alpha-synuclein and Nervous-System-Diseases

The possible usefulness of alpha-synuclein (aSyn) determinations in peripheral tissues (blood cells, salivary gland biopsies, olfactory mucosa, digestive tract, skin) and in biological fluids, except for cerebrospinal fluid (serum, plasma, saliva, feces, urine), as a marker of several diseases, has been the subject of numerous publications. This narrative review summarizes data from studies trying to determine the role of total, oligomeric, and phosphorylated aSyn determinations as a marker of various diseases, especially PD and other alpha-synucleinopathies. In summary, the results of studies addressing the determinations of aSyn in its different forms in peripheral tissues (especially in platelets, skin, and digestive tract, but also salivary glands and olfactory mucosa), in combination with other potential biomarkers, could be a useful tool to discriminate PD from controls and from other causes of parkinsonisms, including synucleinopathies.

Topics: alpha-Synuclein; Biopsy; Body Fluids; Humans; Nervous System Diseases; Synucleinopathies

Many neurodegenerative diseases are derived from the combined consequences of genetic and environmental factors. One of the common features implicated in the neurodegenerative processes is aggregation of disease-specific neuronal proteins. These proteins are accumulated not only directly in neurons, but also indirectly involve glial cells. Whereas the focus of research has been directed towards the impacts of protein aggregation upon neurons, the influence that it exerts on glial cells has been relatively overlooked. Recent studies, however, provide strong evidence on pathogenic responses of glial cells originated from the neuron-derived protein aggregates. Here, we critically examine the latest advancement in investigating how glial cells are activated in neurodegenerative disorders that are associated with α-synuclein aggregates. Often referred to as synucleinopathies, these include Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. To further illustrate, we would discuss paracrine actions of α-synuclein aggregates secreted from neuronal cells in promoting pathogenic reactions from various types of glia and evaluate the non-cell-autonomous mechanism compared to a cell-autonomous one. Such analyses of the impacts of glial responses in neurodegenerative diseases, in the long term, could be further utilized in developing different treatments of the diseases and potentially discovering new drugs.

Topics: alpha-Synuclein; Animals; Humans; Nervous System Diseases; Neuroglia

Knowledge of the physiology of sleep-wake regulation can contribute to an understanding of the pathophysiology and symptoms of neurological diseases and is helpful for initiating specific therapies for sleep-wake cycle stabilization. Based on historically important observations on the close relationship between sleep and neurological diseases, new insights and developments in selected neurological entities are presented in this review article.

Topics: alpha-Synuclein; Brain; Cognition; Germany; Humans; Interdisciplinary Communication; Intersectoral Collaboration; Nervous System Diseases; Orexins; Sleep Wake Disorders; Statistics as Topic

Multiple system atrophy (MSA) is a fatal adult-onset neurodegenerative disorder of uncertain etiology, clinically manifesting with autonomic failure associated with parkinsonism, cerebellar dysfunction, and pyramidal signs in variable combination. The pathological process affects central autonomic, striatonigral, and olivopontocerebellar systems. These show varying degrees of neurodegeneration and underlie the stratification of the heterogenous disorder into MSA-P and MSA-C clinical variants, which correlate to the morphologic phenotypes of striatonigral degeneration and olivopontocerebellar atrophy (MSA-C). The lesions are not limited to these most consistently and severely affected systems but may involve many other parts of the central, peripheral, and autonomic nervous systems, underpinning the multisystem character of MSA. The histological core feature are glial cytoplasmic inclusions (GCIs, Papp-Lantos bodies) in all types of oligodendroglia that contain aggregates of misfolded α-Synuclein (α-Syn). In addition to the ectopic appearance of α-Syn in oligodendrocytes and other cells, oxidative stress, proteasomal and mitochondrial dysfunction, excitotoxiciy, neuroinflammation, metabolic changes, and energy failure are important contributors to the pathogenesis of MSA, as shown by various neurotoxic and transgenic animal models. Although the basic mechanisms of α-Syn-triggered neurodegeneration are not completely understood, neuron-to-oligodendrocyte transfer of α-Syn by prion-like spreading, inducing oligodendroglial and myelin dysfunction associated with chronic neuroinflammation, are suggested finally to lead to a system-specific pattern of neurodegeneration.

