathological; Staining and Labeling

In E. coli cells, rescue of non-native proteins and promotion of native state structure is assisted by the chaperonin GroEL. An important key to this activity lies in the structure of the apical domain of GroEL (GroEL-AD) (residue 191-376), which recognizes and binds non-native protein molecules through hydrophobic interactions. In this study, we investigated the effects of GroEL-AD on the aggregation of various client proteins (α-Synuclein, Aβ42, and GroES) that lead to the formation of distinct protein fibrils in vitro. We found that GroEL-AD effectively inhibited the fibril formation of these three proteins when added at concentrations above a critical threshold; the specific ratio differed for each client protein, reflecting the relative affinities. The effect of GroEL-AD in all three cases was to decrease the concentration of aggregate-forming unfolded client protein or its early intermediates in solution, thereby preventing aggregation and fibrillation. Binding affinity assays revealed some differences in the binding mechanisms of GroEL-AD toward each client. Our findings suggest a possible applicability of this minimal functioning derivative of the chaperonins (the "minichaperones") as protein fibrillation modulators and detectors.

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Chaperonin 10; Chaperonin 60; Escherichia coli Proteins; Humans; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Models, Molecular; Peptide Fragments; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Protein Domains; Protein Folding; Recombinant Proteins

Parkinson's disease (PD) is characterized by the progressive appearance of intraneuronal Lewy aggregates, which are primarily composed of misfolded α-synuclein (α-syn). The aggregates are believed to propagate via neural pathways following a stereotypical pattern, starting in the olfactory bulb (OB) and gut. We hypothesized that injection of fibrillar α-syn into the OB of wild-type mice would recreate the sequential progression of Lewy-like pathology, while triggering olfactory deficits. We demonstrate that injected α-syn fibrils recruit endogenous α-syn into pathological aggregates that spread transneuronally over several months, initially in the olfactory network and later in distant brain regions. The seeded inclusions contain posttranslationally modified α-syn that is Thioflavin S positive, indicative of amyloid fibrils. The spreading α-syn pathology induces progressive and specific olfactory deficits. Thus, we demonstrate that propagating α-syn pathology triggered in the OB is functionally detrimental. Collectively, we have created a mouse model of prodromal PD.

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Disease Progression; Female; Lewy Bodies; Mice; Mice, Inbred C57BL; Neural Pathways; Olfactory Bulb; Olfactory Tubercle; Parkinson Disease; Protein Aggregation, Pathological

The Parkinson's disease-associated protein α-synuclein exhibits significant conformational heterogeneity. Bacterially expressed α-synuclein is known to bind to copper, resulting in the formation of aggregation-prone compact conformations. However, in vivo, α-synuclein undergoes acetylation at its N-terminus. Here the effect of this modification and the pathological H50Q mutation on copper binding and subsequent conformational transitions were investigated by electrospray ionization-ion mobility spectrometry-mass spectrometry. We demonstrate that acetylation perturbs the ability of α-synuclein to bind copper and that the H50Q missense mutation in the presence of N-terminal acetylation prevents copper binding. These modifications and mutations prevent the formation of the most compact conformations and inhibit copper-induced aggregation.

Topics: Acetylation; alpha-Synuclein; Copper; Humans; Mutation, Missense; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization

Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic d,l-α-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated α-synuclein (α-syn) aggregation to toxic oligomers by an "off-pathway" mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-β component region of α-syn, which are responsible for α-syn's membrane intercalation and self-assembly, thus changing the overall conformation of α-syn. CP-2 also remodels α-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α-syn in neuronal cells overexpressing α-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

Topics: alpha-Synuclein; Amyloid; Animals; Humans; Neurons; Parkinson Disease; PC12 Cells; Peptides, Cyclic; Protein Aggregates; Protein Aggregation, Pathological; Rats

Parkinson's disease is one of the most common neurodegenerative disorders of the elderly and ageing hence described to be a major risk factor. Telomere shortening as a result of the inability to fully replicate the ends of linear chromosomes is one of the hallmarks of ageing. The role of telomere dysfunction in neurological diseases and the ageing brain is not clarified and there is an ongoing discussion whether telomere shortening is linked to Parkinson's disease. Here we studied a mouse model of Parkinson's disease (Thy-1 [A30P] α-synuclein transgenic mouse model) in the background of telomere shortening (Terc knockout mouse model). α-synuclein transgenic mice with short telomeres (αSYN(tg/tg) G3Terc(-/-)) developed an accelerated disease with significantly decreased survival. This accelerated phenotype of mice with short telomeres was characterized by a declined motor performance and an increased formation of α-synuclein aggregates. Immunohistochemical analysis and mRNA expression studies revealed that the disease end-stage brain stem microglia showed an impaired response in αSYN(tg/tg) G3Terc(-/-) microglia animals. These results provide the first experimental data that telomere shortening accelerates α-synuclein pathology that is linked to limited microglia function in the brainstem.

Topics: alpha-Synuclein; Animals; Brain Stem; Disease Progression; Humans; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Motor Activity; Parkinsonian Disorders; Phenotype; Postural Balance; Protein Aggregation, Pathological; RNA, Messenger; Telomere Shortening; Time Factors

Topics: alpha-Synuclein; Caspase 1; Humans; Nerve Degeneration; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Proteolysis

Parkinson's disease is a highly debilitating neurodegenerative condition whose pathological hallmark is the presence in nerve cells of proteinacious deposits, known as Lewy bodies, composed primarily of amyloid fibrils of α-synuclein. Several missense mutations in the gene encoding α-synuclein have been associated with familial variants of Parkinson's disease and have been shown to affect the kinetics of the aggregation of the protein. Using a combination of experimental and theoretical approaches, we present a systematic in vitro study of the influence of disease-associated single-point mutations on the individual processes involved in α-synuclein aggregation into amyloid fibrils. We find that lipid-induced fibril production and surface catalyzed fibril amplification are the processes most strongly affected by these mutations and show that familial mutations can induce dramatic changes in the crucial processes thought to be associated with the initiation and spreading of the aggregation of α-synuclein.

Topics: alpha-Synuclein; Amyloid; Humans; Kinetics; Lipids; Mutation; Nerve Tissue Proteins; Parkinson Disease; Protein Aggregation, Pathological

α-Synuclein is a presynaptic protein associated to Parkinson's disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson's disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

Topics: alpha-Synuclein; Humans; Microtubules; Parkinson Disease; Protein Aggregation, Pathological; Protein Binding; Protein Folding; Protein Multimerization; Tubulin

Amyloid beta (Aβ) aggregation is generally associated with Alzheimer's onset. Here, we demonstrate that incubation of dopaminergic SH-SY5Y cells with an Aβ peptide fragment (an 11-mer composed of residues 25-35; Aβ (25-35)) results in elevated intracellular nitrosative stress and induces chemical mutation of protein disulfide isomerase (PDI), an endoplasmic reticulum-resident oxidoreductase chaperone. Furthermore, Aβ (25-35) provokes aggregation of both the minor and major biomarkers of Parkinson's disease, namely, synphilin-1 and α-synuclein, respectively. Importantly, fluorescence studies demonstrate that Aβ (25-35) triggers colocalization of these Parkinsonian biomarkers to form Lewy-body-like aggregates, a key and irreversible milestone in the neurometabolic cascade leading to Parkinson's disease. In addition, fluorescence assays also reveal direct, aggregation-seeding interactions between Aβ (25-35), PDI and α-synuclein, suggesting neuronal pathogenesis occurs via prion-type cross-transfectivity. These data indicate that the introduction of an Alzheimer's-associated biomarker in dopaminergic cells is proliferative, with the percolative effect exercised via dual, independent, Parkinson-pathogenic pathways, one stress-derived and the other prion-like. The results define a novel molecular roadmap for Parkinsonian transfectivity via an Alzheimeric burden and reveal the involvement of PDI in amyloid beta induced Parkinson's.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Apoptosis; Biomarkers; Carrier Proteins; Cell Line, Tumor; Cytosol; Dopaminergic Neurons; Dynamic Light Scattering; Flow Cytometry; Green Fluorescent Proteins; Immunohistochemistry; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Lewy Bodies; Mice; Microscopy, Confocal; Nerve Tissue Proteins; Parkinsonian Disorders; Peptide Fragments; Protein Aggregation, Pathological; Reactive Oxygen Species; Transfection

Parkinson's disease (PD) is the most widespread form of dementia where there is an age related degeneration of dopaminergic neurons in the substantia nigra region of the brain. Accumulation of α-synuclein (αS) protein aggregate, mitochondrial dysfunction, oxidative stress, and neuronal cell death are the pathological hallmarks of PD. In this context, amalgamation of curcumin and piperine having profound cognitive properties, and antioxidant activity seems beneficial. However, the blood-brain barrier (BBB) is the major impediment for delivery of neurotherapeutics to the brain. The present study involves formulation of curcumin and piperine coloaded glyceryl monooleate (GMO) nanoparticles coated with various surfactants with a view to enhance the bioavailability of curcumin and penetration of both drugs to the brain tissue crossing the BBB and to enhance the anti-parkinsonism effect of both drugs in a single platform. In vitro results demonstrated augmented inhibition of αS protein into oligomers and fibrils, reduced rotenone induced toxicity, oxidative stress, and apoptosis, and activation of autophagic pathway by dual drug loaded NPs compared to native counterpart. Further, in vivo studies revealed that our formulated dual drug loaded NPs were able to cross BBB, rescued the rotenone induced motor coordination impairment, and restrained dopaminergic neuronal degeneration in a PD mouse model.

