Multiple System Atrophy Brains Research Articles

Comparing alpha-synuclein-interactomes between multiple systems atrophy and Parkinson's disease reveals unique and shared pathological features.

Primary synucleinopathies, such as Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are neurodegenerative disorders with some shared clinical and pathological features. Aggregates of alpha-synuclein (αsyn) phosphorylated at serine 129 (PSER129) are the hallmark of synucleinopathies, which for PD/DLB are found predominantly in neurons (Neuronal cytoplasmic inclusions "NCIs"), but for MSA, aggregates are primarily found in oligodendroglia (Glial cytoplasmic inclusions "GCIs"). It remains unclear if the distinct pathological presentation of PD/DLB and MSA are manifestations of distinct or shared pathological processes. We hypothesize that the distinct synucleinopathies MSA and PD/DLB share common molecular features. Using the in-situ proximity labeling technique biotinylation by antibody recognition (BAR), we compare aggregated αsyn-interactomes (BAR-PSER129) and total αsyn-interactomes (BAR-MJFR1) between MSA (n=5) and PD/DLB (n=10) in forebrain and midbrain structures. For BAR-PSER129 and BAR-MJFR1 captures, αsyn was the most significantly enriched protein in PD/DLB and MSA. In PD/DLB, BAR-PSER129 identified 194 αsyn-aggregate-interacting proteins, while BAR-MJFR1 identified 245 αsyn interacting proteins. In contrast, in the MSA brain, only 38 and 175 proteins were identified for each capture, respectively. When comparing MSA and PD/DLB, a high overlap (59.5%) was observed between BAR-MJFR1 captured proteins, whereas less overlap (14.4%) was observed for BAR-PSER129. Direct comparison between MSA and PD/DLB revealed 79 PD/DLB-associated proteins and only three MSA-associated proteins (CBR1, CRYAB, and GFAP). Pathway enrichment analysis revealed PD/DLB interactions were dominated by vesicle/SNARE-associated pathways, in contrast to MSA, which strongly enriched for metabolic/catabolic, iron, and cellular oxidant detoxification pathways. A subnetwork of cytosolic antioxidant enzymes called peroxiredoxins drove cellular detoxification pathways in MSA. A common network of 26 proteins, including neuronal-specific proteins (e.g., SNYGR3) with HSPA8 at the core, was shared between MSA and DLB/PD. Extracellular exosome pathways were universally enriched regardless of disease or BAR target protein. Synucleinopathies have divergent and convergent αsyn-aggregate interactions, indicating unique and shared pathogenic mechanisms. MSA uniquely involves oxidant detoxification processes in glial cells, while vesicular processes in neurons dominate PD/DLB. Shared interactions, specifically SNYGR3 (i.e., a neuronal protein), between MSA and PD/DLB suggest neuronal axons origin for both diseases. In conclusion, we provide αsyn aggregates protein interaction maps for two distinct synucleinopathies.

DNAJB6 is expressed in neurons and oligodendrocytes of the human brain.

DNAJB6 is a suppressor of α-synuclein aggregation in vivo and in vitro. DNAJB6 is strongly expressed in the brain, and its overall protein expression is altered in neurodegenerative conditions such as Parkinson's Disease (PD) and Multiple System Atrophy (MSA). These two diseases are characterized by accumulation of aggregated α-synuclein in neurons and oligodendrocytes, respectively. To further explore this, we employed post-mortem normal human brain material to investigate the regional and cell type specific protein expression of DNAJB6. We found that the DNAJB6 protein is ubiquitously expressed across various regions of the brain. Notably, we demonstrate for the first time that DNAJB6 is present in nearly half (41%-53%) of the oligodendrocyte population and in the majority (68%-80%) of neurons. However, DNAJB6 was only sparsely present in other cell types such as astrocytes and microglia. Given that α-synuclein aggregation in oligodendrocytes is a hallmark of MSA, we investigated DNAJB6 presence in MSA brains compared to control brains. We found no significant difference in the percentage of oligodendrocytes where DNAJB6 was present in MSA brains relative to control brains. In conclusion, our results reveal an expression of the DNAJB6 protein across various regions of the human brain, and that DNAJB6 is almost exclusively present in neurons and oligodendrocytes. Since prior studies have shown that PD and MSA brains have altered levels of DNAJB6 relative to control brains, DNAJB6 may be an interesting target for drug development.

