Project description:Multiple system atrophy is a progressive, neurodegenerative disease characterized by parkinsonism, ataxia, autonomic dysfunction, and accumulation of α-synuclein in oligodendrocytes. To understand how α-synuclein aggregates impact oligodendroglial homeostasis, we investigated an oligodendroglial cell model of α-synuclein dependent degeneration and identified responses linked to the NF-κB transcription factor stress system. Coexpression of human α-synuclein and the oligodendroglial protein p25α increased the expression of IκBα mRNA and protein early during the degenerative process and this was dependent on both aggregation and Ser129 phosphorylation of α-synuclein. This response was prodegenerative because blocking IκBα expression by siRNA rescued the cells. IκBα is an inhibitor of NF-κB and acts by binding and retaining NF-κB p65 in the cytoplasm. The protection obtained by silencing IκBα was accompanied by a strong increase in nuclear p65 translocation indicating that NF-κB activation protects against α-synuclein aggregate stress. In the cellular model, two different phenotypes were observed; degenerating cells retracting their microtubules and resilient cells tolerating the coexpression of α-synuclein and p25α. The resilient cells displayed a significant higher nuclear translocation of p65 and activation of the NF-κB system relied on stress elicited by aggregated and Ser129 phosphorylated α-synuclein. To validate the relationship between oligodendroglial α-synuclein expression and IκBα, we analyzed two different lines of transgenic mice expressing human α-synuclein under the control of the oligodendrocytic MBP promotor (intermediate-expresser line 1 and high-expresser line 29). IκBα mRNA expression was increased in both lines and immunofluorescence microscopy and in situ hybridization revealed that IκBα mRNA and protein is expressed in oligodendrocytes. IκBα mRNA expression was demonstrated prior to activation of microglia and astrocytes in line 1. Human brain tissue affected by MSA displayed increased expression of IκBα and NF-κB p65 in some oligodendrocytes containing glial cytoplasmic inclusions. Our data suggest that oligodendroglial IκBα expression and NF-κB are activated early in the course of MSA and their balance contributes to the decision of cellular demise. Favoring oligodendroglial NF-κB activation may represent a therapeutic strategy for this devastating disease.

Project description:Multiple system atrophy (MSA) is characterized by the presence of distinctive glial cytoplasmic inclusions (GCIs) within oligodendrocytes that contain the neuronal protein alpha-synuclein (aSyn) and the oligodendroglia-specific phosphoprotein TPPP/p25α. However, the role of oligodendroglial aSyn and p25α in the formation of aSyn-rich GCIs remains unclear. To address this conundrum, we have applied human aSyn (haSyn) pre-formed fibrils (PFFs) to rat wild-type (WT)-, haSyn-, or p25α-overexpressing oligodendroglial cells and to primary differentiated oligodendrocytes derived from WT, knockout (KO)-aSyn, and PLP-haSyn-transgenic mice. HaSyn PFFs are readily taken up by oligodendroglial cells and can recruit minute amounts of endogenous aSyn into the formation of insoluble, highly aggregated, pathological assemblies. The overexpression of haSyn or p25α accelerates the recruitment of endogenous protein and the generation of such aberrant species. In haSyn PFF-treated primary oligodendrocytes, the microtubule and myelin networks are disrupted, thus recapitulating a pathological hallmark of MSA, in a manner totally dependent upon the seeding of endogenous aSyn. Furthermore, using oligodendroglial and primary cortical cultures, we demonstrated that pathology-related S129 aSyn phosphorylation depends on aSyn and p25α protein load and may involve different aSyn "strains" present in oligodendroglial and neuronal synucleinopathies. Importantly, this hypothesis was further supported by data obtained from human post-mortem brain material derived from patients with MSA and dementia with Lewy bodies. Finally, delivery of haSyn PFFs into the mouse brain led to the formation of aberrant aSyn forms, including the endogenous protein, within oligodendroglia and evoked myelin decompaction in WT mice, but not in KO-aSyn mice. This line of research highlights the role of endogenous aSyn and p25α in the formation of pathological aSyn assemblies in oligodendrocytes and provides in vivo evidence of the contribution of oligodendroglial aSyn in the establishment of aSyn pathology in MSA.

Project description:Accumulation of neuronal α-synuclein is a prominent feature in Parkinson's disease. More recently, such abnormal protein aggregation has been reported to spread from cell to cell and exosomes are considered as important mediators. The focus of such research, however, has been primarily in neurons. Given the increasing recognition of the importance of non-cell autonomous-mediated neurotoxicity, it is critical to investigate the contribution of glia to α-synuclein aggregation and spread. Microglia are the primary phagocytes in the brain and have been well-documented as inducers of neuroinflammation. How and to what extent microglia and their exosomes impact α-synuclein pathology has not been well delineated. We report here that when treated with human α-synuclein preformed fibrils, exosomes containing α-synuclein released by microglia are fully capable of inducing protein aggregation in the recipient neurons. Additionally, when combined with microglial proinflammatory cytokines, these exosomes further increased protein aggregation in neurons. Inhibition of exosome synthesis in microglia reduced α-synuclein transmission. The in vivo significance of these exosomes was demonstrated by stereotaxic injection of exosomes isolated from α-synuclein preformed fibrils treated microglia into the mouse striatum. Phosphorylated α-synuclein was observed in multiple brain regions consistent with their neuronal connectivity. These animals also exhibited neurodegeneration in the nigrostriatal pathway in a time-dependent manner. Depleting microglia in vivo dramatically suppressed the transmission of α-synuclein after stereotaxic injection of preformed fibrils. Mechanistically, we report here that α-synuclein preformed fibrils impaired autophagy flux by upregulating PELI1, which in turn, resulted in degradation of LAMP2 in activated microglia. More importantly, by purifying microglia/macrophage derived exosomes in the CSF of Parkinson's disease patients, we confirmed the presence of α-synuclein oligomer in CD11b+ exosomes, which were able to induce α-synuclein aggregation in neurons, further supporting the translational aspect of this study. Taken together, our study supports the view that microglial exosomes contribute to the progression of α-synuclein pathology and therefore, they may serve as a promising therapeutic target for Parkinson's disease.