Topics: Alkaloids; alpha-Synuclein; Animals; Antiparkinson Agents; Benzodioxoles; Blood-Brain Barrier; Capillary Permeability; Curcumin; Drug Delivery Systems; Drug Therapy, Combination; Liposomes; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Nanoparticles; Neuroprotective Agents; Parkinsonian Disorders; PC12 Cells; Piperidines; Polyunsaturated Alkamides; Protein Aggregation, Pathological; Random Allocation; Rats; Rotenone; Surface-Active Agents

Cell physiology is impaired before protein aggregation and this may be more relevant than inclusions themselves for neurodegeneration. The present study aimed to characterize an animal model to enable the analysis of the cell biology before and after protein aggregation. Ten-month-old Lewis rats were exposed either to 1 or 2 mg/kg/day of rotenone, delivered subcutaneously through mini-pumps, for one month. Hyperphosphorylated TAU, alpha-synuclein, amyloid-beta peptide and protein carbonylation (indicative of oxidative stress) were evaluated in the hippocampus, substantia nigra and locus coeruleus through immunohistochemistry or western blot. It was found that 2 mg/kg/day rotenone increased amyloid-beta peptide, hyperphosphorylation of TAU and alpha-synuclein. Rotenone at 1mg/kg/day did not alter protein levels. Protein carbonylation remained unchanged. This study demonstrated that aged Lewis rats exposed to a low dose of rotenone is a useful model to study cellular processes before protein aggregation, while the higher dose makes a good model to study the effects of protein inclusions.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Blotting, Western; Central Nervous System; Disease Models, Animal; Hippocampus; Immunohistochemistry; Locus Coeruleus; Male; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Protein Carbonylation; Rats, Inbred Lew; Reproducibility of Results; Rotenone; Substantia Nigra

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

Topics: alpha-Synuclein; Amino Acid Substitution; Animals; Cells, Cultured; Copper; Environment; Genetic Predisposition to Disease; Histidine; Humans; Inclusion Bodies; Kinetics; Mutation; Neurons; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation, alpha-Helical; Rats

Misfolded alpha-synuclein (AS) and other neurodegenerative disorder proteins display prion-like transmission of protein aggregation. Factors responsible for the initiation of AS aggregation are unknown. To evaluate the role of amyloid proteins made by the microbiota we exposed aged rats and transgenic C. elegans to E. coli producing the extracellular bacterial amyloid protein curli. Rats exposed to curli-producing bacteria displayed increased neuronal AS deposition in both gut and brain and enhanced microgliosis and astrogliosis compared to rats exposed to either mutant bacteria unable to synthesize curli, or to vehicle alone. Animals exposed to curli producing bacteria also had more expression of TLR2, IL-6 and TNF in the brain than the other two groups. There were no differences among the rat groups in survival, body weight, inflammation in the mouth, retina, kidneys or gut epithelia, and circulating cytokine levels. AS-expressing C. elegans fed on curli-producing bacteria also had enhanced AS aggregation. These results suggest that bacterial amyloid functions as a trigger to initiate AS aggregation through cross-seeding and also primes responses of the innate immune system.

Topics: alpha-Synuclein; Amyloid; Animals; Bacterial Proteins; Caenorhabditis elegans; Escherichia coli; Escherichia coli Proteins; Protein Aggregation, Pathological; Rats; Rats, Inbred F344

In type-2 diabetes (T2D) and Parkinson's disease (PD), polypeptide assembly into amyloid fibers plays central roles: in PD, α-synuclein (aS) forms amyloids and in T2D, amylin [islet amyloid polypeptide (IAPP)] forms amyloids. Using a combination of biophysical methods in vitro we have investigated whether aS, IAPP, and unprocessed IAPP, pro-IAPP, polypeptides can cross-react. Whereas IAPP forms amyloids within minutes, aS takes many hours to assemble into amyloids and pro-IAPP aggregates even slower under the same conditions. We discovered that preformed amyloids of pro-IAPP inhibit, whereas IAPP amyloids promote, aS amyloid formation. Amyloids of aS promote pro-IAPP amyloid formation, whereas they inhibit IAPP amyloid formation. In contrast, mixing of IAPP and aS monomers results in coaggregation that is faster than either protein alone; moreover, pro-IAPP can incorporate aS monomers into its amyloid fibers. From this intricate network of cross-reactivity, it is clear that the presence of IAPP can accelerate aS amyloid formation. This observation may explain why T2D patients are susceptible to developing PD.

Topics: alpha-Synuclein; Amyloidogenic Proteins; Amyloidosis; Animals; Diabetes Mellitus, Type 2; Humans; Islet Amyloid Polypeptide; Microscopy, Atomic Force; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding

α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson's disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

Topics: alpha-Synuclein; Amino Acid Sequence; beta-Synuclein; Binding, Competitive; Hydrogen-Ion Concentration; Lipids; Phosphatidylserines; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Sequence Alignment; Surface Properties

Synucleinopathies are a group of neurodegenerative diseases associated with alpha-synuclein (α-Syn) aggregation. Recently, increasing evidence has demonstrated the existence of different structural characteristics or 'strains' of α-Syn, supporting the concept that synucleinopathies share several common features with prion diseases and possibly explaining how a single protein results in different clinical phenotypes within synucleinopathies. In earlier studies, the different strains were generated through the regulation of solution conditions, temperature, or repetitive seeded fibrillization in vitro. Here, we synthesize homogeneous α-Syn phosphorylated at serine 129 (pS129 α-Syn), which is highly associated with the pathological changes, and demonstrate that phosphorylation at Ser129 induces α-Syn to form a distinct strain with different structures, propagation properties, and higher cytotoxicity compared with the wild-type α-Syn. The results are the first demonstration that post-translational modification of α-Syn can induce different strain formation, offering a new mechanism for strain formation.

Topics: alpha-Synuclein; Cell Line, Tumor; Humans; Phosphorylation; Protein Aggregation, Pathological; Protein Processing, Post-Translational

Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

Topics: alpha-Synuclein; Fluorescence; Models, Biological; Molecular Weight; Mutant Proteins; Nanoparticles; Protein Aggregates; Protein Aggregation, Pathological; Protein Biosynthesis; Protein Multimerization; Single Molecule Imaging; Temperature; Ultracentrifugation

Alpha-synuclein (α-Syn) is a small presynaptic protein of 140 amino acids. Its pathologic intracellular aggregation within the central nervous system yields protein fibrillar inclusions named Lewy bodies that are the hallmarks of Parkinson's disease (PD). In solution, pure α-Syn adopts an intrinsically disordered structure and assembles into fibrils that exhibit considerable morphological heterogeneity depending on their assembly conditions. We recently established tightly controlled experimental conditions allowing the assembly of α-Syn into highly homogeneous and pure polymorphs. The latter exhibited differences in their shape, their structure but also in their functional properties. We have conducted an AFM study at high resolution and performed a statistical analysis of fibrillar α-Syn shape and thermal fluctuations to calculate the persistence length to further assess the nanomechanical properties of α-Syn polymorphs. Herein, we demonstrated quantitatively that distinct polymorphs made of the same protein (wild-type α-Syn) show significant differences in their morphology (height, width and periodicity) and physical properties (persistence length, bending rigidity and axial Young's modulus).