Topics: alpha-Synuclein; Animals; Humans; Multiple System Atrophy; Nervous System Diseases; Neurons; Oligodendroglia; Shy-Drager Syndrome

Electron paramagnetic resonance spectroscopy (EPR) has the potential to give much detail on the structure of the paramagnetic transition ion coordination sites, principally of Cu2+, in a number of proteins associated with central nervous system diseases. Since these sites have been implicated in misfolding/mis-oligomerisation events associated with neurotoxic molecular species and/or the catalysis of damaging redox reactions in neurodegeneration, an understanding of their structure is important to the development of therapeutic agents. Nevertheless EPR, by its nature an in vitro technique, has its limitations in the study of such complex biochemical systems involving self-associating proteins that are sensitive to their chemical environment. These limitations are at the instrumental and theoretical level, which must be understood and the EPR data interpreted in the light of other biophysical and biochemical studies if useful conclusions are to be drawn.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Binding Sites; Copper; Dimerization; Electron Spin Resonance Spectroscopy; Humans; Models, Chemical; Models, Molecular; Nervous System Diseases; Oxidation-Reduction; Protein Conformation; Protein Folding; Spin Labels; Weights and Measures

Topics: alpha-Synuclein; Caveolae; Cholesterol; Humans; Lipid Metabolism; Membrane Microdomains; Membrane Proteins; Nervous System; Nervous System Diseases; Neurodegenerative Diseases

Alpha-synuclein is an abundant neuronal protein that has been linked both to normal synaptic function and to neurodegeneration. Most significantly, mutations in the gene encoding for alpha-synuclein are responsible for Parkinson's disease (PD) in rare familial cases, and the aggregated protein is a major component of Lewy bodies found in sporadic PD. Here we review recent data regarding the structure, the regulation at the transcriptional and posttranslational level, and the physiologic and aberrant functions of alpha-synuclein. We focus in particular on the fibrilization potential of alpha-synuclein and on its link with defects in protein degradation.

Topics: alpha-Synuclein; Animals; Humans; Nerve Tissue Proteins; Nervous System Diseases; Synucleins

Accumulation and toxic conversion to protofibrils of alpha-synuclein has been associated with neurological disorders such as Parkinson's disease (PD), Lewy body disease, multiple system atrophy, neurodegeneration with brain iron accumulation type 1, and Alzheimer's disease. In recent years, modeling these disorders in transgenic (tg) mice and flies has helped improve understanding of the pathogenesis of these diseases and has established the basis for the development of new experimental treatments. Overexpression of alpha-synuclein in tg mice in a region- and cell-specific manner results in degeneration of selective circuitries accompanied by motor deficits and inclusion formation similar to what is found in PD and related disorders. Furthermore, studies in singly and doubly tg mice have shown that toxic conversion and accumulation can be accelerated by alpha-synuclein mutations associated with familial parkinsonism, by amyloid beta peptide 1-42 (Abeta 1-42), and by oxidative stress. In contrast, molecular chaperones such as Hsp70 and close homologues such as alpha-synuclein have been shown to suppress toxicity. Similar studies are underway to evaluate the effects of other modifying genes that might play a role in alpha-synuclein ubiquitination. Among them considerable interest has been placed on the role of molecules associated with familial parkinsonism (Parkin, UCHL-1). Furthermore, studying the targeted overexpression of alpha-synuclein and other modifier genes in the nigrostriatal and limbic system by using regulatable promoters, lentiviral vectors, and siRNA will help improve understanding of the molecular mechanisms involved in selective neuronal vulnerability, and it will aid the development of new treatments.