Project description:Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions. We also identify a KFERQ-like motif in the TPPP/p25A sequence that enables its effective degradation via chaperone-mediated autophagy (CMA) in an in vitro system of rat brain lysosomes. Furthermore, in a MSA-like setting established by addition of human recombinant SNCA pre-formed fibrils (PFFs) as seeds of pathological SNCA, we thoroughly characterize the contribution of CMA and macroautophagy in particular, in the removal of the exogenously added and the seeded oligodendroglial SNCA pathological assemblies. We also show that PFF treatment impairs autophagic flux and that TPPP/p25A exerts an inhibitory effect on macroautophagy, while at the same time CMA is upregulated to remove the pathological SNCA species formed within oligodendrocytes. Finally, augmentation of CMA or macroautophagy accelerates the removal of the engendered pathological SNCA conformations further suggesting that autophagy targeting may represent a successful approach for the clearance of pathological SNCA and/or TPPP/p25A in the context of MSA.Abbreviations: 3MA: 3-methyladenine; ACTB: actin, beta; ALP: autophagy-lysosome pathway; ATG5: autophagy related 5; AR7: atypical retinoid 7; CMA: chaperone-mediated autophagy; CMV: cytomegalovirus; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; Epox: epoxomicin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCIs: glial cytoplasmic inclusions; GFP: green fluorescent protein; HMW: high molecular weight; h: hours; HSPA8/HSC70: heat shock protein 8; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mcherry: monomeric cherry; MFI: mean fluorescence intensity; mRFP: monomeric red fluorescent protein; MSA: multiple system atrophy; OLN: oligodendrocytes; OPCs: oligodendroglial progenitor cells; PBS: phosphate-buffered saline; PC12: pheochromocytoma cell line; PD: Parkinson disease; PFFs: pre-formed fibrils; PIs: protease inhibitors; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; Rap: rapamycin; RFP: red fluorescent protein; Scr: scrambled; SDS: sodium dodecyl sulfate; SE: standard error; siRNAs: small interfering RNAs; SNCA: synuclein, alpha; SQSTM1: sequestosome 1; TPPP: tubulin polymerization promoting protein; TUBA: tubulin, alpha; UPS: ubiquitin-proteasome system; WT: wild type.

Project description:Multiple system atrophy (MSA) is a horrible and unrelenting neurodegenerative disorder with an uncertain etiology and pathophysiology. MSA is a unique proteinopathy in which alpha-synuclein (α-syn) accumulates preferentially in oligodendroglia rather than neurons. Glial cytoplasmic inclusions (GCIs) of α-syn are thought to elicit changes in oligodendrocyte function, such as reduced neurotrophic support and demyelination, leading to neurodegeneration. To date, only a murine model using one of three promoters exist to study this disease. We sought to develop novel rat and nonhuman primate (NHP) models of MSA by overexpressing α-syn in oligodendroglia using a novel oligotrophic adeno-associated virus (AAV) vector, Olig001. To establish tropism, rats received intrastriatal injections of Olig001 expressing GFP. Histological analysis showed widespread expression of GFP throughout the striatum and corpus callosum with >95% of GFP+ cells co-localizing with oligodendroglia and little to no expression in neurons or astrocytes. We next tested the efficacy of this vector in rhesus macaques with intrastriatal injections of Olig001 expressing GFP. As in rats, we observed a large number of GFP+ cells in gray matter and white matter tracts of the striatum and the corpus callosum, with 90-94% of GFP+ cells co-localizing with an oligodendroglial marker. To evaluate the potential of our vector to elicit MSA-like pathology in NHPs, we injected rhesus macaques intrastriatally with Olig001 expressing the α-syn transgene. Histological analysis 3-months after injection demonstrated widespread α-syn expression throughout the striatum as determined by LB509 and phosphorylated serine-129 α-syn immunoreactivity, all of which displayed as tropism similar to that seen with GFP. As in MSA, Olig001-α-syn GCIs in our model were resistant to proteinase K digestion and caused microglial activation. Critically, demyelination was observed in the white matter tracts of the corpus callosum and striatum of Olig001-α-syn but not Olig001-GFP injected animals, similar to the human disease. These data support the concept that this vector can provide novel rodent and nonhuman primate models of MSA.

Project description:Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.

Project description:Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

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Correction to: α-Synuclein in blood exosomes immunoprecipitated using neuronal and oligodendroglial markers distinguishes Parkinson's disease from multiple system atrophy.

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