A novel mouse model for investigating α-synuclein aggregates in oligodendrocytes: implications for the glial cytoplasmic inclusions in multiple system atrophy

The aggregated alpha-synuclein (αsyn) in oligodendrocytes (OLGs) is one of the pathological hallmarks in multiple system atrophy (MSA). We have previously reported that αsyn accumulates not only in neurons but also in OLGs long after the administration of αsyn preformed fibrils (PFFs) in mice. However, detailed spatial and temporal analysis of oligodendroglial αsyn aggregates was technically difficult due to the background neuronal αsyn aggregates. The aim of this study is to create a novel mouse that easily enables sensitive and specific detection of αsyn aggregates in OLGs and the comparable analysis of the cellular tropism of αsyn aggregates in MSA brains. To this end, we generated transgenic (Tg) mice expressing human αsyn-green fluorescent protein (GFP) fusion proteins in OLGs under the control of the 2’, 3’-cyclic nucleotide 3’-phosphodiesterase (CNP) promoter (CNP-SNCAGFP Tg mice). Injection of αsyn PFFs in these mice induced distinct GFP-positive aggregates in the processes of OLGs as early as one month post-inoculation (mpi), and their number and size increased in a centripetal manner. Moreover, MSA-brain homogenates (BH) induced significantly more oligodendroglial αsyn aggregates than neuronal αsyn aggregates compared to DLB-BH in CNP-SNCAGFP Tg mice, suggestive of their potential tropism of αsyn seeds for OLGs. In conclusion, CNP-SNCAGFP Tg mice are useful for studying the development and tropism of αsyn aggregates in OLGs and could contribute to the development of therapeutics targeting αsyn aggregates in OLGs.

Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

Multiple System Atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.

Role of complement activation and disruption of the blood–brain barrier in the pathogenesis of multiple system atrophy

Multiple system atrophy (MSA) is a progressive and sporadic neurodegenerative disorder characterized by the histological appearance of glial cytoplasmic inclusions primarily composed of α-synuclein. Recently, complement-mediated neuroinflammation has been proposed as a key factor in the pathogenesis of numerous neurodegenerative disorders. We conducted immunohistochemical/immunofluorescent assays targeting a number of complements to explore the role of complements in MSA pathogenesis using brain samples from deceased patients and controls. Complement deposition was notably increased in the cerebral vasculature and myelin sheath in the MSA brains. Furthermore, fibrinogen leakage resulting from the disruption of the blood–brain barrier (BBB) was observed, along with the presence of C1q-positive microglia clusters surrounding the MSA brain vessels. These immunohistochemical/immunofluorescent findings suggest that complement activation and BBB disruption play critical roles in MSA progression.

Understanding the Role of CDH4 in Multiple System Atrophy Brain.

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease clinically characterized by parkinsonism, cerebellar ataxia, and autonomic dysfunction. A major pathological feature of MSA is the presence of α-synuclein aggregates in oligodendrocytes, the myelinating cells of the central nervous system. A genome-wide association study revealed that the CDH4 gene is associated with MSA. However, virtually nothing is known about the role of CDH4 in the context of MSA. Our aim was to compare the expression of CDH4 between MSA and control brains, and to investigate its relationship with α-synuclein in oligodendrocytes. RNA and protein were prepared from putamen, motor cortex white matter, cerebellum, and superior occipital cortex tissues collected from MSA (N = 11) and control (N = 13) brains. The expression of CDH4 was measured at mRNA and protein levels by qPCR and western blotting. Oligodendrocyte cells were cultured on plates and transfected with CDH4 cDNA and its impact on α-synuclein was analyzed. Firstly, we found that CDH4 in MSA brain was significantly elevated in the disease-affected motor cortex white matter in MSA (N = 11) compared to controls (N = 13) and unaltered in the disease-unaffected superior occipital cortex. Secondly, we determined that increases in CDH4 expression caused changes in the cellular levels of α-synuclein in oligodendrocytes. When put together, these results provide evidence that support the GWAS association of CDH4 with MSA.

Quantitative cellular changes in multiple system atrophy brains.