Topics: alpha-Synuclein; Animals; Animals, Genetically Modified; Disease Models, Animal; Humans; Nerve Tissue Proteins; Nervous System Diseases; Protein Processing, Post-Translational; Synucleins; Toxins, Biological

Synuclein proteins are produced, in vertebrates, by three genes. They share structural resemblance to apolipoproteins, but are abundant in the neuronal cytosol and present in enriched amounts at presynaptic terminals. Synucleins have been specifically implicated in three diseases:Alzheimer's (AD), Parkinson's (PD) and breast cancer. In AD, a peptide derived from alpha-synuclein forms an intrinsic component of plaque amyloid. In PD, an alpha-synuclein allele is genetically linked to several independent familial cases, and the protein appears to accumulate in Lewy bodies. In breast cancer, increased expression of gamma-synuclein correlates with disease progression. In songbirds, alpha-synuclein expression is correlated with plasticity in the developing song control system. Although the normal function of synucleins is unknown, a role in membrane plasticity seems likely.

Topics: alpha-Synuclein; Animals; gamma-Synuclein; Humans; Molecular Sequence Data; Nerve Degeneration; Nerve Tissue Proteins; Nervous System Diseases; Neuronal Plasticity; Sequence Homology, Amino Acid; Synapses; Synucleins

Idiopathic Parkinson's disease (PD) is a neurodegenerative disorder with a broad spectrum of motor and non-motor symptoms. The neuropathological characteristics of idiopathic PD are the degeneration of dopaminergic neurons in the striatum, and the propagation of aggregates of misfolded α-synuclein in the brain following a specific pattern (Braak et al., 2006). The relationship of this pattern with motor and cognitive symptoms is still equivocal. Therefore, we investigated longitudinally the spatio-temporal patterns of atrophy propagation in PD, their inter-individual variability and associations with clinical symptoms. Magnetic resonance (MR) images of 37 PD patients and 27 controls were acquired at up to 15 time-points per subject, and over observation periods of up to 8.8 years (mean: 3.7 years). MR images were analyzed by Deformation-based Morphometry to measure region volumes and their longitudinal changes. Differences of these regional volume data between patients and controls and their associations with clinical symptoms were calculated. At baseline, group differences in the regional volumes were found mainly in areas of the sensory, motor and orbitofrontal cortices, areas in the frontal operculum, inferior frontal sulcus, hippocampus and entorhinal cortex, and in the substantia nigra, among others. The longitudinal analysis yielded more widespread and more pronounced group differences, with significantly accelerated volume decreases in PD patients in the occipital and temporal lobes, the inferior parietal lobule, as well as in the insula, putamen and nucleus basalis Meynert. The white matter was less affected than the gray matter. Worse clinical scores (MMSE, PDQ-39, UPDRS-III) were in particular associated with volume decreases of cortical areas, amygdala and basal forebrain nuclei, but not of the basal ganglia. The observed longitudinal patterns of accelerated volume decrease in PD patients largely coincide with the pattern of α-synuclein pathology in PD stages 3-5 as proposed by Braak and colleagues. Thus, longitudinal DBM appears to depict already in-vivo the progression of neuropathological changes.

Topics: alpha-Synuclein; Atrophy; Brain; Gray Matter; Humans; Longitudinal Studies; Magnetic Resonance Imaging; Nervous System Diseases; Parkinson Disease

