alpha-synuclein and Amyloidosis

Over the past thirty years, researchers have highlighted the role played by a class of proteins or polypeptides that forms pathogenic amyloid aggregates in vivo, including i) the amyloid Aβ peptide, which is known to form senile plaques in Alzheimer's disease; ii) α-synuclein, responsible for Lewy body formation in Parkinson's disease and iii) IAPP, which is the protein component of type 2 diabetes-associated islet amyloids. These proteins, known as intrinsically disordered proteins (IDPs), are present as highly dynamic conformational ensembles. IDPs can partially (mis) fold into (dys) functional conformations and accumulate as amyloid aggregates upon interaction with other cytosolic partners such as proteins or lipid membranes. In addition, an increasing number of reports link the toxicity of amyloid proteins to their harmful effects on membrane integrity. Still, the molecular mechanism underlying the amyloidogenic proteins transfer from the aqueous environment to the hydrocarbon core of the membrane is poorly understood. This review starts with a historical overview of the toxicity models of amyloidogenic proteins to contextualize the more recent lipid-chaperone hypothesis. Then, we report the early molecular-level events in the aggregation and ion-channel pore formation of Aβ, IAPP, and α-synuclein interacting with model membranes, emphasizing the complexity of these processes due to their different spatial-temporal resolutions. Next, we underline the need for a combined experimental and computational approach, focusing on the strengths and weaknesses of the most commonly used techniques. Finally, the last two chapters highlight the crucial role of lipid-protein complexes as molecular switches among ion-channel-like formation, detergent-like, and fibril formation mechanisms and their implication in fighting amyloidogenic diseases.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Diabetes Mellitus, Type 2; Humans; Intrinsically Disordered Proteins; Lipids; Molecular Chaperones; Peptides

Estimating the amyloid level in yeast Saccharomyces, we found out that the red pigment (product of polymerization of aminoimidazole ribotide) accumulating in ade1 and ade2 mutants leads to drop of the amyloid content. We demonstrated in vitro that fibrils of several proteins grown in the presence of the red pigment stop formation at the protofibril stage and form stable aggregates due to coalescence. Also, the red pigment inhibits reactive oxygen species accumulation in cells. This observation suggests that red pigment is involved in oxidative stress response. We developed an approach to identify the proteins whose aggregation state depends on prion (amyloid) or red pigment presence. These sets of proteins overlap and in both cases involve many different chaperones. Red pigment binds amyloids and is supposed to prevent chaperone-mediated prion propagation. An original yeast-Drosophila model was offered to estimate the red pigment effect on human proteins involved in neurodegeneration. As yeast cells are a natural feed of Drosophila, we could compare the data on transgenic flies fed on red and white yeast cells. Red pigment inhibits aggregation of human Amyloid beta and α-synuclein expressed in yeast cells. In the brain of transgenic flies, the red pigment diminishes amyloid beta level and the area of neurodegeneration. An improvement in memory and viability accompanied these changes. In transgenic flies expressing human α-synuclein, the pigment leads to a decreased death rate of dopaminergic neurons and improves mobility. The obtained results demonstrate yeast red pigment potential for the treatment of neurodegenerative diseases.

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Amyloidosis; Animals; Animals, Genetically Modified; Drosophila; Prions; Protein Aggregates; Saccharomyces cerevisiae

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Molecular Chaperones; Neurodegenerative Diseases; Prions

Amyloid formation is a pathological process associated with a wide range of degenerative disorders, including Alzheimer's disease, Parkinson's disease, and diabetes mellitus type 2. During disease progression, abnormal accumulation and deposition of proteinaceous material are accompanied by tissue degradation, inflammation, and dysfunction. Agents that can interfere with the process of amyloid formation or target already formed amyloid assemblies are consequently of therapeutic interest. In this context, a few endogenous proteins have been associated with an anti-amyloidogenic activity. Here, we review the properties of transthyretin, apolipoprotein E, clusterin, and BRICHOS protein domain which all effectively interfere with amyloid in vitro, as well as displaying a clinical impact in humans or animal models. Their involvement in the amyloid formation process is discussed, which may aid and inspire new strategies for therapeutic interventions.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Amyloidogenic Proteins; Amyloidosis; Animals; Humans; Parkinson Disease

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

Amyloidosis is a concept that implicates disorders and complications that are due to abnormal protein accumulation in different cells and tissues. Protein aggregation-associated diseases are classified according to the type of aggregates and deposition sites, such as neurodegenerative disorders and type 2 diabetes mellitus. Polyphenolic phytochemicals such as curcumin and its derivatives have anti-amyloid effects both in vitro and in animal models; however, the underlying mechanisms are not understood. In this review, we summarized possible mechanisms by which curcumin could interfere with self-assembly processes and reduce amyloid aggregation in amyloidosis. Furthermore, we discuss clinical trials in which curcumin is used as a therapeutic agent for the treatment of diseases linking to protein aggregates.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Clinical Trials as Topic; Creutzfeldt-Jakob Syndrome; Curcumin; Diabetes Mellitus, Type 2; Humans; Huntington Disease; Hypoglycemic Agents; Mitochondria; Neuroprotective Agents; Oxidative Stress; Parkinson Disease; Protein Aggregates; tau Proteins

Synucleinopathies are a heterogeneous group of neurodegenerative diseases with amyloid deposits that contain the α-synuclein (SNCA/α-Syn) protein as a common hallmark. It is astonishing that aggregates of a single protein are able to give rise to a whole range of different disease manifestations. The prion strain hypothesis offers a possible explanation for this conundrum. According to this hypothesis, a single protein sequence is able to misfold into distinct amyloid structures that can cause different pathologies. In fact, a growing body of evidence suggests that conformationally distinct α-Syn assemblies might be the causative agents behind different synucleinopathies. In this review, we provide an overview of research on the strain hypothesis as it applies to synucleinopathies and discuss the potential implications for diagnostic and therapeutic purposes.