Topics: alpha-Synuclein; Elastic Modulus; Parkinson Disease; Protein Aggregation, Pathological; Protein Structure, Quaternary

α-synuclein (aSyn) is associated with both sporadic and familial forms of Parkinson's disease (PD), the second most common neurodegenerative disorder after Alzheimer's disease. In particular, multiplications and point mutations in the gene encoding for aSyn cause familial forms of PD. Moreover, the accumulation of aSyn in Lewy Bodies and Lewy neurites in disorders such as PD, dementia with Lewy bodies, or multiple system atrophy, suggests aSyn misfolding and aggregation plays an important role in these disorders, collectively known as synucleinopathies. The exact function of aSyn remains unclear, but it is known to be associated with vesicles and membranes, and to have an impact on important cellular functions such as intracellular trafficking and protein degradation systems, leading to cellular pathologies that can be readily studied in cell-based models. Thus, understanding the molecular effects of aSyn point mutations may provide important insight into the molecular mechanisms underlying disease onset.We investigated the effect of the recently identified A53E aSyn mutation. Combining in vitro studies with studies in cell models, we found that this mutation reduces aSyn aggregation and increases proteasome activity, altering normal proteostasis.We observed that, in our experimental paradigms, the A53E mutation affects specific steps of the aggregation process of aSyn and different cellular processes, providing novel ideas about the molecular mechanisms involved in synucleinopathies.

Topics: alpha-Synuclein; Cell Line, Tumor; Golgi Apparatus; HEK293 Cells; Humans; Inclusion Bodies; Point Mutation; Protein Aggregation, Pathological; Saccharomyces cerevisiae

The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils.

Topics: alpha-Synuclein; Amyloid; Cathepsin B; Cathepsin D; Humans; Lysosomes; Protein Aggregation, Pathological

Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, β-amyloid and α-synuclein. The constructed systems provided direct visualization of protein-protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development.

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Drug Discovery; Fluorescence; Humans; Islet Amyloid Polypeptide; Models, Molecular; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization

Aggregation of α-Synuclein (α-Syn) in Lewy bodies is largely responsible for the demise and death of dopamine neurons. Oxidative stress associated with the aggregation-induced oxidative damage is considered as a possible origin of the toxicity. However, the cellular mechanism of H2O2 in the aggregation of α-Syn remains a debate, i.e., whether the aggregation is caused by endogenously secreted or exogenous H2O2 from upstream. Here, we report on the development of an ultrasensitive plasmonic assay with a designed nanoplasmonic probe to unravel the role of H2O2 in the aggregation of α-Syn. The nanoplasmonic probe is composed of a Au nanoparticle with surface-attached double-stranded DNA and horseradish peroxidase (HRP). In the presence of H2O2, HRP initiates the polymerization of aniline, which in turn results in the in situ formation of a layer of conducting polymer on the nanoplasmonic probe. By monitoring the associated plasmonic response, we can sensitively detect H2O2 with a remarkably low detection limit of 8 nM. With this ultrasensitive plasmonic assay, we find that exogenous H2O2 plays a dominant role for the aggregation of α-Syn in vitro, whereas the contribution from endogenously secreted H2O2 is negligible.

Topics: alpha-Synuclein; Aniline Compounds; DNA; Gold; HEK293 Cells; Horseradish Peroxidase; Humans; Hydrogen Peroxide; Metal Nanoparticles; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Polymerization; Protein Aggregation, Pathological; Surface Plasmon Resonance

Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes β-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When α-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of α-synuclein aggregates.

Topics: alpha-Synuclein; Cell Line; Enzyme Activation; Gene Knockout Techniques; Gene Order; Genetic Loci; Glucosylceramidase; Humans; Lysosomes; Mutation; Protein Aggregation, Pathological; Protein Binding; Zinc Fingers

α-Synuclein physiologically chaperones SNARE-complex assembly at the synapse but pathologically misfolds into neurotoxic aggregates that are characteristic for neurodegenerative disorders, such as Parkinson's disease, and that may spread from one neuron to the next throughout the brain during Parkinson's disease pathogenesis. In normal nerve terminals, α-synuclein is present in an equilibrium between a cytosolic form that is natively unfolded and monomeric and a membrane-bound form that is composed of an α-helical multimeric species that chaperones SNARE-complex assembly. Although the neurotoxicity of α-synuclein is well established, the relationship between the native conformations of α-synuclein and its pathological aggregation remain incompletely understood; most importantly, it is unclear whether α-synuclein aggregation originates from its monomeric cytosolic or oligomeric membrane-bound form. Here, we address this question by introducing into α-synuclein point mutations that block membrane binding and by then assessing the effect of blocking membrane binding on α-synuclein aggregation and neurotoxicity. We show that membrane binding inhibits α-synuclein aggregation; conversely, blocking membrane binding enhances α-synuclein aggregation. Stereotactic viral expression of wild-type and mutant α-synuclein in the substantia nigra of mice demonstrated that blocking α-synuclein membrane binding significantly enhanced its neurotoxicity in vivo. Our data delineate a folding pathway for α-synuclein that ranges from a physiological multimeric, α-helical, and membrane-bound species that acts as a SNARE-complex chaperone over a monomeric, natively unfolded form to an amyloid-like aggregate that is neurotoxic in vivo.

Topics: alpha-Synuclein; Animals; HEK293 Cells; Humans; Liposomes; Male; Mice; Neurons; Neurotoxicity Syndromes; Point Mutation; Postural Balance; Protein Aggregation, Pathological; Protein Binding; Substantia Nigra

Misfolding and aggregation of α-synuclein (α-syn) into Lewy bodies is associated with a range of neurological disorders, including Parkinson's disease (PD). The cell-to-cell transmission of α-syn pathology has been linked to soluble amyloid oligomer populations that precede Lewy body formation. Oligomers produced in vitro under certain conditions have been demonstrated to induce intracellular aggregation in cell culture models. In the present study, we characterize, by ESI-ion mobility spectrometry (IMS)-MS, a specific population of α-syn oligomers. These MS-compatible oligomers were compared with oligomers with known seeding and pore-forming capabilities and were shown to have the ability to induce intracellular aggregation. Each oligomer type was shown to have distinct epitope profiles that correlated with their toxic gain-of-function. Structurally, the MS compatible oligomers populated a range of species from dimers through to hexamers. Lower-order oligomers were structurally diverse and consistent with unstructured assemblies. Higher-order oligomers were shown to be compact with ring-like structures. The observation of this compact state may explain how this natively disordered protein is able to transfer pathology from cell to cell and avoid degradation by cellular proteases.

Topics: alpha-Synuclein; Calcium Signaling; Cell Line, Tumor; Cell Survival; Computational Biology; Expert Systems; Humans; Intrinsically Disordered Proteins; Models, Molecular; Molecular Weight; Neurons; Protein Aggregation, Pathological; Protein Conformation; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Aggregation and fibril formation of human alpha-Synuclein (αS) are neuropathological hallmarks of Parkinson's disease and other synucleinopathies. The molecular mechanisms of αS aggregation and fibrillogenesis are largely unknown. Several studies suggested a sequence of events from αS dimerization via oligomerization and pre-fibrillar aggregation to αS fibril formation. In contrast to αS, little evidence suggests that γS can form protein aggregates in the brain, and for βS its neurotoxic properties and aggregation propensities are controversially discussed. These apparent differences in aggregation behavior prompted us to investigate the first step in Synuclein aggregation, i.e. the formation of dimers or oligomers, by Bimolecular Fluorescence Complementation in cells. This assay showed some Synuclein-specific limitations, questioning its performance on a single cell level. Nevertheless, we unequivocally demonstrate that all Synucleins can interact with each other in a very similar way. Given the divergent aggregation properties of the three Synucleins this suggests that formation of dimers is not predictive for the aggregation of αS, βS or γS in the aged or diseased brain.