Parkinson's disease and related disorders are devastating neurodegenerative pathologies. Since α-synuclein was identified as a main component of Lewy bodies and neurites, efforts have been made to clarify the pathogenic mechanisms of α-synuclein's detrimental effects. α-synuclein oligomers are the most harmful species and may recruit and activate glial cells. Inflammation is emerging as a bridge between genetic susceptibility and environmental factors co-fostering Parkinson's disease. However, direct evidence linking inflammation to the harmful activities of α-synuclein oligomers or to the Parkinson's disease behavioural phenotype is lacking.. To clarify whether neuroinflammation influences Parkinson's disease pathogenesis, we developed: (i) a 'double-hit' approach in C57BL/6 naive mice where peripherally administered lipopolysaccharides were followed by intracerebroventricular injection of an inactive oligomer dose; (ii) a transgenic 'double-hit' model where lipopolysaccharides were given to A53T α-synuclein transgenic Parkinson's disease mice.. Lipopolysaccharides induced a long-lasting neuroinflammatory response which facilitated the detrimental cognitive activities of oligomers. LPS-activated microglia and astrocytes responded differently to the oligomers with microglia activating further and acquiring a pro-inflammatory M1 phenotype, while astrocytes atrophied. In the transgenic 'double-hit' A53T mouse model, lipopolysaccharides aggravated cognitive deficits and increased microgliosis. Again, astrocytes responded differently to the double challenge. These findings indicate that peripherally induced neuroinflammation potentiates the α-synuclein oligomer's actions and aggravates cognitive deficits in A53T mice.. The fine management of both peripheral and central inflammation may offer a promising therapeutic approach to prevent or slow down some behavioural aspects in α-synucleinopathies.

Topics: alpha-Synuclein; Animals; Astrocytes; Disease Models, Animal; Inflammation; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Nerve Degeneration; Nervous System Diseases; Parkinson Disease; Substantia Nigra

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

Epidemiological studies demonstrated that pesticide exposure, such as rotenone and paraquat, increases the risk of Parkinson's disease (PD). Chronic systemic exposure to rotenone, a mitochondrial complex I inhibitor, could reproduce many features of PD. However, the adoption of the models is limiting because of variability in animal sensitivity and the inability of other investigators to consistently reproduce the PD neuropathology. In addition, most of rotenone models were produced in rats. Here, we tried to establish a high-reproducible rotenone model using C57BL/6J mice. The rotenone mouse model was produced by chronic systemic exposure to a low dose of rotenone (2.5 mg/kg/day) for 4 weeks by subcutaneous implantation of rotenone-filled osmotic mini pump. The rotenone-treated mice exhibited motor deficits assessed by open field, rotarod and cylinder test and gastrointestinal dysfunction. Rotenone treatment decreased the number of dopaminergic neuronal cells in the substantia nigra pars compacta (SNpc) and lesioned nerve terminal in the striatum. In addition, we observed significant reduction of cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) and the intestinal myenteric plexus. Moreover, α-synuclein was accumulated in neuronal soma in the SNpc, DMV and intestinal myenteric plexus in rotenone-treated mice. These data suggest that the low-dose rotenone mouse model could reproduce behavioral and central and peripheral neurodegenerative features of PD and be a useful model for investigation of PD pathogenesis.

Topics: alpha-Synuclein; Animals; Behavior, Animal; Biomarkers; Cholinergic Neurons; Disease Models, Animal; Dopaminergic Neurons; Electron Transport Complex I; Environmental Exposure; Fluorescent Antibody Technique; Insecticides; Male; Mice; Mitochondria; Motor Disorders; Myenteric Plexus; Nervous System Diseases; Parkinson Disease; Rotenone; Substantia Nigra

Visualization of phosphorylated α-synuclein at serine 129 (p-syn) in skin nerves is a promising test for the in vivo diagnosis of synucleinopathies. Here the aim was to establish the intra- and inter-laboratory reproducibility of measurement of intraneural p-syn immunoreactivity in two laboratories with major expertise (Würzburg and Bologna).. In total, 43 patients affected by Parkinson's disease (PD 21 patients), dementia with Lewy bodies (DLB 1), rapid eye movement sleep behaviour disorder (RBD 11), multiple system atrophy (MSA-P 4) and small fibre neuropathy (SFN 6) were enrolled. Skin biopsy was performed at the C7 paravertebral spine region and distal skin sites (thigh or leg). The analysis was standardized in both laboratories and carried out blinded on a single skin section double stained with antibodies to p-syn and the pan-axonal marker protein gene product 9.5. Fifty skin sections were randomly selected for the analysis: 25 from C7 and 25 from distal sites. Differently classified sections were re-evaluated to understand the reasons for the discrepancy.. The intra-laboratory analysis showed an excellent reproducibility both in Würzburg (concordance of classification 100% of sections; K = 1; P < 0.001) and Bologna (96% of sections; K = 0.92; P < 0.001). Inter-laboratory analysis showed reproducibility in 45 sections (90%; K = 0.8; P < 0.001) and a different classification in five sections, which was mainly due to fragmented skin samples or weak fluorescent signals.. Analysis of p-syn showed excellent inter- and intra-laboratory reproducibility supporting the reliability of this technique. The few ascertained discordances were important to further improve the standardization of this technique.