Topics: alpha-Synuclein; Amyloid; Amyloidosis; Animals; Brain; Humans; Protein Conformation; Protein Folding; Synucleinopathies

Amyloids are fibrous cross-β protein aggregates that are capable of proliferation via nucleated polymerization. Amyloid conformation likely represents an ancient protein fold and is linked to various biological or pathological manifestations. Self-perpetuating amyloid-based protein conformers provide a molecular basis for transmissible (infectious or heritable) protein isoforms, termed prions. Amyloids and prions, as well as other types of misfolded aggregated proteins are associated with a variety of devastating mammalian and human diseases, such as Alzheimer's, Parkinson's and Huntington's diseases, transmissible spongiform encephalopathies (TSEs), amyotrophic lateral sclerosis (ALS) and transthyretinopathies. In yeast and fungi, amyloid-based prions control phenotypically detectable heritable traits. Simplicity of cultivation requirements and availability of powerful genetic approaches makes yeast Saccharomyces cerevisiae an excellent model system for studying molecular and cellular mechanisms governing amyloid formation and propagation. Genetic techniques allowing for the expression of mammalian or human amyloidogenic and prionogenic proteins in yeast enable researchers to capitalize on yeast advantages for characterization of the properties of disease-related proteins. Chimeric constructs employing mammalian and human aggregation-prone proteins or domains, fused to fluorophores or to endogenous yeast proteins allow for cytological or phenotypic detection of disease-related protein aggregation in yeast cells. Yeast systems are amenable to high-throughput screening for antagonists of amyloid formation, propagation and/or toxicity. This review summarizes up to date achievements of yeast assays in application to studying mammalian and human disease-related aggregating proteins, and discusses both limitations and further perspectives of yeast-based strategies.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Amyotrophic Lateral Sclerosis; Animals; Humans; Parkinson Disease; Prion Diseases; Prions; Recombinant Fusion Proteins; Saccharomyces cerevisiae; tau Proteins

Alpha-Synuclein is found in the neuronal cells but its native function is not well known. While α -synuclein is an intrinsically disordered protein that adopts a helical conformation upon membrane binding, numerous studies have shown that oligomeric β-forms of this protein are cytotoxic. This response to misfolded species contributes to Parkinson's Disease etiology and symptoms. The resulting amyloid fibrils are an established diagnostic in Parkinson's Disease. In this review, we focus on strategies that have been used to inhibit the amyloidogenesis of α -synuclein either by stabilizing the native state, or by redirecting the pathway to less toxic aggregates. Small molecules such as polyphenols, peptides as well as large proteins have proven effective at protecting cells against the cytotoxicity of α-synuclein. These strategies may lead to the development of therapeutic agents that could prove useful in combating this disease.

Topics: alpha-Synuclein; Amyloid; Amyloidosis; Animals; Humans; Mutation; Parkinson Disease; Protein Folding

Neurodegenerative diseases constitute a set of pathological conditions originating from the slow, irreversible and systematic cell loss within the various regions of the brain and/or the spinal cord. Neurodegenerative diseases are proteinopathies associated with misbehavior and disarrangement of a specific protein, affecting its processing, functioning, and/or folding. Many proteins associated with human neurodegenerative diseases are intrinsically disordered; i.e., they lack stable tertiary and/or secondary structure under physiological conditions in vitro. Intrinsically disordered proteins (IDPs) have broad presentation in nature. Functionally, they complement ordered proteins, being typically involved in regulation, signaling and control. Structures and functions of IDPs are intensively modulated by alternative splicing and posttranslational modifications. It is recognized now that nanoimaging offers a set of tools to analyze protein misfolding and self-assembly via monitoring the aggregation process, to visualize protein aggregates, and to analyze properties of these aggregates. The major goals of this review are to show the interconnections between intrinsic disorder and human neurodegenerative diseases and to overview a recent progress in development of novel nanoimaging tools to follow protein aggregation.

Topics: alpha-Synuclein; Alzheimer Disease; Amino Acid Sequence; Amyloid; Amyloidosis; Computational Biology; Down Syndrome; Humans; Lewy Body Disease; Models, Molecular; Nerve Tissue Proteins; Neurodegenerative Diseases; Parkinson Disease; Prion Diseases; Protein Conformation

Amyloidogenesis defines a condition in which a soluble and innocuous protein turns to insoluble protein aggregates known as amyloid fibrils. This protein suprastructure derived via chemically specific molecular self-assembly process has been commonly observed in various neurodegenerative disorders such as Alzheimer's, Parkinson's, and Prion diseases. Although the major culprit for the cellular degeneration in the diseases remains unsettled, amyloidogenesis is considered to be etiologically involved. Recent recognition of fibrillar polymorphism observed mostly from in vitro amyloidogeneses may indicate that multiple mechanisms for the amyloid fibril formation would be operated. Nucleation-dependent fibrillation is the prevalent model for assessing the self-assembly process. Following thermodynamically unfavorable seed formation, monomeric polypeptides bind to the seeds by exerting structural adjustments to the template, which leads to accelerated amyloid fibril formation. In this review, we propose another in vitro model of amyloidogenesis named double-concerted fibrillation. Here, two consecutive assembly processes of monomers and subsequent oligomeric species are responsible for the amyloid fibril formation of alpha-synuclein, a pathological component of Parkinson's disease, following structural rearrangement within the oligomers which then act as a growing unit for the fibrillation. [BMB reports 2009; 42(9): 541-551].

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloidosis; Humans; Parkinson Disease

Neurodegenerative diseases are characterized by selective and progressive loss of specific populations of neurons, which determines the clinical presentation. The same neuronal populations can be affected in a number of different disorders. Given that the clinical presentation reflects the particular population of neurons that are targets of the disease process, it is clear that for any given clinical syndrome, more than one neurodegenerative disease can account for the clinical syndrome. Because of this clinical ambiguity, for the purpose of this brief review neurodegenerative disorders are classified according to the underlying molecular pathology rather than their clinical presentation. The major neurodegenerative diseases can be classified into amyloidoses, tauopathies, alpha-synucleinopathies and TDP-43 proteinopathies.