Topics: alpha-Synuclein; beta-Synuclein; Cells, Cultured; gamma-Synuclein; HEK293 Cells; HeLa Cells; Humans; Microscopy, Fluorescence; Neoplasm Proteins; Prognosis; Protein Aggregates; Protein Aggregation, Pathological; Protein Isoforms; Protein Multimerization; Synucleins

Accumulating evidence suggests that deposition of neurotoxic α-synuclein aggregates in the brain during the development of neurodegenerative diseases like Parkinson's disease can be curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates. Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol is limited owing to its instability. As chemical modifications in curcumin confiscate this limitation, such efforts are intensively performed to discover molecules with similar but enhanced stability and superior properties. This study focuses on the inhibitory effect of two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their derivatives against α-synuclein aggregation, fibrillization and toxicity. Employing biochemical, biophysical and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting fibrillization and disrupting preformed fibrils but also preventing formation of A11 conformation in the protein that imparts toxic effects. Compounds 3 and 15 also decreased neurotoxicity associated with fast aggregating A53T mutant form of α-synuclein. These two analogues of curcumin described here may therefore be useful therapeutic inhibitors for the treatment of α-synuclein amyloidosis and toxicity in Parkinson's disease and other synucleinopathies.

Topics: alpha-Synuclein; Curcumin; Dose-Response Relationship, Drug; Humans; Kinetics; Models, Biological; Mutation; Neurodegenerative Diseases; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization; Structure-Activity Relationship

Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloidogenic Proteins; Humans; Ligands; Parkinson Disease; Protein Aggregation, Pathological; Protein Structure, Secondary; Pyridones

Loss-of-function mutations in GBA1, which cause the autosomal recessive lysosomal storage disease, Gaucher disease (GD), are also a key genetic risk factor for the α-synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase and reductions in this enzyme result in the accumulation of the glycolipid substrates glucosylceramide and glucosylsphingosine. Deficits in autophagy and lysosomal degradation pathways likely contribute to the pathological accumulation of α-synuclein in PD. In this report we used conduritol-β-epoxide (CBE), a potent selective irreversible competitive inhibitor of glucocerebrosidase, to model reduced glucocerebrosidase activity in vivo, and tested whether sustained glucocerebrosidase inhibition in mice could induce neuropathological abnormalities including α-synucleinopathy, and neurodegeneration.. Our data demonstrate that daily systemic CBE treatment over 28 days caused accumulation of insoluble α-synuclein aggregates in the substantia nigra, and altered levels of proteins involved in the autophagy lysosomal system. These neuropathological changes were paralleled by widespread neuroinflammation, upregulation of complement C1q, abnormalities in synaptic, axonal transport and cytoskeletal proteins, and neurodegeneration.. A reduction in brain GCase activity has been linked to sporadic PD and normal aging, and may contribute to the susceptibility of vulnerable neurons to degeneration. This report demonstrates that systemic reduction of GCase activity using chemical inhibition, leads to neuropathological changes in the brain reminiscent of α-synucleinopathy.. These data reveal a link between reduced glucocerebrosidase and the development of α-synucleinopathy and pathophysiological abnormalities in mice, and support the development of GCase therapeutics to reduce α-synucleinopathy in PD and related disorders.

Topics: alpha-Synuclein; Animals; Autophagy; Axonal Transport; Cerebral Cortex; Complement Activation; Complement C1q; Glucosylceramidase; Inositol; Male; Mice; Microglia; Parkinson Disease, Secondary; Protein Aggregation, Pathological; Proteins; Synaptic Transmission

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.

Topics: alpha-Synuclein; Amyloid; Heme; Humans; Parkinson Disease; Protein Aggregation, Pathological; Protein Multimerization

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson's disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced.

Topics: alpha-Synuclein; Animals; Electron Transport Complex I; Humans; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondrial Diseases; Mutation; Neurons; Oxidative Stress; Parkinson Disease; Protein Aggregation, Pathological; Reactive Oxygen Species

Vector based over-expression of α-synuclein is a newly developed method to establish animal Parkinson's disease (PD) model. In this paper, we inject the rat brain with recombinant adeno-associated virus (rAAV) to express α-synuclein wild-type and A53T mutation, and compared the degeneration of dopaminergic neurons between them.. The rAAV vectors were injected into the substantia nigra pars compacta (SNpc) of rat brain. In different time point, immunohistochemistry was used to detect the expression of α-synuclein. The expression level was lower in the 3rd and 6th week and increased from the 9th week. α-synuclein spread around the neurons in SNpc in the 12th week. The loss of dopaminergic neurons was increasing along the expression of α-synuclein, and damage extent was more serious in the A53T group than the WT group. In the A53T group, there were more insoluble inclusions can be detected, and the phosphorylation of α-synuclein was also higher.. The result of comparison between the two types of α-synuclein showed that A53T mutated α-synuclein was more effective to establish PD model, and the model based A53T mutated α-synuclein was a suitable model to early-onset PD.

Topics: Age Factors; alpha-Synuclein; Amino Acid Substitution; Animals; Apomorphine; Brain; Disease Models, Animal; Dopaminergic Neurons; Immunohistochemistry; Male; Mutant Proteins; Mutation, Missense; Parkinsonian Disorders; Phosphorylation; Protein Aggregation, Pathological; Rats; Rats, Transgenic; Rats, Wistar; Recombinant Proteins; Torsion Abnormality

Molecular chaperones facilitate the folding and assembly of proteins and inhibit their aberrant aggregation. They thus offer several opportunities for biomedical and biotechnological applications, as for example they can often prevent protein aggregation more effectively than other therapeutic molecules, including small molecules and antibodies. Here we present a method of designing molecular chaperones with enhanced activity against specific amyloidogenic substrates while leaving unaltered their functions toward other substrates. The method consists of grafting onto a molecular chaperone a peptide designed to bind specifically an epitope in the target substrate. We illustrate this strategy by describing Hsp70 variants with increased affinities for α-synuclein and Aβ42 but otherwise unaltered affinities for other substrates. These designed variants inhibit protein aggregation and disaggregate preformed fibrils significantly more effectively than wild-type Hsp70 indicating that the strategy presented here provides a possible route for tailoring rationally molecular chaperones for specific purposes.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Drug Design; Genetic Variation; HSP70 Heat-Shock Proteins; Humans; Models, Molecular; Molecular Chaperones; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation; Protein Engineering; Protein Interaction Domains and Motifs; Recombinant Proteins

Parkinson's disease (PD) is an age-related movement disorder characterized by a progressive degeneration of dopaminergic neurons in the midbrain. Although the presence of amyloid deposits of α-synuclein (α-syn) is the main pathological feature, PD brains also present a severe permanent inflammation, which largely contributes to neuropathology. Although α-syn has recently been implicated in this process, the molecular mechanisms underlying neuroinflammation remain unknown. In the present study, we investigated the ability of different α-syn aggregates to trigger inflammatory responses. We showed that α-syn induced inflammation through activation of Toll-like receptor 2 (TLR2) and the nucleotide oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome only when folded as amyloid fibrils. Oligomeric species, thought to be the primary species responsible for the disease, were surprisingly unable to trigger the same cascades. As neuroinflammation is a key player in PD pathology, these results put fibrils back to the fore and rekindles discussions about the primary toxic species contributing to the disease. Our data also suggest that the inflammatory properties of α-syn fibrils are linked to their intrinsic structure, most probably to their cross-β structure. Since fibrils of other amyloids induce similar immunological responses, we propose that the canonical fibril-specific cross-β structure represents a new generic motif recognized by the innate immune system.

Topics: alpha-Synuclein; Amyloid; Carrier Proteins; Cell Line; Humans; Immunity, Innate; Inflammasomes; Inflammation; Interleukin-1beta; NLR Family, Pyrin Domain-Containing 3 Protein; Parkinson Disease; Protein Aggregation, Pathological; Protein Structure, Secondary; Signal Transduction; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Bacterial Proteins; Blotting, Western; Caenorhabditis elegans; Circular Dichroism; Escherichia coli; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; HSP70 Heat-Shock Proteins; Humans; Luminescent Proteins; Microscopy, Confocal; Microscopy, Electron, Transmission; Parkinsonian Disorders; Protein Aggregates; Protein Aggregation, Pathological; RNA Interference

The accumulation of mis-folded and/or abnormally modified proteins is a major characteristic of many neurodegenerative diseases. In Lewy body disease (LBD), which includes Parkinson's disease and dementia with Lewy bodies, insoluble α-synuclein is widely deposited in the presynaptic terminals as well as in the neuronal cytoplasm in distinct brain regions. It is well known that the autophagy-lysosome system serves as an efficient degradation pathway for abnormal molecules within cells. To test the possibility that activated autophagy can degrade abnormal molecules, we investigated the effect of trehalose on abnormal aggregation of α-synuclein in a model of LBD. Trehalose is a natural disaccharide composed of two glucose units and functions as an autophagy inducer. Consistent with previous studies, trehalose increased level of the autophagosomal protein LC3, especially a lipidated form LC3-II in cultured cells and mice brain. Also, trehalose increased levels of several chaperon molecules, such as HSP90 and SigmaR1, in the brains of LBD model mice. Further studies revealed that level of detergent-insoluble α-synuclein was suppressed in mice following oral administration of trehalose, despite an apparent alteration was not observed regarding abnormal aggregation of α-synuclein. These results suggest that the oral intake of trehalose modulates propensity of molecules prior to aggregation formation.