Topics: Adult; Aged; Aged, 80 and over; alpha-Synuclein; Biopsy; Female; Humans; Immunohistochemistry; Male; Middle Aged; Multiple System Atrophy; Nervous System Diseases; Parkinson Disease; Peripheral Nerves; Phosphorylation; REM Sleep Behavior Disorder; Reproducibility of Results; Skin

Mutations in the

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Dopaminergic Neurons; Gene Knock-In Techniques; Male; Mice; Mutation; Nervous System Diseases; Neurodegenerative Diseases; Neuropathology; Parkinson Disease; Protein Transport; tau Proteins; Vesicular Transport Proteins

Topics: alpha-Synuclein; Amyloid beta-Peptides; Antigens, CD; Cerebral Cortex; Chorea; Female; Glial Fibrillary Acidic Protein; Humans; Middle Aged; Nervous System Diseases; Prions

Compelling evidence suggests that accumulation and aggregation of alpha-synuclein (α-syn) contribute to the pathogenesis of Parkinson's disease (PD). Here, we describe a novel Bacterial Artificial Chromosome (BAC) transgenic model, in which we have expressed wild-type human α-syn fused to green fluorescent protein (GFP), under control of the mouse α-syn promoter. We observed a widespread and high expression of α-syn-GFP in multiple brain regions, including the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and the ventral tegmental area, the olfactory bulb as well as in neocortical neurons. With increasing age, transgenic mice exhibited reductions in amphetamine-induced locomotor activity in the open field, impaired rotarod performance and a reduced striatal dopamine release, as measured by amperometry. In addition, they progressively developed deficits in an odor discrimination test. Western blot analysis revealed that α-syn-GFP and phospho-α-syn levels increased in multiple brain regions, as the mice grew older. Further, we observed, by immunohistochemical staining for phospho-α-syn and in vivo by two-photon microscopy, the formation of α-syn aggregates as the mice aged. The latter illustrates that the model can be used to track α-syn aggregation in vivo. In summary, this novel BAC α-syn-GFP model mimics a unique set of aspects of PD progression combined with the possibility of tracking α-syn aggregation in neocortex of living mice. Therefore, this α-syn-GFP-mouse model can provide a powerful tool that will facilitate the study of α-syn biology and its involvement in PD pathogenesis.

Topics: Aging; alpha-Synuclein; Amphetamine; Animals; Discrimination, Psychological; Dopamine; Dopamine Agents; Dopaminergic Neurons; Green Fluorescent Proteins; Humans; Locomotion; Mice; Mice, Transgenic; Movement Disorders; Neocortex; Nervous System Diseases; Olfaction Disorders; Smell; Substantia Nigra; Ventral Tegmental Area