Topics: alpha-Synuclein; Amyloidosis; Gene Expression; Humans; Neurodegenerative Diseases; Neurons; Pathology, Molecular; Syndrome; Tauopathies; TDP-43 Proteinopathies

Protein aggregation is a prominent feature of many neurodegenerative diseases, such as Alzheimer's, Huntington's, and Parkinson's diseases, as well as spongiform encephalopathies and systemic amyloidoses. These diseases are sometimes called protein misfolding diseases, but the latter term begs the question of what is the "folded" state of proteins for which normal structure and function are unknown. Amyloid consists of linear, unbranched protein or peptide fibrils of approximately 100 A diameter. These fibrils are composed of a wide variety of proteins that have no sequence homology, and no similarity in three-dimensional structures--and yet, as fibrils, they share a common secondary structure, the beta-sheet. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidation of fibril structure. Recent advances in solid-state NMR spectroscopy and other biophysical techniques have led to the partial elucidation of fibril structure. Surprisingly at the time, for beta-amyloid, a set of 39-43-amino-acid peptides believed to play a pathogenic role in Alzheimer's disease, the beta-sheets are parallel with all amino acids of the sheets in-register. Since the time of those observations, however, it has become clear that there is no universal structure for amyloid fibrils. While many of the amyloid fibrils described thus far have a parallel beta-sheet structure, some have antiparallel beta-sheets, and other, more subtle structural differences among amyloids exist as well. Amyloids demonstrate conformational plasticity, the ability to adopt more than one stable tertiary fold. Conformational plasticity could account for "strain" differences in prions, and for the fact that a single polypeptide can form different fibril types with conformational differences at the atomic level. More recent data now indicate that the fibrils may not be the most potent or proximate mediators of cyto- and neurotoxicity. This damage is not confined to cell death, but also includes more subtle forms of damage, such as disruption of synaptic plasticity in the central nervous system. Rather than fibrils, prefibrillar aggregates, variously called "micelles," "protofibrils," or ADDLs (beta-amyloid-derived diffusible ligands in the case of beta-amyloid) may be the more proximate mediators of cell damage. These are soluble oligomers of aggregating peptides or proteins, but their structure is very challenging to study, because they are generally diffi

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloid; Amyloidosis; Animals; Brain; Cell Membrane; Humans; Models, Molecular; Molecular Sequence Data; Parkinson Disease; Protein Denaturation

Alpha-synuclein is a major component of Lewy bodies in Parkinson's disease and is found associated with several other forms of dementia. As with other neurodegenerative diseases, the ability of alpha-synuclein to aggregate and form fibrillar deposits seems central to its pathology. We have defined a sequence within the NAC region of alpha-synuclein that is necessary for aggregation. Exploitation of chemically modified analogues of this peptide may produce inhibitors of aggregation.

Topics: alpha-Synuclein; Amino Acid Sequence; Amyloidosis; Animals; Humans; Molecular Sequence Data; Nerve Tissue Proteins; Parkinson Disease; Protein Structure, Quaternary; Synucleins

A paradigm shift in understanding Parkinson's disease (PD) and related disorders is emerging from studies showing that alpha-synuclein (AS) gene mutations cause familial PD; AS is abnormally nitrated, phosphorylated, and ubiquitinated; AS forms neuronal and glial inclusions; AS fibrillizes in vitro; and AS transgenic animals develop neurodegeneration with AS amyloid inclusions. Thus, PD and related synucleinopathies are brain amyloidoses that may share similar mechanisms and targets for drug discovery.

Topics: alpha-Synuclein; Amyloidosis; Animals; Brain; Humans; Inclusion Bodies; Nerve Tissue Proteins; Parkinsonian Disorders; Synucleins

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. While the classic clinical-neuropathological features of PD have been well established, mechanisms underlying brain degeneration in PD are unknown, and only partially effective symptomatic treatments for PD exist. Further, there are no therapeutic interventions that prevent PD or block the progression of this relentless neurodegenerative disorder. However, dramatic new insights into the role of alpha-synuclein (AS) in the pathobiology of PD have emerged recently, and this has led to the development of transgenic animal models of PD-like AS pathologies. Continuing advances in this research direction should advance understanding of PD and accelerate discovery of more effective therapies for this and related synucleinopathies.

Topics: alpha-Synuclein; Amyloidosis; Humans; Nerve Tissue Proteins; Neurodegenerative Diseases; Parkinson Disease; Synucleins

Neuronal death in Parkinson's disease (PD), one of the most common neurodegenerative disorders in the adult and aging population is probably caused by misfolding of synaptic proteins such as alpha-synuclein. Although, some treatments are currently available to control some of the symptoms of PD, none of these approaches directly addresses the mechanisms of disease. With the advent of new experimental animal models for this disorder, the potential for development and discovery of new treatment has been significantly bolstered. Among them, overexpression of alpha-synuclein results in motor deficits. dopaminergic loss and formation of inclusion bodies. Co-expression of mutant amyloid precursor protein, accelerates alpha-synuclein aggregation and enhances the neurodegenerative pathology in these mice, providing a unique model where to investigate the interactions between Abeta1-42 and alpha-synuclein and to develop treatments for combined Alzheimer's disease and PD. Development of anti-parkinsonian treatments based on these models includes: (i) anti-aggregation or pro-degradation compounds, (ii) neuroprotective compounds, and (iii) neurotrophic agents. Among them, we characterized beta-synuclein, the non-amyloidogenic homologue of alpha-synuclein, as an inhibitor of aggregation of alpha-synuclein. Our results raise the intriguing possibility that beta-synuclein might be a natural negative regulator of alpha-synuclein aggregation, and that a similar class of endogenous factors might regulate the aggregation state of other molecules involved in neurodegeneration. Such an anti-amyloidogenic property of beta-synuclein might also provide a novel strategy for the treatment of neurodegenerative disorders.

Topics: alpha-Synuclein; Amyloidosis; Animals; Animals, Genetically Modified; beta-Synuclein; Cell Aggregation; Disease Models, Animal; Humans; Nerve Tissue Proteins; Parkinson Disease, Secondary; Protein Binding; Protein Folding; Synucleins

The human α-synuclein protein, identified as one of the main markers of Parkinson's disease, is a 140-amino acid thermostable protein that can easily be overexpressed in E. coli. The purification protocol determines the ability of the protein to assemble into amyloid fibrils of well-defined structures. Here, we describe the purification and assembly protocols to obtain three well-characterized amyloid forms (ribbon, fibrils, and fibril-91) used to assess their activity in biochemical and cellular assays or to investigate their atomic structure by cryo-electron microscopy and solid-state NMR.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Cryoelectron Microscopy; Escherichia coli; Humans; Parkinson Disease

Protein aggregates, hereunder amyloid fibrils, can undergo a maturation process, whereby early formed aggregates undergo a structural and physicochemical transition leading to more mature species. In the case of amyloid-related diseases, such maturation confers distinctive biological properties of the aggregates, which may account for a range of diverse pathological subtypes. Here, we present a protocol for the preparation of α-synuclein amyloid fibrils differing in the level of their maturation. We utilize widely accessible biophysical techniques to characterize the structure and morphology and a simple thermal treatment procedure to test their thermodynamic stability. Their biological properties are probed by means of binding to native plasma membrane sheets originating from mammalian cell lines.

