Glial Cytoplasmic Inclusions Research Articles

Cerebral mitochondrial electron transport chain dysfunction in multiple system atrophy and Parkinson\u2019s disease

Multiple system atrophy (MSA) is a neurodegenerative disease characterised by glial cytoplasmic inclusions (GCIs), containing α-synuclein. Mutated COQ2, encoding an enzyme essential for co-enzyme Q10 (CoQ10) biosynthesis, has been associated with MSA. CoQ10 is an electron carrier in the mitochondrial electron transport chain (ETC) and antioxidant. It has been shown to be deficient in MSA brain tissue, thus implicating mitochondrial dysfunction in MSA. To investigate mitochondrial dysfunction in MSA further we examined ETC activity in MSA and control brain tissue, compared with Parkinson’s disease (PD) where mitochondrial dysfunction is known to be important. Using cerebellar and occipital white matter ETC complex I, II/III and IV activities were measured spectrophotometrically, selected individual components of the ETC were assessed by immunoblotting and cellular complex IV activity was analysed by enzyme histochemistry. We show decreased complex II/III activity with increased complex I and IV activity in MSA cerebellar white matter. This corresponds with the deficit in CoQ10 previously described in MSA and reflects the high regional pathological burden of GCIs. This study highlights mitochondrial dysfunction in MSA pathogenesis, suggests an influence on selective regional vulnerability to disease and points to shared disease mechanisms in α-synucleinopathies.

Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

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.

Abundance of Synaptic Vesicle-Related Proteins in Alpha-Synuclein-Containing Protein Inclusions Suggests a Targeted Formation Mechanism.

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

Wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation

Multiple system atrophy (MSA) is a fatal adult-onset neurodegenerative disease that is characterized by varying degrees of cerebellar dysfunction and Parkinsonism. The neuropathological hallmark of MSA is alpha-synuclein (AS)-positive glial cytoplasmic inclusions (GCIs). Although severe neuronal loss (NL) is also observed in MSA, neuronal inclusions (NIs) are rare compared to GCIs, such that the pathological mechanism of NL in MSA is unclear. GCIs and NIs are late-stage pathology features relative to AS oligomers and may not represent early pathological changes in MSA. To reveal the early pathology of MSA, it is necessary to examine the early aggregation of AS, i.e., AS oligomers. Here, we adopted a proximity ligation assay (PLA) to examine the distribution of AS oligomers in brain tissue samples from patients with MSA and other diseases. Surprisingly, MSA brains showed a widespread distribution and abundant accumulation of oligomeric AS in neurons as well as oligodendrocytes of the neocortex. In several regions, oligomeric AS signal intensity was higher in cases with MSA than in cases with Parkinson’s disease. In contrast to previous studies, AS–PLA revealed abundant AS oligomer accumulation in Purkinje cells in MSA brains, identifying oligomeric AS accumulation as a possible cause of Purkinje cell loss. This wide distribution of AS oligomers in MSA brain neurons has not been described previously and indicates a pathological mechanism of NL in MSA.

Multiple system atrophy.

Multiple system atrophy (MSA) is a sporadic, adult-onset, relentlessly progressive neurodegenerative disorder, clinically characterized by various combinations of autonomic failure, parkinsonism and ataxia. The neuropathological hallmark of MSA are glial cytoplasmic inclusions consisting of misfolded α-synuclein. Selective atrophy and neuronal loss in striatonigral and olivopontocerebellar systems underlie the division into two main motor phenotypes of MSA-parkinsonian type and MSA-cerebellar type. Isolated autonomic failure and REM sleep behavior disorder are common premotor features of MSA. Beyond the core clinical symptoms, MSA manifests with a number of non-motor and motor features. Red flags highly specific for MSA may provide clues for a correct diagnosis, but in general the diagnostic accuracy of the second consensus criteria is suboptimal, particularly in early disease stages. In this chapter, the authors discuss the historical milestones, etiopathogenesis, neuropathological findings, clinical features, red flags, differential diagnosis, diagnostic criteria, imaging and other biomarkers, current treatment, unmet needs and future treatments for MSA.

Anle138b modulates α-synuclein oligomerization and prevents motor decline and neurodegeneration in a mouse model of multiple system atrophy.