Multiple system atrophy (MSA) is a neurodegenerative disorder characterised by a combined symptomatology of parkinsonism, cerebellar ataxia, autonomic failure and corticospinal dysfunction. In brains of MSA patients, the hallmark lesion is the aggregation of misfolded alpha-synuclein in oligodendrocytes. Even though the underlying pathological mechanisms remain poorly understood, the evidence suggests that alpha-synuclein aggregation in oligodendrocytes may contribute to the neurodegeneration seen in MSA. The primary aim of this review is to summarise the published stereological data on the total number of neurons and glial cell subtypes (oligodendrocytes, astrocytes and microglia) and volumes in brains from MSA patients. Thus, we include in this review exclusively the reports of unbiased quantitative data from brain regions including the neocortex, nuclei of the cerebrum, the brainstem and the cerebellum. Furthermore, we compare and discuss the stereological results in the context of imaging findings and MSA symptomatology. In general, the stereological results agree with the common neuropathological findings of neurodegeneration and gliosis in brains from MSA patients and support a major loss of nigrostriatal neurons in MSA patients with predominant parkinsonism (MSA-P), as well as olivopontocerebellar atrophy in MSA patients with predominant cerebellar ataxia (MSA-C). Surprisingly, the reports indicate only a minor loss of oligodendrocytes in sub-cortical regions of the cerebrum (glial cells not studied in the cerebellum) and negligible changes in brain volumes. In the past decades, the use of stereological methods has provided a vast amount of accurate information on cell numbers and volumes in the brains of MSA patients. Combining different techniques such as stereology and diagnostic imaging (e.g. MRI, PET and SPECT) with clinical data allows for a more detailed interdisciplinary understanding of the disease and illuminates the relationship between neuropathological changes and MSA symptomatology.

The G51D SNCA mutation generates a slowly progressive α-synuclein strain in early-onset Parkinson’s disease

Unique strains of α-synuclein aggregates have been postulated to underlie the spectrum of clinical and pathological presentations seen across the synucleinopathies. Whereas multiple system atrophy (MSA) is associated with a predominance of oligodendroglial α-synuclein inclusions, α-synuclein aggregates in Parkinson’s disease (PD) preferentially accumulate in neurons. The G51D mutation in the SNCA gene encoding α-synuclein causes an aggressive, early-onset form of PD that exhibits clinical and neuropathological traits reminiscent of both PD and MSA. To assess the strain characteristics of G51D PD α-synuclein aggregates, we performed propagation studies in M83 transgenic mice by intracerebrally inoculating patient brain extracts. The properties of the induced α-synuclein aggregates in the brains of injected mice were examined using immunohistochemistry, a conformational stability assay, and by performing α-synuclein seed amplification assays. Unlike MSA-injected mice, which developed a progressive motor phenotype, G51D PD-inoculated animals remained free of overt neurological illness for up to 18 months post-inoculation. However, a subclinical synucleinopathy was present in G51D PD-inoculated mice, characterized by the accumulation of α-synuclein aggregates in restricted regions of the brain. The induced α-synuclein aggregates in G51D PD-injected mice exhibited distinct properties in a seed amplification assay and were much more stable than those present in mice injected with MSA extract, which mirrored the differences observed between human MSA and G51D PD brain samples. These results suggest that the G51D SNCA mutation specifies the formation of a slowly propagating α-synuclein strain that more closely resembles α-synuclein aggregates associated with PD than MSA.

Epsin2, a novel target for multiple system atrophy therapy via α-synuclein/FABP7 propagation.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by the accumulation of misfolded α-synuclein (αSyn) and myelin disruption. However, the mechanism underlying αSyn accumulation in MSA brains remains unclear. Here, we aimed to identify epsin-2 as a potential regulator of αSyn propagation in MSA brains. In the MSA mouse model, PLP-hαSyn mice, and FABP7/αSyn hetero-aggregate-injected mice, we initially discovered that fatty acid-binding protein 7 (FABP7) is related to MSA development and forms hetero-aggregates with αSyn, which exhibit stronger toxicity than αSyn aggregates. Moreover, the injected FABP7/αSyn hetero-aggregates in mice selectively accumulated only in oligodendrocytes and Purkinje neurons, causing cerebellar dysfunction. Furthermore, bioinformatic analyses of whole blood from MSA patients and FABP7 knockdown mice revealed that epsin-2, a protein expressed in both oligodendrocytes and Purkinje cells, could potentially regulate FABP7/αSyn hetero-aggregate propagation via clathrin-dependent endocytosis. Lastly, adeno-associated virus type 5-dependent epsin-2 knockdown mice exhibited decreased levels of αSyn aggregate accumulation in Purkinje neurons and oligodendrocytes, as well as improved myelin levels and Purkinje neuron function in the cerebellum and motor performance. These findings suggest that epsin-2 plays a significant role in αSyn accumulation in MSA, and we propose epsin-2 as a novel therapeutic target for MSA.