Topics: alpha-Synuclein; Amyloid; Amyloidosis; Animals; Biophysics; Humans; Mammals; Protein Aggregates

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Phase Transition; Prion Proteins; Prions

The aggregation of α-synuclein (α-Syn) is a key pathological hallmark of Parkinson's disease (PD). α-Syn undergoes liquid-liquid phase separation (LLPS) to drive amyloid aggregation. How the LLPS of α-Syn is regulated remains largely unknown. Here, we discovered that the C-terminal region modulates α-Syn phase separation through electrostatic interactions. The wild-type (WT) and PD disease-related truncated α-Syn can co-exist in the condensates. The truncated α-Syn could dramatically promote WT α-Syn phase separation. Further studies demonstrated that the truncated α-Syn accelerated WT α-Syn turning to amyloid aggregates by modulation of phase separation. Together, our findings disclose the role of the C-terminal domain in the LLPS of α-Syn and pave the path for understanding the mechanism of truncated α-Syn in PD pathology.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Parkinson Disease

The division of amyloid fibril particles through fragmentation is implicated in the progression of human neurodegenerative disorders such as Parkinson's disease. Fragmentation of amyloid fibrils plays a crucial role in the propagation of the amyloid state encoded in their three-dimensional structures and may have an important role in the spreading of potentially pathological properties and phenotypes in amyloid-associated diseases. However, despite the mechanistic importance of fibril fragmentation, the relative stabilities of different types or different polymorphs of amyloid fibrils toward fragmentation remain to be quantified. We have previously developed an approach to compare the relative stabilities of different types of amyloid fibrils toward fragmentation. In this study, we show that controlled sonication, a widely used method of mechanical perturbation for amyloid seed generation, can be used as a form of mechanical perturbation for rapid comparative assessment of the relative fragmentation stabilities of different amyloid fibril structures. This approach is applied to assess the relative fragmentation stabilities of amyloid formed in vitro from wild type (WT) α-synuclein and two familial mutant variants of α-synuclein (A30P and A53T) that generate morphologically different fibril structures. Our results demonstrate that the fibril fragmentation stabilities of these different α-synuclein fibril polymorphs are all highly length dependent but distinct, with both A30P and A53T α-synuclein fibrils displaying increased resistance towards sonication-induced fibril fragmentation compared with WT α-synuclein fibrils. These conclusions show that fragmentation stabilities of different amyloid fibril polymorph structures can be diverse and suggest that the approach we report here will be useful in comparing the relative stabilities of amyloid fibril types or fibril polymorphs toward fragmentation under different biological conditions.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Parkinson Disease

The aggregation of α-synuclein (α-Syn) is a critical pathological hallmark of Parkinson's disease (PD). Prevention of α-Syn aggregation has become a key strategy for treating PD. Recent studies have suggested that α-Syn undergoes liquid-liquid phase separation (LLPS) to facilitate nucleation and amyloid formation. Here, we examined the modulation of α-Syn aggregation by myricetin, a polyhydroxyflavonol compound, under the conditions of LLPS. Unexpectedly, neither the initial morphology nor the phase-separated fraction of α-Syn was altered by myricetin. However, the dynamics of α-Syn condensates decreased upon myricetin binding. Further studies showed that myricetin dose-dependently inhibits amyloid aggregation in the condensates by delaying the liquid-to-solid phase transition. In addition, myricetin could disassemble the preformed α-Syn amyloid aggregates matured from the condensates. Together, our study shows that myricetin inhibits α-Syn amyloid aggregation in the condensates by retarding the liquid-to-solid phase transition and reveals that α-Syn phase transition can be targeted to inhibit amyloid aggregation.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Flavonoids; Humans; Parkinson Disease

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

Alpha-synuclein (αSyn) is a protein involved in neurodegenerative disorders including Parkinson's disease. Amyloid formation of αSyn can be modulated by the 'P1 region' (residues 36-42). Here, mutational studies of P1 reveal that Y39A and S42A extend the lag-phase of αSyn amyloid formation in vitro and rescue amyloid-associated cytotoxicity in C. elegans. Additionally, L38I αSyn forms amyloid fibrils more rapidly than WT, L38A has no effect, but L38M does not form amyloid fibrils in vitro and protects from proteotoxicity. Swapping the sequence of the two residues that differ in the P1 region of the paralogue γSyn to those of αSyn did not enhance fibril formation for γSyn. Peptide binding experiments using NMR showed that P1 synergises with residues in the NAC and C-terminal regions to initiate aggregation. The remarkable specificity of the interactions that control αSyn amyloid formation, identifies this region as a potential target for therapeutics, despite their weak and transient nature.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Animals; Caenorhabditis elegans; Humans; Parkinson Disease

The progress of neurodegenerative disorders correlates with the spread of their associated amyloidogenic proteins. Here, we investigated whether amyloid entry into nonconstitutive neurons could drive cross-toxic outcomes. Amyloid β (Aβ) was stereotaxically introduced into the rodent midbrain tegmentum, where it is not endogenously expressed. Postinfusion, rodent motor and sensorimotor capacities were assessed by standard behavioral tests at 3, 6, 9, and 12 months. The longitudinal study revealed no behavioral abnormalities. However, Aβ insult provoked intraneuronal inclusions positive for phosphorylated α-synuclein in dopaminergic neurons and were seen throughout the midbrain, a pathognomonic biomarker suggesting Parkinson's pathogenesis. These findings not only underscore the cross-toxic potential of amyloid proteins but also provide a mechanism by which they disrupt homeostasis in nonconstitutive neurons and cause neuronal corruption, injury, and demise. This study may help reconcile the large incidence of neurodegenerative comorbidity observed clinically.