Topics: Administration, Oral; alpha-Synuclein; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain; Disease Models, Animal; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Lewy Body Disease; Maltose; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Molecular Chaperones; Protein Aggregation, Pathological; Receptors, sigma; Sigma-1 Receptor; Solubility; Trehalose

Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.

Topics: alpha-Synuclein; Amyloid; Electron Microscope Tomography; HSC70 Heat-Shock Proteins; HSP110 Heat-Shock Proteins; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; In Vitro Techniques; Kinetics; Molecular Chaperones; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization; Solubility

Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recent multicenter genetic studies have revealed that mutations in the glucocerebrosidase 1 (GBA1) gene, which are responsible for Gaucher's disease, are strong risk factors for PD and DLB. However, the mechanistic link between the functional loss of glucocerebrosidase (GCase) and the toxicity of αSyn in vivo is not fully understood. In this study, we employed Drosophila models to examine the effect of GCase deficiency on the neurotoxicity of αSyn and its molecular mechanism. Behavioral and histological analyses showed that knockdown of the Drosophila homolog of GBA1 (dGBA1) exacerbates the locomotor dysfunction, loss of dopaminergic neurons and retinal degeneration of αSyn-expressing flies. This phenotypic aggravation was associated with the accumulation of proteinase K (PK)-resistant αSyn, rather than with changes in the total amount of αSyn, raising the possibility that glucosylceramide (GlcCer), a substrate of GCase, accelerates the misfolding of αSyn. Indeed, in vitro experiments revealed that GlcCer directly promotes the conversion of recombinant αSyn into the PK-resistant form, representing a toxic conformational change. Similar to dGBA1 knockdown, knockdown of the Drosophila homolog of β-galactosidase (β-Gal) also aggravated locomotor dysfunction of the αSyn flies, and its substrate GM1 ganglioside accelerated the formation of PK-resistant αSyn. Our findings suggest that the functional loss of GCase or β-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration.

Topics: alpha-Synuclein; Animals; beta-Galactosidase; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Endopeptidase K; Gene Knockdown Techniques; Glucosylceramidase; Glucosylceramides; Humans; Male; Parkinsonian Disorders; Protein Aggregation, Pathological; Protein Folding

Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. An ability to specifically and selectively control protein function via conformational modulation is an important goal for development of novel therapeutics and studies of protein mechanism in biological networks and disease. Here we applied a second-harmonic generation-based technique for studying protein conformation in solution and in real time to the intrinsically disordered, Parkinson disease related protein α-synuclein. From a fragment library, we identified small molecule modulators that bind to monomeric α-synuclein in vitro and significantly reduce α-synuclein aggregation in a neuronal cell culture model. Our results indicate that the conformation of α-synuclein is linked to the aggregation of protein in cells. They also provide support for a therapeutic strategy of targeting specific conformations of the protein to suppress or control its aggregation.

Topics: alpha-Synuclein; Antiparkinson Agents; Cell Line, Tumor; Humans; Ligands; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Small Molecule Libraries

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson's disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.

Topics: alpha-Synuclein; Animals; Escherichia coli Proteins; Humans; Membrane Transport Proteins; Mice; Molecular Chaperones; Nuclear Magnetic Resonance, Biomolecular; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization; Protein Structure, Secondary; Recombinant Fusion Proteins

Pathology in Parkinson's disease is linked to self-association of α-Synuclein (αS) into pathogenic oligomeric species and highly ordered amyloid fibrils. Developing effective therapeutic strategies against this debilitating disease is critical and βS, a pre-synaptic protein that co-localizes with αS, can act as an inhibitor of αS assembly. Despite the potential importance of βS as an inhibitor of αS, the nature, location and specificity of the molecular interactions between these two proteins is unknown. Here we use NMR paramagnetic relaxation enhancement experiments, to demonstrate that βS interacts directly with αS in a transient dimer complex with high specificity and weak affinity. Inhibition of αS by βS arises from transient αS/βS heterodimer species that exist primarily in head- to- tail configurations while αS aggregation arises from a more heterogeneous and weaker range of transient interactions that include both head-to-head and head-to-tail configurations. Our results highlight that intrinsically disordered proteins can interact directly with one another at low affinity and that the transient interactions that drive inhibition versus aggregation are distinct by virtue of their plasticity and specificity.

Topics: alpha-Synuclein; Amino Acid Sequence; beta-Synuclein; Binding Sites; Models, Biological; Molecular Sequence Data; Neurodegenerative Diseases; Nuclear Magnetic Resonance, Biomolecular; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Multimerization; Protein Transport; Sequence Alignment

Topics: alpha-Synuclein; Biomedical Research; Humans; Nerve Degeneration; Prions; Protein Aggregation, Pathological

Impaired olfaction is an early symptom in Parkinson disease (PD), although the exact cause is as yet unknown. Here, we investigated the link between PD-related mutant α-Synuclein (α-SYN) pathology and olfactory deficit, by examining the integration of adult-born neurons in the olfactory bulb (OB) of A30P α-SYN overexpressing mice. To this end, we chose to label one well-known vulnerable subpopulation of adult-born cells, the dopaminergic neurons. Using in vivo two-photon imaging, we followed the dynamic process of neuronal turnover in transgenic A30P α-SYN and wild-type mice over a period of 2.5 months. Our results reveal no difference in the number of cells that reach, and possibly integrate into, the glomerular layer in the OB. However, in mutant transgenic mice these new neurons have a significantly shortened survival, resulting in an overall reduction in the addition of neurons to the glomerular layer over time. We therefore propose unstable integration and impaired homeostasis of functional new neurons as a likely contributor to odour discrimination deficits in mutant α-SYN mice.

Topics: alpha-Synuclein; Animals; Dopamine; Dopaminergic Neurons; Lentivirus; Mice; Mice, Inbred C57BL; Mice, Transgenic; Olfaction Disorders; Olfactory Bulb; Parkinson Disease; Protein Aggregation, Pathological; Radiography; Smell

The anti-epileptic agent zonisamide (ZNS) has been shown to exert protective effects in neurotoxin-based mouse models of Parkinson disease. However, it is unknown whether ZNS can attenuate toxicity of familial Parkinson's disease-causing gene products. In this study, we investigated the effects of ZNS on neurodegeneration induced by expression of A53T α-synuclein in the rat substantia nigra using a recombinant adeno-associated virus vector. Expression of A53T α-synuclein yielded severe loss of nigral dopamine neurons and striatal dopamine nerve terminals from 2 weeks to 4 weeks after viral injection. Oral administration of ZNS (40 mg/kg/day) significantly delayed the pace of degeneration at 4 weeks after viral injection as compared with the vehicle group. This effect lasted until 8 weeks after viral injection, the final point of observation. ZNS treatment had no impact on the survival of nigrostriatal dopamine neurons in rats expressing green fluorescent protein. Quantification of striatal Ser129-phosphorylated α-synuclein-positive aggregates showed that these aggregates rapidly formed from 2 weeks to 4 weeks after viral injection. This increase was closely correlated with loss of nigrostriatal dopamine neurons. However, ZNS treatment failed to alter the number of all striatal Ser129-phosphorylated α-synuclein-positive aggregates, including small dot-like and large round structures. The number of these aggregates was almost constant at 4 weeks and 8 weeks after viral injection, although ZNS persistently prevented loss of nigrostriatal dopamine neurons during this period. Also, ZNS treatment did not affect the number of striatal aggregates larger than 10 µm in diameter. These data show that ZNS attenuates α-synuclein-induced toxicity in a manner that is independent of the formation and maturation of α-synuclein aggregates in an in vivo model of familial Parkinson's disease, suggesting that ZNS may protect nigrostriatal dopamine neurons by modulating cellular damage or a cell death pathway commonly caused by neurotoxins and α-synuclein.