ABSTRACTBackgroundMSA is a fatal neurodegenerative disease characterized by autonomic failure and severe motor impairment. Its main pathological hallmark is the accumulation of α‐synuclein in oligodendrocytes, leading to glial and neuronal dysfunction and neurodegeneration. These features are recapitulated in the PLP‐hαSyn mouse model expressing human α‐synuclein in oligodendrocytes. At present, there is no effective disease‐modifying therapy. Previous experiments have shown that the aggregation inhibitor, anle138b, reduces neurodegeneration and behavioral deficits in mouse models of other proteinopathies.ObjectivesTo test the therapeutic potential of anle138b in a mouse model of MSA.MethodsTwo‐month‐old PLP‐hαSyn mice were fed over a period of 4 months with pellets containing anle138b at two different doses (0.6 and 2 g/kg) and compared to healthy controls and PLP‐hαSyn mice fed with placebo pellets. At the end of the treatment, behavioral and histological analyses were performed.ResultsWe observed a reversal of motor function to healthy control levels when PLP‐hαSyn mice were treated with both doses of anle138b. Histological and molecular analyses showed a significant reduction in α‐synuclein oligomers and glial cytoplasmic inclusions in animals fed with anle138b compared to nontreated mice. These animals also present preservation of dopaminergic neurons and reduction in microglial activation in SN correlating with the α‐synuclein reduction observed.ConclusionsAnle138b reduces α‐synuclein accumulation in PLP‐hαSyn mice, leading to neuroprotection, reduction of microglial activation, and preservation of motor function supporting the use of anle138b in a future clinical trial for MSA. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy.

SummaryMultiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets.

NLRP3 Inflammasome-Related Proteins Are Upregulated in the Putamen of Patients With Multiple System Atrophy.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia, and autonomic dysfunction. Microglial infiltration is an important mediator in MSA. The nucleotide-binding domain, leucine-rich repeats-containing family, pyrin domain-containing-3 (NLRP3) inflammasome complex, comprising NLRP3, apoptotic speck protein containing a caspase recruitment domain (ASC), and cysteine aspartic acid protease 1 (Caspase 1), regulates microglial inflammation in several neurodegenerative diseases. However, its role in MSA remains unknown. This study aimed to investigate the role of the NLRP3 inflammasome in MSA. Immunohistochemical staining of postmortem brains from 11 cases of MSA, 5 of Parkinson disease, and 6 age-matched controls were assessed. The relationships among α-synuclein deposition, microglial infiltration, and NLRP3 inflammasome-related proteins (NLRP3, ASC, and Caspase 1) were quantitatively analyzed. Double-labeling immunofluorescence staining confirmed colocalization of NLRP3 inflammasome-related proteins and Cluster of Differentiation 68. We demonstrated that the density of microglia expressing NLRP3 inflammasome-related proteins was increased in the putamina of MSA cases and was significantly related to the deposition of phosphorylated α-synuclein-positive glial cytoplasmic inclusions, tyrosine hydroxylase-positive fiber loss, and gliosis of glial fibrillary acidic protein-positive astrocytes. Our study suggests that the NLRP3 inflammasome is significantly upregulated and correlates with the neurodegenerative process in MSA.

TIGAR inclusion pathology is specific for Lewy body diseases

BackgroundWe previously reported up-regulation of tigarb (the zebrafish orthologue of human TIGAR, TP53 – Induced Glycolysis and Apoptosis Regulator) in a zebrafish pink1−/− model of Parkinson’s disease (PD). Genetic inactivation of tigarb led to the rescue of dopaminergic neurons and mitochondrial function in pink−/− zebrafish. The aim of this study was to determine the relevance of TIGAR for human PD, investigate its disease specificity and identify relevant upstream and downstream mechanisms. Materials and methodsTIGAR Immunohistochemistry, using a range of antibodies, was undertaken for detailed assessment of TIGAR in formalin-fixed, paraffin-embedded tissue from post mortem brains of PD patients and other neurodegenerative disorders (n = 10 controls, 10 PD cases, 10 dementia with Lewy bodies, 5 motor neurone disease (MND), 3 multiple system atrophy (MSA)) and complemented by immunohistochemistry for p53, hexokinase I (HK-I) and hexokinase II (HK-II; n = 4 control, 4 PD, and 4 dementia with Lewy bodies). ResultsTIGAR was detected in Lewy bodies and Lewy neurites in the substantia nigra of sporadic PD and Dementia with Lewy bodies (DLB) patients. Staining of adjacent sections and double staining confirmed the presence of TIGAR alongside alpha-synuclein in these LB and neurites. In contrast, TIGAR-positive aggregates were not seen in cortical Lewy bodies. TIGAR protein was also absent in both TDP-43-positive inclusions in MND and glial cytoplasmic inclusions in MSA. Subsequent investigation of the TIGAR-upstream regulator p53 and the downstream targets HK-I and HK-II in PD brains suggested a possible mild increase in HK-I. ConclusionsTIGAR protein, is present in SN Lewy bodies of both sporadic PD and DLB. The absence of TIGAR protein in the pathological inclusions of MND or MSA suggests disease specificity and further raises the possibility that TIGAR may be involved in PD pathogenesis.

Dynactin is involved in Lewy body pathology.