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Amyloidogenic Proteins; Amyloidosis; Animals; Biomarkers; Brain; Dopaminergic Neurons; Longitudinal Studies; Rodentia

Alpha-synuclein is a key protein involved in the development and progression of Parkinson's disease and other synucleinopathies. The intrinsically disordered nature of alpha-synuclein hinders the computational screening of new drug candidates for the treatment of these neurodegenerative diseases. In the present work, replica exchange molecular dynamics simulations of the full-length alpha-synuclein together with low-molecular ligands were utilized to predict the binding site and effect on the amyloid-like conversion of the protein. This approach enabled an accurate prediction of the binding sites for three tested compounds (fasudil, phthalocyanine tetrasulfonate, and spermine), giving good agreement with data from experiments by other groups. Lots of information about the binding and protein conformational ensemble enabled the suggestion of a putative effect of the ligands on the amyloid-like conversion of alpha-synuclein and the mechanism of anti- and pro-amyloid activity of the tested compounds. Therefore, this approach looks promising for testing new drug candidates for binding with alpha-synuclein or other intrinsically disordered proteins and at the same time the estimation of the effect on protein behavior, including amyloid-like aggregation.

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Intrinsically Disordered Proteins; Ligands; Protein Conformation; Spermine

Topics: alpha-Synuclein; Alzheimer Disease; Amyloidosis; Brain; Humans; Lewy Bodies; Lewy Body Disease

The role of the environment in amyloid formation based on the fuzzy oil drop model (FOD) is discussed here. This model assumes that the hydrophobicity distribution within a globular protein is consistent with a 3D Gaussian (3DG) distribution. Such a distribution is interpreted as the idealized effect of the presence of a polar solvent-water. A chain with a sequence of amino acids (which are bipolar molecules) determined by evolution recreates a micelle-like structure with varying accuracy. The membrane, which is a specific environment with opposite characteristics to the polar aquatic environment, directs the hydrophobic residues towards the surface. The modification of the FOD model to the FOD-M form takes into account the specificity of the cell membrane. It consists in "inverting" the 3DG distribution (complementing the Gaussian distribution), which expresses the exposure of hydrophobic residues on the surface. It turns out that the influence of the environment for any protein (soluble or membrane-anchored) is the result of a consensus factor expressing the participation of the polar environment and the "inverted" environment. The ratio between the proportion of the aqueous and the "reversed" environment turns out to be a characteristic property of a given protein, including amyloid protein in particular. The structure of amyloid proteins has been characterized in the context of prion, intrinsically disordered, and other non-complexing proteins to cover a wider spectrum of molecules with the given characteristics based on the FOD-M model.

Topics: Algorithms; alpha-Synuclein; Amyloidogenic Proteins; Amyloidosis; Computer Simulation; Humans; Hydrophobic and Hydrophilic Interactions; Immunoglobulin G; Models, Molecular; Models, Theoretical; Prealbumin; Protein Conformation; Protein Folding; tau Proteins

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

Alzheimer's disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of β-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of β-sheet structures in different monogenic and bigenic cellular models of Alzheimer's disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-β, α-synuclein) and (amyloid-β, Tau) neuron-like cells display changes in β-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer's disease.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Amyloidosis; Animals; Cell Line, Tumor; Disease Models, Animal; Humans; Mice; Microscopy, Fluorescence; Neuroblastoma; Neurodegenerative Diseases; Neurons; Protein Conformation; Protein Structure, Secondary; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Synchrotrons

Protein misfolding diseases are a group of devastating disorders characterized by structural conversion of a soluble protein into an amyloid-like aggregate. Typically, the structural conversion occurs by misfolding of a single disease-associated protein, such as α-synuclein (αS) in Parkinson's disease, amyloid-β in Alzheimer's disease, and prion protein (PrP) in transmissible spongiform encephalopathies (TSEs). However, accumulating evidence has implicated that cross-interactions between heterologous amyloidogenic proteins dramatically impact on amyloidogenesis and disease pathology. Here we show αS in a monomeric state can suppress amyloidogenesis of PrP in vitro. Thioflavin-T assays and transmission electron miscopy revealed that monomeric αS inhibits the nucleation step of amyloidogenesis without inhibiting the growing step. Surface plasmon resonance and co-sedimentation assays neither detected interaction between αS and monomeric PrP nor fibrillar PrP. These results suggested that αS suppress amyloidogenesis of PrP by binding to a transiently accumulated intermediate, such as a partially unfolded state. Moreover, we found that oligomeric αS, which was recently suggested to interact with PrP, also did not interact with PrP. Taken together, our study revealed a chaperon-like activity of αS against PrP amyloidogenesis, suggesting a possible involvement of αS in the pathology of TSEs.

Topics: alpha-Synuclein; Amyloidosis; Humans; Molecular Chaperones; Prion Proteins; Recombinant Proteins

Amyloid fibrils are formed by denatured proteins when the supersaturation of denatured proteins is broken by agitation, such as ultrasonication, or by seeding, although the detailed mechanism of how solubility and supersaturation regulate amyloid formation remains unclear. To further understand the mechanism of amyloid formation, we examined α-synuclein (α-syn) amyloid formation at varying concentrations of SDS, LPA, heparin, or NaCl at pH 7.5. Amyloid fibrils were formed below or around the critical micelle concentrations (CMCs) of SDS (2.75 mM) and LPA (0.24 mM), although no fibrils were formed above the CMCs. On the other hand, amyloid fibrils were formed with 0.01-2.5 mg/mL of heparin and 0.5-1.0 M NaCl, and amyloid formation was gradually suppressed at higher concentrations of heparin and NaCl. To reproduce these concentration-dependent effects of additives, we constructed two models: (i) the ligand-binding-dependent solubility-modulation model and (ii) the cosolute-dependent direct solubility-modulation model, both of which were used by Tanford and colleagues to analyze the additive-dependent conformational transitions of proteins. The solubility of α-syn was assumed to vary depending on the concentration of additives either by the decreased solubility of the additive-α-syn complex (model i) or by the direct regulation of α-syn solubility (model ii). Both models well reproduced additive-dependent bell-shaped profiles of acceleration and inhibition observed for SDS and LPA. As for heparin and NaCl, participation of amorphous aggregates at high concentrations of additives was suggested. The models confirmed that solubility and supersaturation play major roles in driving amyloid formation