Epigenetic age acceleration is associated with oligodendrocyte proportions in MSA and control brain tissue.

Epigenetic clocks are widely applied as surrogates for biological age in different tissues and/or diseases, including several neurodegenerative diseases. Despite white matter (WM) changes often being observed in neurodegenerative diseases, no study has investigated epigenetic ageing in white matter. We analysed the performances of two DNA methylation-based clocks, DNAmClockMulti and DNAmClockCortical , in post-mortem WM tissue from multiple subcortical regions and the cerebellum, and in oligodendrocyte-enriched nuclei. We also examined epigenetic ageing in control and multiple system atrophy (MSA) (WM and mixed WM and grey matter), as MSA is a neurodegenerative disease comprising pronounced WM changes and α-synuclein aggregates in oligodendrocytes. Estimated DNA methylation (DNAm) ages showed strong correlations with chronological ages, even in WM (e.g., DNAmClockCortical , r = [0.80-0.97], p < 0.05). However, performances and DNAm age estimates differed between clocks and brain regions. DNAmClockMulti significantly underestimated ages in all cohorts except in the MSA prefrontal cortex mixed tissue, whereas DNAmClockCortical tended towards age overestimations. Pronounced age overestimations in the oligodendrocyte-enriched cohorts (e.g., oligodendrocyte-enriched nuclei, p = 6.1 × 10-5 ) suggested that this cell type ages faster. Indeed, significant positive correlations were observed between estimated oligodendrocyte proportions and DNAm age acceleration estimated by DNAmClockCortical (r > 0.31, p < 0.05), and similar trends were obtained with DNAmClockMulti . Although increased age acceleration was observed in MSA compared with controls, no significant differences were detected upon adjustment for possible confounders (e.g., cell-type proportions). Our findings show that oligodendrocyte proportions positively influence epigenetic age acceleration across brain regions and highlight the need to further investigate this in ageing and neurodegeneration.

Phosphorylation of Tau at Threonine 231 in Patients With Multiple System Atrophy and in a Mouse Model.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder pathologically characterized by the presence of glial cytoplasmic inclusions (GCIs). Some MSA patients exhibit motor deficits with accompanying cognitive impairment. Of note, some patients suffering from MSA with longer disease duration have AT8-positive signals, which correspond to phosphorylated tau (P-tau) at 202/205 (P-tau202/205). However, P-tau sites other than the AT8 antibody epitope antibody are less well studied. Here, we focused on the effect of α-synuclein (Syn) expression on the phosphorylation of tau in MSA model mice. Among the 6 kinds of antibodies against P-tau, we confirmed that antibodies against P-tau at 231 (P-tau231) were phospho-specific and found that P-tau231 level was increased in parallel with disease progression in MSA model mice. Additional studies of human brains revealed that P-tau231 was mainly expressed in the temporal cortex in MSA brains and that its expression level was significantly higher in MSA patients than in controls. Immunohistochemical analysis showed that anti-P-tau231-, but not AT8, antibodies mainly immunolabeled hippocampal CA2/3 pyramidal neurons, and some GCIs in MSA. These data suggest that P-tau231 occurs in MSA differently from P-tau202/205.

Pathological substrate of memory impairment in multiple system atrophy.

Synaptic dysfunction in Parkinson's disease is caused by propagation of pathogenic α-synuclein between neurons. Previously, in multiple system atrophy (MSA), pathologically characterised by ectopic deposition of abnormal α-synuclein predominantly in oligodendrocytes, we demonstrated that the occurrence of memory impairment was associated with the number of α-synuclein-positive neuronal cytoplasmic inclusions (NCIs) in the hippocampus. In the present study, we aimed to investigate how abnormal α-synuclein in the hippocampus can lead to memory impairment. We performed pathological and biochemical analyses using a mouse model of adult-onset MSA and human cases (MSA, N = 25; Parkinson's disease, N = 3; Alzheimer's disease, N = 2; normal controls, N = 11). In addition, the MSA model mice were examined behaviourally and physiologically. In the MSA model, inducible human α-synuclein was first expressed in oligodendrocytes and subsequently accumulated in the cytoplasm of excitatory hippocampal neurons (NCI-like structures) and their presynaptic nerve terminals with the development of memory impairment. α-Synuclein oligomers increased simultaneously in the hippocampus of the MSA model. Hippocampal dendritic spines also decreased in number, followed by suppression of long-term potentiation. Consistent with these findings obtained in the MSA model, post-mortem analysis of human MSA brain tissues showed that cases of MSA with memory impairment developed more NCIs in excitatory hippocampal neurons along with α-synuclein oligomers than those without. Our results provide new insights into the role of α-synuclein oligomers as a possible pathological cause of memory impairment in MSA.