Dynactin forms a protein complex with dynein that retrogradely transports cargo along microtubules. Dysfunction of this dynein-dynactin complex causes several neurodegenerative diseases such as Perry syndrome, motor neuron diseases and progressive supranuclear palsy. Recently, we reported colocalization of phosphorylated α-synuclein (p-SNCA) and the largest subunit of dynactin (DCTN1) in Lewy body (LB)-like structures in Perry syndrome. Previous reports have not focused on the relationship between dynactin and synucleinopathies. Thus, we examined autopsied human brains from patients with Parkinson's disease, dementia with LBs, and multiple system atrophy using immunohistochemistry for p-SNCA, DCTN1, dynactin 2 (DCTN2, dynamitin) and dynein cytoplasmic 1 intermediate chain 1 (DYNC1I1). We also examined microtubule affinity-regulating kinases (MARKs), which phosphorylate microtubule-associated proteins and trigger microtubule disruption. Both brainstem-type and cortical LBs were immunopositive for DCTN1, DCTN2, DYNC1I1 and p-MARK and their staining often overlapped with p-SNCA. Lewy neurites were also immunopositive for DCTN1, DCTN2 and DYNC1I1. However, p-SNCA-positive inclusions of multiple system atrophy, which included both glial and neuronal cytoplasmic inclusions, were immunonegative for DCTN1, DCTN2, DYNC1I1 and p-MARK. Thus, immunohistochemistry for dynein-dynactin complex molecules, especially DCTN1, can clearly distinguish LBs from neuronal cytoplasmic inclusions. Our results suggest that dynactin is closely associated with LB pathology.

Limited effects of dysfunctional macroautophagy on the accumulation of extracellularly derived \u03b1-synuclein in oligodendroglia: implications for MSA pathogenesis

BackgroundThe progressive neurodegenerative disorder multiple system atrophy (MSA) is characterized by α-synuclein-positive (oligodendro-) glial cytoplasmic inclusions (GCIs). A connection between the abnormal accumulation of α-synuclein in GCIs and disease initiation and progression has been postulated. Mechanisms involved in the formation of GCIs are unclear. Abnormal uptake of α-synuclein from extracellular space, oligodendroglial overexpression of α-synuclein, and/or dysfunctional protein degradation including macroautophagy have all been discussed. In the current study, we investigated whether dysfunctional macroautophagy aggravates accumulation of extracellular α-synuclein in the oligodendroglia.ResultsWe show that oligodendroglia uptake monomeric and fibrillar extracellular α-synuclein. Blocking macroautophagy through bafilomycin A1 treatment or genetic knockdown of LC3B does not consistently change the level of incorporated α-synuclein in oligodendroglia exposed to extracellular soluble/monomeric or fibrillar α-synuclein, however leads to higher oxidative stress in combination with fibrillar α-synuclein treatment. Finally, we detected no evidence for GCI-like formation resulting from dysfunctional macroautophagy in oligodendroglia using confocal microscopy.ConclusionIn summary, isolated dysfunctional macroautophagy is not sufficient to enhance abnormal accumulation of uptaken α-synuclein in vitro, but may lead to increased production of reactive oxygen species in the presence of fibrillar α-synuclein. Multiple complementary pathways are likely to contribute to GCI formation in MSA.

TDP-43 pathology in multiple system atrophy: colocalization of TDP-43 and α-synuclein in glial cytoplasmic inclusions.

This study aimed to assess clinicopathologic features of transactive response DNA-binding protein of 43 kDa (TDP-43) pathology and its risk factors in multiple system atrophy (MSA). Paraffin-embedded sections of the amygdala and basal forebrain from 186 autopsy-confirmed MSA cases were screened with immunohistochemistry for phospho-TDP-43. In cases having TDP-43 pathology, additional brain regions were assessed. Immunohistochemical and immunofluorescence double-staining and immunogold electron microscopy (IEM) were performed to evaluate colocalization of TDP-43 and α-synuclein. Genetic risk factors for TDP-43 pathology were also analysed. Immunohistochemistry showed various morphologies of TDP-43 pathology in 13 cases (7%), such as subpial astrocytic inclusions, neuronal inclusions, dystrophic neurites, perivascular inclusions and glial cytoplasmic inclusions (GCIs). Multivariable logistic regression models revealed that only advanced age, but not concurrent Alzheimer's disease, argyrophilic grain disease or hippocampal sclerosis, was an independent risk factor for TDP-43 pathology in MSA (OR: 1.11, 95% CI: 1.04-1.19, P = 0.002). TDP-43 pathology was restricted to the amygdala in eight cases and extended to the hippocampus in two cases. The remaining three cases had widespread TDP-43 pathology. Immunohistochemical and immunofluorescence double-staining and IEM revealed colocalization of α-synuclein and TDP-43 in GCIs with granule-coated filaments. Pilot genetic studies failed to show associations between risk variants of TMEM106B or GRN and TDP-43 pathology. TDP-43 pathology is rare in MSA and occurs mainly in the medial temporal lobe. Advanced age is a risk factor for TDP-43 pathology in MSA. Colocalization of TDP-43 and α-synuclein in GCIs suggests possible direct interaction between the two molecules.