Topics: alpha-Synuclein; Animals; Cell Count; Dependovirus; Disease Models, Animal; Dopaminergic Neurons; Isoxazoles; Male; Mice; Neuroprotective Agents; Parkinson Disease; Protein Aggregation, Pathological; Rats; Substantia Nigra; Time Factors; Zonisamide

Gaucher disease, a prevalent lysosomal storage disease (LSD), is caused by insufficient activity of acid β-glucosidase (GCase) and the resultant glucosylceramide (GC)/glucosylsphingosine (GS) accumulation in visceral organs (Type 1) and the central nervous system (Types 2 and 3). Recent clinical and genetic studies implicate a pathogenic link between Gaucher and neurodegenerative diseases. The aggregation and inclusion bodies of α-synuclein with ubiquitin are present in the brains of Gaucher disease patients and mouse models. Indirect evidence of β-amyloid pathology promoting α-synuclein fibrillation supports these pathogenic proteins as a common feature in neurodegenerative diseases. Here, multiple proteins are implicated in the pathogenesis of chronic neuronopathic Gaucher disease (nGD). Immunohistochemical and biochemical analyses showed significant amounts of β-amyloid and amyloid precursor protein (APP) aggregates in the cortex, hippocampus, stratum and substantia nigra of the nGD mice. APP aggregates were in neuronal cells and colocalized with α-synuclein signals. A majority of APP co-localized with the mitochondrial markers TOM40 and Cox IV; a small portion co-localized with the autophagy proteins, P62/LC3, and the lysosomal marker, LAMP1. In cultured wild-type brain cortical neural cells, the GCase-irreversible inhibitor, conduritol B epoxide (CBE), reproduced the APP/α-synuclein aggregation and the accumulation of GC/GS. Ultrastructural studies showed numerous larger-sized and electron-dense mitochondria in nGD cerebral cortical neural cells. Significant reductions of mitochondrial adenosine triphosphate production and oxygen consumption (28-40%) were detected in nGD brains and in CBE-treated neural cells. These studies implicate defective GCase function and GC/GS accumulation as risk factors for mitochondrial dysfunction and the multi-proteinopathies (α-synuclein-, APP- and Aβ-aggregates) in nGD.

Topics: alpha-Synuclein; Amyloid beta-Protein Precursor; Animals; beta-Glucosidase; Cells, Cultured; Cerebral Cortex; Corpus Striatum; Disease Models, Animal; Enzyme Inhibitors; Gaucher Disease; Gene Expression Regulation; Hippocampus; Humans; Inositol; Lysosomal Membrane Proteins; Membrane Transport Proteins; Mice; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Neurons; Prostaglandin-Endoperoxide Synthases; Protein Aggregation, Pathological; Substantia Nigra

The liposoluble insecticide rotenone is commonly used as a mitochondrial complex I inhibitor to replicate Parkinson's disease (PD) pathological features. However, there was no assessment of the spatial learning and memory abilities in chronic rotenone-induced PD models. In the present study, by rotarod test and Thioflavine T staining, we first noted the impairment of motor coordination in rotenone-treated group for 3 months, as well as alpha-synuclein inclusions in the nigral dopaminergic neurons in C57BL/6 mice with intragastrical delivery of rotenone (5 mg/Kg) for 3 months rather than 1 month. We then evaluated spatial learning and memory abilities by Morris water maze task in this model. The results showed escape latency reduced in rotenone-intoxicated mice for 3 months, indicating an improvement of learning ability. However, it was delayed slightly but not significantly in rotenone-intoxicated mice for 1 month. Similarly, we demonstrated that spatial memory ability was enhanced in 3-month-treatment group, but impaired in 1-month-treatment group. There were no proliferating cell nuclear antigen and doublecortin positive cells in the hippocampus by double immunofluorescent staining, indicating the absence of hippocampal neurogenesis in rotenone-intoxicated mice. These results suggest that spatial learning and memory abilities are disturbed in chronic rotenone-intoxicated PD model.

Topics: alpha-Synuclein; Animal Experimentation; Animals; Insecticides; Memory; Mice; Neurogenesis; Pars Compacta; Protein Aggregation, Pathological; Psychomotor Performance; Rotenone; Spatial Learning

Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD.

Topics: alpha-Synuclein; Animals; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Gene Expression Regulation; Humans; Lewy Bodies; Mice; Mice, Transgenic; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Proteolysis; Signal Transduction; Synapses

Pathologic aggregation of α-synuclein is a central process in the pathogenesis of Parkinson's disease. The α-synuclein gene (SNCA) encodes at least 4 different α-synuclein isoforms through alternative splicing (SNCA140, SNCA126, SNCA112, SNCA98). Differential expression of α-synuclein isoforms has been shown in Lewy body diseases. In contrast to the canonical α-synuclein isoform of 140 amino acid residues (SNCA140), which has been investigated in detail, little is known about the properties of the 3 alternative isoforms. We have investigated the aggregation properties of all 4 isoforms in cultured cells and analyzed fibril-formation of 3 isoforms (SNCA140, SNCA126, and SNCA98) in vitro by electron microscopy. Each of the 3 alternative isoforms aggregates significantly less than the canonical isoform SNCA140. Electron microscopy showed that SNCA140 formed the well-known relatively straight fibrils while SNCA126 formed shorter fibrils, which were arranged in parallel fibril bundles and SNCA98 formed annular structures. Expression analysis of α-synuclein isoforms in different human brain regions demonstrated low expression levels of the alternative isoforms in comparison to the canonical SNCA140 isoform. These findings demonstrate that α-synuclein isoforms differ qualitatively and quantitatively in their aggregation properties. The biological consequences of these findings remain to be explored in vitro and in vivo.

Topics: alpha-Synuclein; Amino Acid Sequence; Brain; HEK293 Cells; Humans; Molecular Sequence Data; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Isoforms

Autophagy is a major degradation pathway for abnormal aggregated proteins and organelles that cause various neurodegenerative diseases. Current evidence suggests a central role for autophagy in pathogenesis of Parkinson's disease, and that dysfunction in the autophagic system may lead to α-synuclein accumulation. In the present study, we investigated whether mesenchymal stem cells (MSCs) would enhance autophagy and thus exert a neuroprotective effect through the modulation of α-synuclein in parkinsonian models. In MPP(+)-treated neuronal cells, coculture with MSCs increased cellular viability, attenuated expression of α-synuclein, and enhanced the number of LC3-II-positive autophagosomes compared with cells treated with MPP(+) only. In an MPTP-treated animal model of Parkinson's disease, MSC administration significantly increased final maturation of late autophagic vacuoles, fusion with lysosomes. Moreover, MSC administration significantly reduced the level of α-synuclein in dopaminergic neurons, which was elevated in MPTP-treated mice. These results suggest that MSC treatment significantly enhances autophagolysosome formation and may modulate α-synuclein expression in parkinsonian models, which may lead to increased neuronal survival in the presence of neurotoxins.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 1-Methyl-4-phenylpyridinium; alpha-Synuclein; Animals; Autophagy; Cell Survival; Cells, Cultured; Disease Models, Animal; Dopaminergic Neurons; Humans; Male; Mesenchymal Stem Cells; Mice, Inbred C57BL; Neurotoxins; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological

A novel mutation in the α-Synuclein (α-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-Syn(G51D) behaves similarly to α-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregation.

Topics: alpha-Synuclein; Brain; Buffers; Cell Differentiation; Cell Line; Cell Membrane; Cell Nucleus; Cells, Cultured; Humans; Inclusion Bodies; Mitochondria; Mutation; Neuroblastoma; Neurons; Nuclear Envelope; Parkinson Disease; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Structure, Secondary; Protein Transport; Saccharomyces cerevisiae; Sodium Dodecyl Sulfate; Subcellular Fractions; Unilamellar Liposomes

Abnormal aggregation of α-synuclein (α-syn) is central to the pathogenesis of Parkinson's disease (PD). Histone deacetylase 6 (HDAC6) was previously shown to control major cell response pathways to the cytotoxic ubiquitinated aggregates in some protein aggregation diseases. Whether it influences the aggregation process of α-syn in PD models and its related mechanisms are not completely known. Here, we characterized the expression and function of HDAC6 in the ubiquitin-proteasome system impairment-induced PD model. Our results showed that HDAC6 inhibition further exacerbated the nigrostriatal dopamine neurodegeneration and upregulated α-syn oligomers levels, whereas HDAC6 overexpression in vitro showed the opposite effects. More importantly, we provided evidence for the first time that HDAC6 regulating α-syn oligomers levels were related to its ability to trigger the heat shock response in a heat shock protein 90-dependent manner. HDAC6 mediated the dissociation of heat shock protein 90-heat shock factor 1-containing complex, and the activation of heat shock factor 1, which led to the expression of major molecular chaperones to prevent the deleterious α-syn aggregation. Thus, we propose that HDAC6 appears as a key modulator of cell protective response to the cytotoxic α-syn aggregates and may serve as a potential target for therapy development in PD.