Brain proteome profiling implicates the complement and coagulation cascade in multiple system atrophy brain pathology.

BackgroundMultiple system atrophy (MSA) is a rare, progressive, neurodegenerative disorder presenting glia pathology. Still, disease etiology and pathophysiology are unknown, but neuro-inflammation and vascular disruption may be contributing factors to the disease progression. Here, we performed an ex vivo deep proteome profiling of the prefrontal cortex of MSA patients to reveal disease-relevant molecular neuropathological processes. Observations were validated in plasma and cerebrospinal fluid (CSF) of novel cross-sectional patient cohorts.MethodsBrains from 45 MSA patients and 30 normal controls (CTRLs) were included. Brain samples were homogenized and trypsinized for peptide formation and analyzed by high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS). Results were supplemented by western blotting, immuno-capture, tissue clearing and 3D imaging, immunohistochemistry and immunofluorescence. Subsequent measurements of glial fibrillary acid protein (GFAP) and neuro-filament light chain (NFL) levels were performed by immunoblotting in plasma of 20 MSA patients and 20 CTRLs. Finally, we performed a proteome profiling of 144 CSF samples from MSA and CTRLs, as well as other parkinsonian disorders. Data were analyzed using relevant parametric and non-parametric two-sample tests or linear regression tests followed by post hoc tests corrected for multiple testing. Additionally, high-throughput bioinformatic analyses were applied.ResultsWe quantified more than 4,000 proteins across samples and identified 49 differentially expressed proteins with significantly different abundances in MSA patients compared with CTRLs. Pathway analyses showed enrichment of processes related to fibrinolysis and complement cascade activation. Increased fibrinogen subunit β (FGB) protein levels were further verified, and we identified an enriched recognition of FGB by IgGs as well as intra-parenchymal accumulation around blood vessels. We corroborated blood–brain barrier leakage by a significant increase in GFAP and NFL plasma levels in MSA patients that correlated to disease severity and/or duration. Proteome profiling of CSF samples acquired during the disease course, confirmed increased total fibrinogen levels and immune-related components in the soluble fraction of MSA patients. This was also true for the other atypical parkinsonian disorders, dementia with Lewy bodies and progressive supra-nuclear palsy, but not for Parkinson’s disease patients.ConclusionOur results implicate activation of the fibrinolytic cascade and immune system in the brain as contributing factors in MSA associated with a more severe disease course.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-022-04378-z.

Autophagy in α-Synucleinopathies-An Overstrained System.

Alpha-synucleinopathies comprise progressive neurodegenerative diseases, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). They all exhibit the same pathological hallmark, which is the formation of α-synuclein positive deposits in neuronal or glial cells. The aggregation of α-synuclein in the cell body of neurons, giving rise to the so-called Lewy bodies (LBs), is the major characteristic for PD and DLB, whereas the accumulation of α-synuclein in oligodendroglial cells, so-called glial cytoplasmic inclusions (GCIs), is the hallmark for MSA. The mechanisms involved in the intracytoplasmic inclusion formation in neuronal and oligodendroglial cells are not fully understood to date. A possible mechanism could be an impaired autophagic machinery that cannot cope with the high intracellular amount of α-synuclein. In fact, different studies showed that reduced autophagy is involved in α-synuclein aggregation. Furthermore, altered levels of different autophagy markers were reported in PD, DLB, and MSA brains. To date, the trigger point in disease initiation is not entirely clear; that is, whether autophagy dysfunction alone suffices to increase α-synuclein or whether α-synuclein is the pathogenic driver. In the current review, we discuss the involvement of defective autophagy machinery in the formation of α-synuclein aggregates, propagation of α-synuclein, and the resulting neurodegenerative processes in α-synucleinopathies.

Is Multiple System Atrophy a Prion-like Disorder?