To develop a cutaneous biomarker for Parkinson disease (PD).. Twenty patients with PD and 14 age- and sex-matched control subjects underwent examinations, autonomic testing, and skin biopsies at the distal leg, distal thigh, and proximal thigh. α-Synuclein deposition and the density of intraepidermal, sudomotor, and pilomotor nerve fibers were measured. α-Synuclein deposition was normalized to nerve fiber density (the α-synuclein ratio). Results were compared with examination scores and autonomic function testing.. Patients with PD had a distal sensory and autonomic neuropathy characterized by loss of intraepidermal and pilomotor fibers (p < 0.05 vs controls, all sites) and morphologic changes to sudomotor nerve fibers. Patients with PD had greater α-synuclein deposition and higher α-synuclein ratios compared with controls within pilomotor nerves and sudomotor nerves (p < 0.01, all sites) but not sensory nerves. Higher α-synuclein ratios correlated with Hoehn and Yahr scores (r = 0.58-0.71, p < 0.01), with sympathetic adrenergic function (r = -0.40 to -0.66, p < 0.01), and with parasympathetic function (r = -0.66 to -0.77, p > 0.01).. We conclude that α-synuclein deposition is increased in cutaneous sympathetic adrenergic and sympathetic cholinergic fibers but not sensory fibers of patients with PD. Higher α-synuclein deposition is associated with greater autonomic dysfunction and more advanced PD. These data suggest that measures of α-synuclein deposition in cutaneous autonomic nerves may be a useful biomarker in patients with PD.

Topics: Aged; alpha-Synuclein; Autonomic Pathways; Case-Control Studies; Female; Heart Rate; Humans; Male; Middle Aged; Nerve Fibers; Nervous System Diseases; Neurologic Examination; Parkinson Disease; Posture; Respiration; Skin; Statistics as Topic

Rapid eye movement sleep behaviour disorder (RBD) is characterized by loss of muscle atonia during rapid eye movement sleep and is associated with dream enactment behaviour. RBD is often associated with α-synuclein pathology, and we examined if there is a relationship of RBD with cholinergic neuronal loss in the pedunculopontine/laterodorsal tegmental nucleus (PPN/LDT), compared to catecholaminergic neurones in a neighbouring nucleus, the locus coeruleus (LC).. This retrospective study utilized human brain banked tissues of 11 Lewy body disease (LBD) cases with RBD, 10 LBD without RBD, 19 Alzheimer's disease (AD) and 10 neurologically normal controls. Tissues were stained with choline acetyl transferase immunohistochemistry to label neurones of PPN/LDT and tyrosine hydroxylase for the LC. The burden of tau and α-synuclein pathology was measured in the same regions with immunohistochemistry.. Both the LC and PPN/LDT were vulnerable to α-synuclein pathology in LBD and tau pathology in AD, but significant neuronal loss was only detected in these nuclei in LBD. Greater cholinergic depletion was found in both LBD groups, regardless of RBD status, when compared with normals and AD. There were no differences in either degree of neuronal loss or burden of α-synuclein pathology in LBD with and without RBD.. Whether decreases in brainstem cholinergic neurones in LBD contribute to RBD is uncertain, but our findings indicate these neurones are highly vulnerable to α-synuclein pathology in LBD and tau pathology in AD. The mechanism of selective α-synuclein-mediated neuronal loss in these nuclei remains to be determined.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Brain Stem; Catecholamines; Cholinergic Agents; Female; Humans; Lewy Body Disease; Locus Coeruleus; Male; Nervous System Diseases; Neurons; REM Sleep Behavior Disorder; Retrospective Studies

α-Synuclein (αSN) in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD) and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN), which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occurred in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are strikingly similar to those evoked by expression of human wildtype or mutant forms.

Topics: alpha-Synuclein; Animals; Axons; Blotting, Western; Brain; Female; Gene Expression; Hippocampus; Humans; Immunohistochemistry; In Situ Hybridization; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Immunoelectron; Motor Activity; Nervous System Diseases; Neuromuscular Junction; Neurons; Phosphorylation; Spinal Cord; Ubiquitin