Topics: alpha-Synuclein; Animals; Brain; Cells, Cultured; Disease Models, Animal; DNA-Binding Proteins; Heat Shock Transcription Factors; Histone Deacetylase 6; Histone Deacetylases; HSP90 Heat-Shock Proteins; Male; Mice, Inbred C57BL; Molecular Chaperones; Molecular Targeted Therapy; Parkinson Disease; Protein Aggregation, Pathological; Transcription Factors; Ubiquitination

Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to β-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity.

Topics: 14-3-3 Proteins; alpha-Synuclein; Amyloidosis; Humans; Kinetics; Protein Aggregation, Pathological; Protein Binding; Protein Isoforms; Signal Transduction

Over the last two decades, the identification of missense mutations in the α-synuclein (α-Syn) gene SNCA in families with inherited Parkinson disease (PD) has reinforced the central role of α-Syn in PD pathogenesis. Recently, a new missense mutation (H50Q) in α-Syn was described in patients with a familial form of PD and dementia. Here we investigated the effects of this novel mutation on the biophysical properties of α-Syn and the consequences for its cellular function. We found that the H50Q mutation affected neither the structure of free or membrane-bound α-Syn monomer, its interaction with metals, nor its capacity to be phosphorylated in vitro. However, compared with the wild-type (WT) protein, the H50Q mutation accelerated α-Syn fibrillization in vitro. In cell-based models, H50Q mutation did not affect α-Syn subcellular localization or its ability to be phosphorylated by PLK2 and GRK6. Interestingly, H50Q increased α-Syn secretion from SHSY5Y cells into culture medium and induced more mitochondrial fragmentation in hippocampal neurons. Although the transient overexpression of WT or H50Q did not induce toxicity, both species induced significant cell death when added to the culture medium of hippocampal neurons. Strikingly, H50Q exhibited more toxicity, suggesting that the H50Q-related enhancement of α-Syn aggregation and secretion may play a role in the extracellular toxicity of this mutant. Together, our results provide novel insight into the mechanism by which this newly described PD-associated mutation may contribute to the pathogenesis of PD and related disorders.

Topics: alpha-Synuclein; Animals; Cell Death; Cell Line; Cells, Cultured; Humans; Lipid Metabolism; Metals; Mice; Mutant Proteins; Mutation, Missense; Neurons; Parkinson Disease; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Quaternary; Recombinant Proteins

α-Synuclein is a key pathogenic protein that aggregates in hallmark lesions in Parkinson's disease and other α-synucleinopathies. Prior in vitro studies demonstrated that it is a substrate for cross-linking by transglutaminase 2 (TG2) into higher-order species. Here we investigated whether this increased aggregation occurs in vivo and whether TG2 exacerbates α-synuclein toxicity in Mus musculus and Saccharomyces cerevisiae. Compared with α-synuclein transgenic (Syn(Tg)) mice, animals double transgenic for human α-synuclein and TG2 (TG2(Tg)/Syn(Tg)) manifested greater high-molecular-weight insoluble species of α-synuclein in brain lysates and developed α-synuclein aggregates in the synaptic vesicle fraction. In addition, larger proteinase K-resistant aggregates developed, along with increased thioflavin-S-positive amyloid fibrils. This correlated with an exaggerated neuroinflammatory response, as seen with more astrocytes and microglia. Further neuronal damage was suggested by greater morphological disruption of nerve fibers and a trend toward decreased c-Fos immunoreactive neurons. Finally, the performance of TG2(Tg)/Syn(Tg) animals on motor behavioral tasks was worse relative to Syn(Tg) mice. Greater toxicity of α-synuclein was also demonstrated in yeast cells coexpressing TG2. Our findings demonstrate that TG2 promotes the aggregation of α-synuclein in vivo and that this is associated with aggravated toxicity of α-synuclein and its downstream neuropathologic consequences.

Topics: alpha-Synuclein; Animals; Astrocytes; Brain; GTP-Binding Proteins; Humans; Locomotion; Mice; Mice, Inbred C57BL; Nerve Fibers; Protein Aggregates; Protein Aggregation, Pathological; Protein Glutamine gamma Glutamyltransferase 2; Saccharomyces cerevisiae; Synaptic Vesicles; Transglutaminases

Loss-of-function mutations in the PINK1 gene lead to recessive forms of Parkinson's disease. Animal models with depleted PINK1 expression have failed to reproduce significant nigral dopaminergic neurodegeneration and clear alpha-synuclein pathology, main characteristics of the disease. In this study, we investigated whether alpha-synuclein pathology is altered in the absence of PINK1 in cell culture and in vivo. We observed that downregulation of PINK1 enhanced alpha-synuclein aggregation and apoptosis in a neuronal cell culture model for synucleinopathy. Silencing of PINK1 expression in mouse substantia nigra using recombinant adeno-associated viral vectors did not induce dopaminergic neurodegeneration in a long-term study up to 10 months, nor did it enhance or accelerate dopaminergic neurodegeneration after alpha-synuclein overexpression. However, in PINK1 knockout mice, overexpression of alpha-synuclein in the substantia nigra resulted in enhanced dopaminergic neurodegeneration as well as significantly higher levels of alpha-synuclein phosphorylation at serine 129 at 4 weeks postinjection. In conclusion, our results demonstrate that total loss of PINK1 leads to an increased sensitivity to alpha-synuclein-induced neuropathology and cell death in vivo.

Topics: alpha-Synuclein; Animals; Apoptosis; Cells, Cultured; Disease Progression; Down-Regulation; Gene Expression; Humans; Mice, Knockout; Mutation; Neurodegenerative Diseases; Neurons; Parkinson Disease; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Protein Kinases; Substantia Nigra

There is striking evidence that heat shock protein 70 (Hsp70) negatively regulates α-synuclein aggregation, which plays a significant role in the formation and progression of Parkinson disease (PD). However, how the Hsp70 in neurons fails to prevent or even reverse α-synuclein aggregation and toxicity in PD still remains to be determined. In the present study, we constructed an α-synuclein-overexpressed human neuroblastoma cell line, SH-SY5Y-Syn, in which the blockage of Hsp70 promoted α-synuclein aggregation. And we also found that miR-16-1 downregulated Hsp70 and promoted α-synuclein aggregation in the SH-SY5Y-Syn cells. This study revealed a novel regulatory mechanism of Hsp70 expression, which might contribute to the PD development.

Topics: alpha-Synuclein; Cell Line, Tumor; Down-Regulation; HSP70 Heat-Shock Proteins; Humans; MicroRNAs; Parkinson Disease; Protein Aggregation, Pathological

In the present chapter, we discuss the key findings on αsyn (α-synuclein) oligomers from a biophysical point of view. Current structural methods cannot provide a high-resolution structure of αsyn oligomers due to their size, heterogeneity and tendency to aggregate. However, a low-resolution structure of a stable αsyn oligomer population is emerging based on compelling data from different research groups. αsyn oligomers are normally observed during the formation of amyloid fibrils and we discuss how they are connected to this process. Another important topic is the interaction of αsyn oligomers and membranes, and we will discuss the evidence which suggests that this interaction might be essential in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Finally, we present a remarkable example of how small molecules are able to stabilize non-amyloid oligomers and how this might be a potential strategy to inhibit the inherent toxicity of αsyn oligomers. A major challenge is to link the very complex oligomerization pathways seen in clever experiments in vitro with what actually happens in the cell. With the tremendous developments in optical microscopy in mind, we believe that it will be possible to make this link very soon.