Topics: alpha-Synuclein; Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Solubility

Many neurodegenerative diseases are characterized by the accumulation of abnormal protein aggregates in the brain. In Parkinson's disease (PD), α-synuclein (α-syn) forms such aggregates called Lewy bodies (LBs). Recently, it has been reported that aggregates of α-syn with a cross-β structure are capable of propagating within the brain in a prionlike manner. However, the presence of cross-β sheet-rich aggregates in LBs has not been experimentally demonstrated so far. Here, we examined LBs in thin sections of autopsy brains of patients with PD using microbeam X-ray diffraction (XRD) and found that some of them gave a diffraction pattern typical of a cross-β structure. This result confirms that LBs in the brain of PD patients contain amyloid fibrils with a cross-β structure and supports the validity of in vitro propagation experiments using artificially formed amyloid fibrils of α-syn. Notably, our finding supports the concept that PD is a type of amyloidosis, a disease featuring the accumulation of amyloid fibrils of α-syn.

Topics: alpha-Synuclein; Amyloid; Amyloidosis; Animals; Brain; Disease Models, Animal; Disease Susceptibility; Humans; Lewy Bodies; Mice; Parkinson Disease; Plaque, Amyloid; X-Ray Diffraction

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Diabetes Mellitus, Type 2; Humans; Metals; Periodicals as Topic; Protein Binding; Protein Multimerization

Protein aggregation is a hallmark of several degenerative diseases, including Alzheimer's disease, Parkinson's disease and familial amyloidosis (Finnish type) (FAF). A method to isolate and detect amyloids is desired for the diagnosis of amyloid diseases. Here, we report the synthesis of pentameric thiophene amyloid ligand (p-FTAA) linked to agarose resin for selective purification of amyloid aggregates produced in vitro and in vivo. Using amyloid fibrils produced in vitro from α-synuclein, gelsolin, and Aβ

Topics: Acetates; alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Amyloidosis; Animals; Corneal Dystrophies, Hereditary; Gelsolin; Humans; Mice; Peptide Fragments; Protein Aggregates; Sepharose; Thiophenes

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

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

The Nomenclature Committee of the International Society of Amyloidosis (ISA) met during the XVth Symposium of the Society, 3 July-7 July 2016, Uppsala, Sweden, to assess and formulate recommendations for nomenclature for amyloid fibril proteins and the clinical classification of the amyloidoses. An amyloid fibril must exhibit affinity for Congo red and with green, yellow or orange birefringence when the Congo red-stained deposits are viewed with polarized light. While congophilia and birefringence remain the gold standard for demonstration of amyloid deposits, new staining and imaging techniques are proving useful. To be included in the nomenclature list, in addition to congophilia and birefringence, the chemical identity of the protein must be unambiguously characterized by protein sequence analysis when possible. In general, it is insufficient to identify a mutation in the gene of a candidate amyloid protein without confirming the variant changes in the amyloid fibril protein. Each distinct form of amyloidosis is uniquely characterized by the chemical identity of the amyloid fibril protein that deposits in the extracellular spaces of tissues and organs and gives rise to the disease syndrome. The fibril proteins are designated as protein A followed by a suffix that is an abbreviation of the parent or precursor protein name. To date, there are 36 known extracellular fibril proteins in humans, 2 of which are iatrogenic in nature and 9 of which have also been identified in animals. Two newly recognized fibril proteins, AApoCII derived from apolipoprotein CII and AApoCIII derived from apolipoprotein CIII, have been added. AApoCII amyloidosis and AApoCIII amyloidosis are hereditary systemic amyloidoses. Intracellular protein inclusions displaying some of the properties of amyloid, "intracellular amyloid" have been reported. Two proteins which were previously characterized as intracellular inclusions, tau and α-synuclein, are now recognized to form extracellular deposits upon cell death and thus have been included in Table 1 as ATau and AαSyn.

Topics: alpha-Synuclein; Amyloidogenic Proteins; Amyloidosis; Apolipoprotein C-II; Apolipoprotein C-III; Biomarkers; Birefringence; Coloring Agents; Congo Red; Gene Expression; Guidelines as Topic; Humans; Prealbumin; Protein Precursors; Sequence Analysis, Protein; Staining and Labeling; tau Proteins; Terminology as Topic

In order to further evaluate the parameters whereby intracerebral administration of recombinant α-synuclein (αS) induces pathological phenotypes in mice, we conducted a series of studies where αS fibrils were injected into the brains of M83 (A53T) and M47 (E46K) αS transgenic (Tg) mice, and non-transgenic (nTg) mice. Using multiple markers to assess αS inclusion formation, we find that injected fibrillar human αS induced widespread cerebral αS inclusion formation in the M83 Tg mice, but in both nTg and M47 Tg mice, induced αS inclusion pathology is largely restricted to the site of injection. Furthermore, mouse αS fibrils injected into nTg mice brains also resulted in inclusion pathology restricted to the site of injection with no evidence for spread. We find no compelling evidence for extensive spread of αS pathology within white matter tracts, and we attribute previous reports of white matter tract spreading to cross-reactivity of the αS pSer129/81A antibody with phosphorylated neurofilament subunit L. These studies suggest that, with the exception of the M83 Tg mice which appear to be uniquely susceptible to induction of inclusion pathology by exogenous forms of αS, there are significant barriers in mice to widespread induction of αS pathology following intracerebral administration of amyloidogenic αS.