Little is known about key pathological events preceding overt neuronal degeneration in Parkinson's disease (PD) and alpha-synucleinopathy. Recombinant adeno-associated virus 2-mediated delivery of mutant (A53T) human alpha-synuclein into the substantia nigra (SN) under a neuron-specific synapsin promoter resulted in protracted neurodegeneration with significant dopaminergic (DA) neuron loss by 17 weeks. As early as 4 weeks, there was an increase in a dopamine metabolite, DOPAC and histologically, DA axons in the striatum were dystrophic with degenerative bulbs. Before neuronal loss, significant changes were identified in levels of proteins relevant to synaptic transmission and axonal transport in the striatum and the SN. For example, striatal levels of rabphilin 3A and syntaxin were reduced. Levels of anterograde transport motor proteins (KIF1A, KIF1B, KIF2A, and KIF3A) were decreased in the striatum, whereas retrograde motor proteins (dynein, dynamitin, and dynactin1) were increased. In contrast to reduced levels in the striatum, KIF1A and KIF2A levels were elevated in the SN. There were dramatic changes in cytoskeletal protein levels, with actin levels increased and alpha-/gamma-tubulin levels reduced. In addition to these alterations, a neuroinflammatory response was observed at 8 weeks in the striatum, but not in the SN, demonstrated by increased levels of Iba-1, activated microglia and increased levels of proinflammatory cytokines, including IL-1beta, IFN-gamma and TNF-alpha. These results demonstrate that changes in proteins relevant to synaptic transmission and axonal transport coupled with neuroinflammation, precede alpha-synuclein-mediated neuronal death. These findings can provide ideas for antecedent biomarkers and presymptomatic interventions in PD.

Topics: alpha-Synuclein; Animals; Axonal Transport; Carrier Proteins; Cell Death; Dependovirus; Disease Models, Animal; Dopamine; Female; Genetic Vectors; Humans; Inflammation; Nervous System Diseases; Neurons; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Substantia Nigra

The microtubule-associated protein tau (MAPT) and alpha-synuclein (SNCA) genes play central roles in neurodegenerative disorders. Mutations in each gene cause familial disease, whereas common genetic variation at both loci contributes to susceptibility to sporadic neurodegenerative disease. Here, we demonstrate exquisite gene regulation of the human MAPT and SNCA transgene loci and functional complementation in neuronal cell cultures and organotypic brain slices using the herpes simplex virus type 1 (HSV-1) amplicon-based infectious bacterial artificial chromosome (iBAC) vector to express complete loci >100 kb. Cell cultures transduced by iBAC vectors carrying a 143 kb MAPT or 135 kb SNCA locus expressed the human loci similar to the endogenous gene. We focused on analysis of the iBAC-MAPT vector carrying the complete MAPT locus. On transduction into neuronal cultures, multiple MAPT transcripts were expressed from iBAC-MAPT under strict developmental and cell type-specific control. In primary neurons from Mapt(-/-) mice, the iBAC-MAPT vector expressed the human tau protein, as detected by enzyme-linked immunosorbent assay and immunocytochemistry, and restored sensitivity of Mapt(-/-) neurons to Abeta peptide treatment in dissociated neuronal cultures and in organotypic slice cultures. The faithful retention of gene expression and phenotype complementation by the system provides a novel method to analyze neurological disease genes.

Topics: alpha-Synuclein; Animals; Cells, Cultured; Chromosomes, Artificial, Bacterial; Genetic Therapy; Genetic Vectors; Humans; Immunohistochemistry; Mice; Nervous System Diseases; Neurons; Rats; tau Proteins; Transgenes