Multiple system atrophy (MSA) is a rapidly progressive, fatal neurodegenerative disease of uncertain aetiology that belongs to the family of α-synucleinopathies. It clinically presents with parkinsonism, cerebellar, autonomic, and motor impairment in variable combinations. Pathological hallmarks are fibrillary α-synuclein (αSyn)-rich glial cytoplasmic inclusions (GCIs) mainly involving oligodendroglia and to a lesser extent neurons, inducing a multisystem neurodegeneration, glial activation, and widespread demyelinization. The neuronal αSyn pathology of MSA has molecular properties different from Lewy bodies in Parkinson’s disease (PD), both of which could serve as a pool of αSyn (prion) seeds that could initiate and drive the pathogenesis of synucleinopathies. The molecular cascade leading to the “prion-like” transfer of “strains” of aggregated αSyn contributing to the progression of the disease is poorly understood, while some presented evidence that MSA is a prion disease. However, this hypothesis is difficult to reconcile with postmortem analysis of human brains and the fact that MSA-like pathology was induced by intracerebral inoculation of human MSA brain homogenates only in homozygous mutant 53T mice, without production of disease-specific GCIs, or with replication of MSA prions in primary astrocyte cultures from transgenic mice expressing human αSyn. Whereas recent intrastriatal injection of Lewy body-derived or synthetic human αSyn fibrils induced PD-like pathology including neuronal αSyn aggregates in macaques, no such transmission of αSyn pathology in non-human primates by MSA brain lysate has been reported until now. Given the similarities between αSyn and prions, there is a considerable debate whether they should be referred to as “prions”, “prion-like”, “prionoids”, or something else. Here, the findings supporting the proposed nature of αSyn as a prion and its self-propagation through seeding as well as the transmissibility of neurodegenerative disorders are discussed. The proof of disease causation rests on the concordance of scientific evidence, none of which has provided convincing evidence for the classification of MSA as a prion disease or its human transmission until now.

Robust \u03b1-synuclein pathology in select brainstem neuronal populations is a potential instigator of multiple system atrophy

Multiple system atrophy (MSA) is an insidious middle age-onset neurodegenerative disease that clinically presents with variable degrees of parkinsonism and cerebellar ataxia. The pathological hallmark of MSA is the progressive accumulation of glial cytoplasmic inclusions (GCIs) in oligodendrocytes that are comprised of α-synuclein (αSyn) aberrantly polymerized into fibrils. Experimentally, MSA brain samples display a high level of seeding activity to induce further αSyn aggregation by a prion-like conformational mechanism. Paradoxically, αSyn is predominantly a neuronal brain protein, with only marginal levels expressed in normal or diseased oligodendrocytes, and αSyn inclusions in other neurodegenerative diseases, including Parkinson’s disease and Dementia with Lewy bodies, are primarily found in neurons. Although GCIs are the hallmark of MSA, using a series of new monoclonal antibodies targeting the carboxy-terminal region of αSyn, we demonstrate that neuronal αSyn pathology in MSA patient brains is remarkably abundant in the pontine nuclei and medullary inferior olivary nucleus. This neuronal αSyn pathology has distinct histological properties compared to GCIs, which allows it to remain concealed to many routine detection methods associated with altered biochemical properties of the carboxy-terminal domain of αSyn. We propose that these previously underappreciated sources of aberrant αSyn could serve as a pool of αSyn prion seeds that can initiate and continue to drive the pathogenesis of MSA.

Extracellular Alpha-Synuclein Promotes a Neuroinhibitory Secretory Phenotype in Astrocytes.

Multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are α-synucleinopathies that exhibit widespread astrogliosis as a component of the neuroinflammatory response. Munc18, a protein critical to vesicle exocytosis, was previously found to strongly mark morphologically activated astrocytes in brain tissue of MSA patients. Immunofluorescence of MSA, DLB and normal brain tissue sections was combined with cell culture and co-culture experiments to investigate the relationship between extracellular α-synuclein and the transition to a secretory astrocyte phenotype. Increased Munc18-positive vesicles were resolved in activated astrocytes in MSA and DLB tissue compared to controls, and they were also significantly upregulated in the human 1321N1 astrocytoma cell line upon treatment with α-synuclein, with parallel increases in GFAP expression and IL-6 secretion. In co-culture experiments, rat primary astrocytes pretreated with α-synuclein inhibited the growth of neurites of co-cultured primary rat neurons and upregulated chondroitin sulphate proteoglycan. Taken together, these results indicate that the secretory machinery is significantly upregulated in the astrocyte response to extracellular α-synuclein and may participate in the release of neuroinhibitory and proinflammatory factors in α-synucleinopathies.