Topics: alpha-Synuclein; Amyloidosis; Animals; Brain; Cells, Cultured; Escherichia coli; Gene Transfer Techniques; Humans; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Nerve Fibers, Myelinated; Neural Pathways; Neurofilament Proteins; Neuroglia; Neurons; Recombinant Proteins; Species Specificity

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

An amyloid form of the protein α-synuclein is the major component of the intraneuronal inclusions called Lewy bodies, which are the neuropathological hallmark of Parkinson's disease (PD). α-Synuclein is known to associate with anionic lipid membranes, and interactions between aggregating α-synuclein and cellular membranes are thought to be important for PD pathology. We have studied the molecular determinants for adsorption of monomeric α-synuclein to planar model lipid membranes composed of zwitterionic phosphatidylcholine alone or in a mixture with anionic phosphatidylserine (relevant for plasma membranes) or anionic cardiolipin (relevant for mitochondrial membranes). We studied the adsorption of the protein to supported bilayers, the position of the protein within and outside the bilayer, and structural changes in the model membranes using two complementary techniques-quartz crystal microbalance with dissipation monitoring, and neutron reflectometry. We found that the interaction and adsorbed conformation depend on membrane charge, protein charge, and electrostatic screening. The results imply that α-synuclein adsorbs in the headgroup region of anionic lipid bilayers with extensions into the bulk but does not penetrate deeply into or across the hydrophobic acyl chain region. The adsorption to anionic bilayers leads to a small perturbation of the acyl chain packing that is independent of anionic headgroup identity. We also explored the effect of changing the area per headgroup in the lipid bilayer by comparing model systems with different degrees of acyl chain saturation. An increase in area per lipid headgroup leads to an increase in the level of α-synuclein adsorption with a reduced water content in the acyl chain layer. In conclusion, the association of α-synuclein to membranes and its adsorbed conformation are of electrostatic origin, combined with van der Waals interactions, but with a very weak correlation to the molecular structure of the anionic lipid headgroup. The perturbation of the acyl chain packing upon monomeric protein adsorption favors association with unsaturated phospholipids preferentially found in the neuronal membrane.

Topics: Adsorption; alpha-Synuclein; Amyloidosis; Crystallography, X-Ray; Humans; Hydrophobic and Hydrophilic Interactions; Lewy Bodies; Lipid Bilayers; Membrane Lipids; Neurons; Neutron Diffraction; Parkinson Disease; Phospholipids; Static Electricity

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.

Topics: Acetophenones; alpha-Synuclein; Amyloid; Amyloid Neuropathies, Familial; Amyloidosis; Animals; Biomarkers; Cell Nucleus; Cell Survival; Chromatin; Cricetinae; Extracellular Space; Hep G2 Cells; Humans; Immunoglobulin Light-chain Amyloidosis; Lung; Mutation, Missense; NADPH Oxidases; Neutrophils; Onium Compounds; Pancreatic Elastase; Peptide Fragments; Prealbumin; Protein Structure, Quaternary; Proteolysis; Reactive Oxygen Species; Skin

α-Synuclein (αSYN) is genetically and neuropathologically linked to a spectrum of neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and related disorders. Cognitive impairment is recapitulated in several αSYN transgenic mouse lines. However, the mechanisms of dysfunction in affected neurons are largely unknown. Here we measured neuronal activity induced gene products in the limbic system of αSYN transgenic mice upon fear conditioning (FC). Induction of the synaptic plasticity marker c-Fos was significantly reduced in the amygdala and hippocampus of (Thy1)-h[A30P]αSYN transgenic mice in an age-dependent manner. Similarly, the neuronal activity inducible polo-like kinase 2 (Plk2) that can phosphorylate αSYN at the pathological site serine-129 was up-regulated in both brain regions upon FC. Plk2 inductions were also significantly impaired in aged (Thy1)-h[A30P]αSYN transgenic mice, both in the amygdala and hippocampus. Plk2 inductions in the amygdala after FC were paralleled by a small but significant increase in the number of neuronal cell bodies immunopositive for serine-129 phosphorylated αSYN in young but not aged (Thy1)-h[A30P]αSYN transgenic mice. In addition, we observed in the aged hippocampus a distinct type of apparently unmodified transgenic αSYN profiles resembling synaptic accumulations of αSYN. Thus, the cognitive decline observed in aged αSYN transgenic mice might be due to impairment of neurotransmission and synaptic plasticity in the limbic system by distinct αSYN species.

Topics: alpha-Synuclein; Amygdala; Amyloidosis; Animals; Cognition Disorders; Cohort Studies; Conditioning, Classical; Fear; Gene Expression Regulation; Hippocampus; Humans; Limbic System; Male; Mice; Mice, Transgenic; Neuronal Plasticity; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-fos; Silver Staining; Synapses; Synaptic Transmission; Time Factors

To understand the role of a crowded physiological environment in the pathogenesis of neurodegenerative diseases, we report the following. 1) The formation of fibrous aggregates of the human Tau fragment Tau-(244-441), when hyperphosphorylated by glycogen synthase kinase-3beta, is dramatically facilitated by the addition of crowding agents. 2) Fibril formation of nonphosphorylated Tau-(244-441) is only promoted moderately by macromolecular crowding. 3) Macromolecular crowding dramatically accelerates amyloid formation by human prion protein. A sigmoidal equation has been used to fit these kinetic data, including published data of human alpha-synuclein, yielding lag times and apparent rate constants for the growth of fibrils for these amyloidogenic proteins. These biochemical data indicate that crowded cell-like environments significantly accelerate the nucleation step of fibril formation of human Tau fragment/human prion protein/human alpha-synuclein (a significant decrease in the lag time). These results can in principle be predicted based on some known data concerning protein concentration effects on fibril formation both in vitro and in vivo. Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of neurodegenerative diseases by accelerating amyloidogenic protein misfolding and inducing human prion fibril fragmentation, which is considered to be an essential step in prion replication.