HtrA2/Omi is a mitochondrial serine protease that is released into the cytosol and promotes apoptotic processes by binding to several members of the inhibitors of apoptosis protein family. HtrA2/Omi knockout mice show a parkinsonian phenotype, and mutations in the gene encoding HtrA2/Omi have been identified as susceptibility factors for Parkinson disease (PD). These results suggest that HtrA2/Omi may be involved in the pathogenesis of PD. We performed immunohistochemical studies of HtrA2/Omi on brains from patients with alpha-synuclein-related disorders, including PD, dementia with Lewy bodies (DLB), and multiple-system atrophy (MSA); patients with other neurodegenerative diseases; and controls. HtrA2/Omi is expressed in normal brain tissue, and there was some anti-HtrA2/Omi immunostaining of neurons in normal brains as well as those with other neurodegenerative diseases. In PD and DLB brains, both classic (i.e. brainstem-type) and cortical Lewy bodies were intensely immunostained; pale bodies were also strongly immunopositive for HtrA2/Omi. In MSA brains, numerous glial cytoplasmic inclusions, neuronal cytoplasmic inclusions, and dystrophic neurites were also intensely immunoreactive for HtrA2/Omi. These results suggest that widespread accumulation of HtrA2/Omi may occur in pathologic alpha-synuclein-containing inclusions in brains with PD, DLB, or MSA and that HtrA2/Omi may be associated with the pathogenesis of alpha-synucleinopathies.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Animals; Autopsy; Blotting, Western; Brain; Brain Chemistry; Female; Fluorescent Antibody Technique, Indirect; High-Temperature Requirement A Serine Peptidase 2; Humans; Immunohistochemistry; Inclusion Bodies; Lewy Bodies; Lewy Body Disease; Male; Mice; Mice, Knockout; Middle Aged; Mitochondrial Proteins; Multiple System Atrophy; Nervous System Diseases; Neuroglia; Neurons; Parkinson Disease; Serine Endopeptidases

Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of "Lewy body" inclusions enriched with aggregated forms of alpha-synuclein, a presynaptic protein. Although alpha-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged alpha-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of alpha-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that alpha-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of alpha-synuclein contribute to neurologic disorders such as Parkinson's disease.

Topics: alpha-Synuclein; Amino Acid Sequence; Animals; Base Sequence; Cells, Cultured; Mitochondria; Molecular Sequence Data; Nerve Degeneration; Nervous System Diseases; Neurons; Oxidative Stress; Parkinson Disease; PC12 Cells; Protein Processing, Post-Translational; Rats; Rotenone; Tandem Mass Spectrometry

Neuronal and oligodendrocytic aggregates of fibrillar alpha-synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise viability of affected cells. Here, we report that a 12-amino acid stretch ((71)VTGVTAVAQKTV(82)) in the middle of the hydrophobic domain of human alpha-synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human beta-synuclein is highly homologous to alpha-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of alpha-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of alpha-synuclein filaments, because it is resistant to proteolytic digestion in alpha-synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human alpha-synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human alpha-synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit alpha-synuclein fibrillization and might arrest disease progression.

Topics: alpha-Synuclein; Amino Acid Sequence; beta-Synuclein; Humans; Molecular Sequence Data; Nerve Tissue Proteins; Nervous System Diseases; Peptide Fragments; Protein Binding; Protein Conformation; Recombinant Proteins; Synucleins

Recently, we have shown that the precursor of the non-Abeta component of Alzheimer's disease amyloid (NACP), also known as alpha-synuclein, is a major component of Lewy bodies (LBs) as well as neuronal and glial cytoplasmic inclusions in multiple system atrophy (MSA). To elucidate whether the accumulation of NACP is specific to LB disease and MSA, we further studied 83 autopsied cases with various neurological disorders, using anti-NACP antibodies. In LB disease, NACP immunoreactivity was present in all of the LBs and Lewy neurites in both the central and peripheral nervous systems, the pale bodies in the substantia nigra, and dystrophic neurites in the hippocampal CA2/3 region. Immunoelectron microscopy revealed that the reaction product was localized within filamentous structures and associated granular structures. In MSA, NACP immunoreactivity was found in the intracytoplasmic inclusions of both neuronal and oligodendroglial cells, neuronal intranuclear inclusions, and swollen neuronal processes. No NACP immunoreactivity was found in a variety of other neuronal or glial inclusions in other disorders, including Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, motor neuron disease and triplet-repeat diseases. These findings strongly suggest that the accumulation of NACP is a cytopathological feature common to LB disease and MSA.

Topics: alpha-Synuclein; Cellular Senescence; Humans; Immunohistochemistry; Lewy Bodies; Microscopy, Immunoelectron; Multiple System Atrophy; Nerve Degeneration; Nerve Tissue Proteins; Nervous System Diseases; Parkinson Disease; Synucleins