Topics: alpha-Synuclein; Amyloid; Antiparkinson Agents; Humans; Models, Molecular; Mutation; Parkinson Disease; Protein Aggregation, Pathological; Protein Folding; Protein Structure, Quaternary

Chaperone-mediated autophagy (CMA) is involved in wild-type α-synuclein degradation in Parkinson's disease (PD), and LAMP2A and Hsc 70 have recently been indicated to be deregulated by microRNAs. To recognize the regularory role of miR-320a in CMA and the possible role in α-synuclein degradation, in the present study, we examined the targeting and regulating role of miR-320 in Hsc 70 expression. We first constructed an α-synuclein-overexpressed human neuroblastoma cell line, SH-SY5Y-Syn(+), stably over-expressing wild-type α-synuclein and sensitive to an autophagy inhibitor, which exerted no effect on the expression of LAMP2A and Hsc 70. Then we evaluated the influence on the CMA by miR-320a in the SH-SY5Y-Syn(+) cells. It was shown that miR-320a mimics transfection of specifically targeted Hsc 70 and reduced its expression at both mRNA and protein levels, however, the other key CMA molecule, LAMP2A was not regulated by miR-320a. Further, the reduced Hsc 70 attenuated the α-synuclein degradation in the SH-SY5Y-Syn(+) cells, and induced a significantly high level of α-synuclein accumulation. In conclusion, we demonstrate that miR-320a specifically targeted the 3' UTR of Hsc 70, decreased Hsc 70 expression at both protein and mRNA levels in α-synuclein-over-expressed SH-SY5Y cells, and resulted in significant α-synuclein intracellular accumulation. These results imply that miR-320a might be implicated in the α-synuclein aggravation in PD.

Topics: 3' Untranslated Regions; alpha-Synuclein; Autophagy; Cell Line, Tumor; HSC70 Heat-Shock Proteins; Humans; Lysosomal-Associated Membrane Protein 2; MicroRNAs; Protein Aggregation, Pathological; Proteolysis; RNA, Messenger

The fibrillization of α-synuclein (α-syn) is involved in Parkinson's disease, a neurodegenerative disorder that affects four million people in the world. The amino acid sequence 71-82 of this protein (VTGVTAVAQKTV) has appeared to be essential for fibril formation. In the present study, we have investigated the secondary structure and thermal stability of the peptide fragment 71-82, α-syn71-82, as a function of concentration and temperature, as well as its interactions with phospholipid model membranes using various spectroscopic techniques. The data show that α-syn71-82 is mainly disordered in solution with the presence of a few β-sheet structure elements. The peptide reversibly forms intermolecular β-sheets with increasing concentration and decreasing temperature, suggesting that it is subjected to a thermodynamic equilibrium between a monomeric and an oligomeric form. This equilibrium seems to be affected by the presence of zwitterionic membranes. Conversely, the influence of the peptide on zwitterionic lipid bilayers is small and concentration-dependent. By contrast, α-syn71-82 is strongly affected by anionic vesicles. The peptide indeed exhibits a dramatic conformational change, reflecting an extensive and irreversible self-aggregation, the majority of the amino acids being involved in a parallel β-sheet conformation. The aggregates appear to be located near the membrane surface but do not perturb significantly the membrane order. Comparing these results with the literature, it appears that α-syn71-82 shares several general properties and structural similarities with its parent protein. These common points suggest that the sequence 71-82 may overall contribute to the behavior and properties of α-syn.

Topics: alpha-Synuclein; Amyloid; Circular Dichroism; Humans; Lipid Bilayers; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Osmolar Concentration; Peptide Fragments; Phosphatidylcholines; Phosphatidylglycerols; Protein Aggregation, Pathological; Protein Conformation; Protein Interaction Domains and Motifs; Protein Stability; Protein Structure, Secondary; Protein Unfolding; Solubility; Temperature

α-Synuclein (α-Syn) oligomerization and amyloid formation are associated with Parkinson's disease (PD) pathogenesis. Studying familial α-Syn mutants associated with early onset PD has therapeutic importance. Here we report the aggregation kinetics and other biophysical properties of a newly discovered PD associated Finnish mutation (A53E). Our in vitro study demonstrated that A53E attenuated α-Syn aggregation and amyloid formation without altering the major secondary structure and initial oligomerization tendency. Further, A53E showed reduced membrane binding affinity compared to A53T and WT. The present study would help to delineate the role of A53E mutation in early onset PD pathogenesis.

Topics: alpha-Synuclein; Amino Acid Substitution; Amyloid; Circular Dichroism; Finland; Fluorescent Dyes; Humans; Kinetics; Lipid Bilayers; Microscopy, Atomic Force; Mutation; Parkinson Disease; Phosphatidylcholines; Phosphatidylethanolamines; Protein Aggregation, Pathological; Protein Structure, Secondary; Recombinant Proteins; Spectrometry, Fluorescence; Surface Plasmon Resonance; Surface Properties

α-Synuclein is an intrinsically disordered protein whose aggregation is implicated in Parkinson's disease. A second member of the synuclein family, β-synuclein, shares significant sequence similarity with α-synuclein but is much more resistant to aggregation. β-Synuclein is missing an 11-residue stretch in the central non-β-amyloid component region that forms the core of α-synuclein amyloid fibrils, yet insertion of these residues into β-synuclein to produce the βSHC construct does not markedly increase the aggregation propensity. To investigate the structural basis of these different behaviors, quantitative nuclear magnetic resonance data, in the form of paramagnetic relaxation enhancement-derived interatomic distances, are combined with molecular dynamics simulations to generate ensembles of structures representative of the solution states of α-synuclein, β-synuclein, and βSHC. Comparison of these ensembles reveals that the differing aggregation propensities of α-synuclein and β-synuclein are associated with differences in the degree of residual structure in the C-terminus coupled to the shorter separation between the N- and C-termini in β-synuclein and βSHC, making protective intramolecular contacts more likely.

Topics: alpha-Synuclein; Amino Acid Sequence; beta-Synuclein; Humans; Molecular Dynamics Simulation; Molecular Sequence Data; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary; Sequence Alignment

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

Topics: alpha-Synuclein; Cell Line; Humans; Lewy Bodies; Lysosomes; Mutagenesis, Site-Directed; Parkinson Disease; Phosphorylation; Point Mutation; Protein Aggregation, Pathological

We studied α-synuclein (αS) aggregation in giant vesicles, and observed dramatic membrane disintegration, as well as lipid incorporation into micrometer-sized suprafibrillar aggregates. In the presence of dye-filled vesicles, dye leakage and fibrillization happen concurrently. However, growing fibrils do not impair the integrity of phospholipid vesicles that have a low affinity for αS. Seeding αS aggregation accelerates dye leakage, indicating that oligomeric species are not required to explain the observed effect. The evolving picture suggests that fibrils that appear in solution bind membranes and recruit membrane-bound monomers, resulting in lipid extraction, membrane destabilization and the formation of lipid-containing suprafibrillar aggregates.

Topics: alpha-Synuclein; Cell Membrane; Humans; Lipid Bilayers; Protein Aggregation, Pathological; Protein Binding; Protein Structure, Secondary

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer's disease, Parkinson's disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.

Topics: Alcohol Dehydrogenase; alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Animals; Caseins; Catalase; gamma-Crystallins; Hemin; Humans; Oxidation-Reduction; Peptide Fragments; Protein Aggregation, Pathological; Protein Folding; Protein Structure, Secondary

α-synuclein is an abundant presynaptic protein that is important for regulation of synaptic vesicle trafficking, and whose misfolding plays a key role in Parkinson's disease. While α-synuclein is disordered in solution, it folds into a helical conformation when bound to synaptic vesicles. Stabilization of helical, folded α-synuclein might therefore interfere with α-synuclein-induced neurotoxicity. Here we show that several small molecules, which delay aggregation of α-synuclein in solution, including the Parkinson's disease drug selegiline, fail to interfere with misfolding of vesicle-bound α-synuclein. In contrast, the porphyrin phtalocyanine tetrasulfonate directly binds to vesicle-bound α-synuclein, stabilizes its helical conformation and thereby delays pathogenic misfolding and aggregation. Our study suggests that small-molecule-mediated stabilization of helical vesicle-bound α-synuclein opens new possibilities to target Parkinson's disease and related synucleinopathies.

Topics: alpha-Synuclein; Antiparkinson Agents; Humans; Indoles; Parkinson Disease; Protein Aggregation, Pathological; Protein Folding; Protein Stability; Protein Structure, Secondary; Selegiline; Synaptic Vesicles