Topics: alpha-Synuclein; Amyloid; Amyloidosis; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Kinetics; Phosphorylation; Prions; Protein Folding; tau Proteins

A recent report showed that the accumulation of alpha-synuclein (alpha-syn) was detected in the brains of one-third of Alzheimer's disease and Down syndrome patients. However, the relationship between amyloid-beta protein (Abeta) and alpha-syn remains unclear. We analyzed the relation between the mutation of presenilin-1 (PS-1) and the pathological features of beta-amyloidosis and alpha-synucleinopathy. We generated doubly transgenic mice overexpressing mutant beta-amyloid precursor protein (betaAPP; Tg2576) and mutant PS-1 (PS1L286Vtg; line 198) and analyzed 19 double Tg betaAPP(+)/PS(+) mice at 5-23 months (young to old), 23 age-matched single Tg betaAPP(+)/PS(-) mice, and 11 non-Tg littermates. Immunohistochemical comparison was performed in these three groups by counting the area and the number of alpha-syn- or phosphorylated alpha-syn (palpha-syn)-positive dystrophic neurites per plaque (ASPDN, pASPDN). The acceleration of Abeta pathology was found with earlier onset and exaggerated numbers in double Tg betaAPP(+)/PS(+) compared with single Tg betaAPP(+)/PS(-) mouse brains. The accumulation of ASPDN and pASPDN was also accelerated in double Tg betaAPP(+)/PS(+) compared with single Tg betaAPP(+)/PS(-) mouse brains, especially in pASPDN. The number and area of alpha-syn and palpha-syn, and the ratio of palpha-syn positive neurites were significantly higher in double Tg betaAPP(+)/PS(+) than in single Tg betaAPP(+)/PS(-) mouse brains in middle-aged and old groups. Additional overexpression of mutant PS-1 accelerated Abeta-induced alpha-synucleinopathy and further facilitated the phosphorylation of alpha-syn, suggesting a direct association between mutant PS-1 and phosphorylation of alpha-syn.

Topics: alpha-Synuclein; Amyloid beta-Protein Precursor; Amyloidosis; Animals; Brain; Cerebral Cortex; Immunohistochemistry; Mice; Mice, Transgenic; Mutation; Neurites; Phosphorylation; Presenilin-1; Time Factors

Amyloidosis producing insoluble fibrillar protein aggregates is the common pathological feature of various neurodegenerative disorders such as Parkinson's and Alzheimer's diseases in which alpha-synuclein and amyloid beta/A4 protein (Abeta) participate to form Lewy bodies and senile plaques, respectively. To develop a novel analytical tool for amyloidosis, resveratrol, the major phenolic constituent of red wine and isolatable from grapevines, was employed to monitor the amyloids of alpha-synuclein and Abeta. Specific interaction to the amyloids enhanced the intrinsic fluorescence of resveratrol at 395 nm with an advent of new shoulder peak at 440 nm following an excitation at 320 nm. An increase in the resveratrol binding fluorescence was proportional to the quantity of amyloids. Typical sigmoidal kinetics of the amyloidosis of alpha-synuclein assessed with the thioflavin-T binding fluorescence or the beta-sheet content was fully reproduced by the resveratrol binding fluorescence. The resveratrol binding to the amyloids became saturated as the dye concentration increased, whereas the enhanced thioflavin-T binding fluorescence was quenched by the unbound thioflavin-T at the high dye concentration. Because resveratrol does not require any adjustment of the amyloid/dye ratio to obtain optimal amyloid binding fluorescence, and it exerts a higher quantum yield than does thioflavin-T, resveratrol is suggested to be a specific and more reliable fluorescent probe to determine the amyloids quantitatively.

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Amyloidosis; Benzothiazoles; Humans; Protein Structure, Quaternary; Resveratrol; Spectrometry, Fluorescence; Stilbenes; Thiazoles

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.

Topics: alpha-Synuclein; Amino Acid Substitution; Amyloid beta-Peptides; Amyloidosis; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Nerve Tissue Proteins; Neurodegenerative Diseases; Peptide Fragments; Peptides; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Synucleins; tau Proteins

Intracytoplasmic filamentous aggregates, such as neurofibrillary tangles in Alzheimer's disease and Lewy bodies in Parkinson's disease, are composed of the proteins tau and alpha-synuclein, respectively. These pathological inclusions are linked directly to the etiology and mechanisms of disease in a wide spectrum of neurodegenerative disorders, termed 'tauopathies' and 'synucleinopathies'. Emerging evidence indicates that there is frequent overlap of the pathological and clinical features of patients with tauopathies and synucleinopathies, thereby re-enforcing the notion that these disorders might be linked mechanistically. Indeed, several lines of investigation suggest that tau and alpha-synuclein might constitute a unique class of unstructured proteins that assemble predominantly into homopolymeric (rather than heteropolymeric) fibrils, which deposit mainly in separate amyloid inclusions, but occasionally deposit together. Thus, the ability of tau and alpha-synuclein to affect each other directly or indirectly might contribute to the overlap in the clinical and pathological features of tauopathies and synucleinopathies.

Topics: alpha-Synuclein; Amyloid; Amyloidosis; Animals; Brain; Humans; Inclusion Bodies; Models, Neurological; Nerve Tissue Proteins; Neurodegenerative Diseases; Neurofibrillary Tangles; Neurons; Synucleins; tau Proteins

Protein misfolding and aggregation have been linked to several human diseases, including Alzheimer's disease, Parkinson's disease, and systemic amyloidosis, by mechanisms that are not yet completely understood. The hallmark of most of these diseases is the formation of highly ordered and beta-sheet-rich aggregates referred to as amyloid fibrils. Fibril formation by WT transthyretin (TTR) or TTR variants has been linked to the etiology of systemic amyloidosis and familial amyloid polyneuropathy, respectively. Similarly, amyloid fibril formation by alpha-synuclein (alpha-syn) has been linked to neurodegeneration in Parkinson's disease, a movement disorder characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Here we show that consecutive cycles of compression-decompression under aggregating conditions lead to reversible dissociation of TTR and alpha-syn fibrils. The high sensitivity of amyloid fibrils toward high hydrostatic pressure (HHP) indicates the existence of packing defects in the fibril core. In addition, through the use of HHP we are able to detect differences in stability between fibrils formed from WT TTR and the familial amyloidotic polyneuropathy-associated variant V30M. The fibrils formed by WT alpha-syn were less susceptible to pressure denaturation than the Parkinson's disease-linked variants, A30P and A53T. This finding implies that fibrils of alpha-syn formed from the variants would be more easily dissolved into small oligomers by the cellular machinery. This result has physiological importance in light of the current view that the pathogenic species are the small aggregates rather the mature fibrils. Finally, the HHP-induced formation of fibrils from TTR is relatively fast (approximately 60 min), a quality that allows screening of antiamyloidogenic drugs.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid; Amyloidosis; Genetic Variation; Humans; Hydrostatic Pressure; In Vitro Techniques; Macromolecular Substances; Models, Molecular; Nerve Tissue Proteins; Parkinson Disease; Prealbumin; Synucleins; Water