BCAS1-positive immature oligodendrocytes are affected by the \u03b1-synuclein-induced pathology of multiple system atrophy

Multiple system atrophy (MSA) is pathologically characterized by the presence of fibrillar α-synuclein-immunoreactive inclusions in oligodendrocytes. Although the myelinating process of oligodendrocytes can be observed in adult human brains, little is known regarding the presence of α-synuclein pathology in immature oligodendrocytes and how their maturation and myelination are affected in MSA brains. Recently, breast carcinoma amplified sequence 1 (BCAS1) has been found to be specifically expressed in immature oligodendrocytes undergoing maturation and myelination. Here, we analyzed the altered dynamics of oligodendroglial maturation in both MSA brains and primary oligodendroglial cell cultures which were incubated with α-synuclein pre-formed fibrils. The numbers of BCAS1-expressing oligodendrocytes that displayed a matured morphology negatively correlated with the density of pathological inclusions in MSA brains but not with that in Parkinson’s disease and diffuse Lewy body disease. In addition, a portion of the BCAS1-expressing oligodendrocyte population showed cytoplasmic inclusions, which were labeled with antibodies against phosphorylated α-synuclein and cleaved caspase-9. Further in vitro examination indicated that the α-synuclein pre-formed fibrils induced cytoplasmic inclusions in the majority of BCAS1-expressing oligodendrocytes. In contrast, the majority of BCAS1-non-expressing mature oligodendrocytes did not develop inclusions on day 4 after maturation induction. Furthermore, exposure of α-synuclein pre-formed fibrils in the BCAS1-positive phase caused a reduction in oligodendroglial cell viability. Our results indicated that oligodendroglial maturation and myelination are impaired in the BCAS1-positive phase of MSA brains, which may lead to the insufficient replacement of defective oligodendrocytes. In vitro, the high susceptibility of BCAS1-expressing primary oligodendrocytes to the extracellular α-synuclein pre-formed fibrils suggests the involvement of insufficient oligodendroglial maturation in MSA disease progression and support the hypothesis that the BCAS1-positive oligodendrocyte lineage cells are prone to take up aggregated α-synuclein in vivo.

Gut Microbiome-Modified Polyphenolic Compounds Inhibit α-Synuclein Seeding and Spreading in α-Synucleinopathies

Misfolding, aggregation and deposition of α-synuclein (α-syn) are major pathologic characteristics of Parkinson’s disease (PD) and the related synucleinopathy, multiple system atrophy (MSA). The spread of α-syn pathology across brain regions is thought to play a key role in the onset and progression of clinical phenotypes. Thus, there is increasing interest in developing strategies that target and attenuate α-syn aggregation and spread. Recent studies of brain-penetrating polyphenolic acids, namely, 3-hydroxybenzoic acid (3-HBA), 3,4-dihydroxybenzoic acid (3,4-diHBA), and 3-(3-hydroxyphenyl)propionic acid (3-HPPA) that are derived from gut microbiota metabolism of dietary polyphenols, show in vitro ability to effectively modulate α-syn misfolding, oligomerization, and mediate aggregated α-syn neurotoxicity. Here we investigate whether 3-HBA, 4-hydroxybenzoic acid (4-HBA), 3,4-diHBA, or 3-HPPA interfere with α-syn spreading in a cell-based system. Using HEK293 cells overexpressing α-syn-A53T-CFP/YFP, we assessed α-syn seeding activity using Fluorescence Resonance Energy Transfer (FRET) to detect and quantify α-syn aggregation. We demonstrated that 3-HPPA, 3,4-diHBA, 3-HBA, and 4-HBA significantly attenuated intracellular α-syn seeding aggregation. To determine whether our compounds could inhibit brain-derived seeding activity, we utilized insoluble α-syn extracted from post-mortem MSA or PD brain specimens. We found that 3-HPPA effectively attenuated MSA-induced aggregation of monomer into high molecular weight aggregates capable of inducing intracellular aggregation. Outcomes from our studies suggest interactions between gut microbiome and certain dietary factors may form the basis for effective therapies that modulate pathologic α-syn propagation. Collectively, our findings provide the basis for future developments of probiotic, prebiotic, or synbiotic approaches for modulating the onset and/or progression of α-synucleinopathies.

α-Synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.