Cellular milieu imparts distinct pathological α-synuclein strains in α-synucleinopathies.

In Lewy body (LB) diseases, including Parkinson’s disease (PD), without and with dementia (PDD), dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD) patients with LB co-pathology1, α-synuclein (α-Syn) aggregates in neurons as LBs and Lewy neurites (LNs)2, while in multiple system atrophy (MSA), α-Syn mainly accumulates in oligodendrocytes as glial cytoplasmic inclusions (GCIs)3. Here, we report that pathological α-Syn in GCIs and LBs (GCI-α-Syn and LB-α-Syn) are conformationally and biologically distinct. GCI-α-Syn forms more compact structures and is ~1,000-fold more potent than LB-α-Syn in seeding α-Syn aggregation, consistent with the highly aggressive nature of MSA. Surprisingly, GCI-α-Syn and LB-α-Syn show no cell type preference in seeding α-Syn pathology, raising the question of why they demonstrate different cell type distributions in LB disease versus MSA. Strikingly, we found that oligodendrocytes but not neurons transform misfolded α-Syn into a GCI-like strain, highlighting that distinct α-Syn strains are generated by different intracellular milieus. Moreover, GCI-α-Syn maintains its high seeding activity when propagated in neurons. Thus, α-Syn strains are determined by both misfolded seeds and intracellular environments.

TDP-43 pathology in anterior temporal pole cortex in aging and Alzheimer\u2019s disease

TDP-43 pathology was investigated in the anterior temporal pole cortex (ATPC) and orbital frontal cortex (OFC), regions often degenerated in frontotemporal lobar degenerations (FTLD), in aging and Alzheimer’s disease (AD). Diagnosis of dementia in the 1160 autopsied participants from 3 studies of community-dwelling elders was based on clinical evaluation and cognitive performance tests which were used to create summary measures of the five cognitive domains. Neuronal and glial TDP-43 cytoplasmic inclusions were quantitated in 8 brain regions by immunohistochemistry, and used in ANOVA and regression analyses. TDP-43 pathology was present in 547 (49.4%) participants in whom ATPC (41.9%) was the most frequently involved neocortical region and in 15.5% of these cases, ATPC was the only neocortical area with TDP-43 pathology suggesting not only that ATPC is involved early by TDP-43 but that ATPC may represent an intermediate stage between mesial temporal lobe involvement by TDP-43 and the last stage with involvement of other neocortical areas. To better study this intermediary neocortical stage, and to integrate with other staging schemes, our previous 3 stage distribution of TDP-43 pathology was revised to a 5 stage distribution scheme with stage 1 showing involvement of the amygdala only; stage 2 showed extension to hippocampus and/or entorhinal cortex; stage 3 showed extension to the ATPC; stage 4 – showed extension to the midtemporal cortex and/or OFC and finally in stage 5, there was extension to the midfrontal cortex. Clinically, cases in stages 2 to 5 had impaired episodic memory, however, stage 3 was distinct from stage 2 since stage 3 cases had significantly increased odds of dementia. The proportion of cases with hippocampal sclerosis increased progressively across the stages with stage 5 showing the largest proportion of hippocampal sclerosis cases. Stage 5 cases differed from other stages by having impairment of semantic memory and perceptual speed, in addition to episodic memory impairment. These data suggest that of the regions studied, TDP-43 pathology in the ATPC is an important early neocortical stage of TDP-43 progression in aging and AD while extension of TDP-43 pathology to the midfrontal cortex is a late stage associated with more severe and global cognitive impairment.

Immunohistochemical and Molecular Investigations Show Alteration in the Inflammatory Profile of Multiple System Atrophy Brain.

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterized by aggregation of α-synuclein in oligodendrocytes to form glial cytoplasmic inclusions. According to the distribution of neurodegeneration, MSA is subtyped as striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA), or as combination of these 2 (mixed MSA). In the current study, we aimed to investigate regional microglial populations and gene expression in the 3 different MSA subtypes. Microscopy with microglial marker Iba-1 combined with either proinflammatory marker CD68 or anti-inflammatory marker Arginase-1 was analyzed in control, SND, and OPCA cases (n = 5) using paraffin embedded sections. Western immunoblotting and cytokine array were used to determine protein expression in MSA and control brain regions. Gene expression was investigated using the NanoString nCounter Human Inflammation panel v2 mRNA Expression Assay. Analysis of neuropathological subtypes of MSA demonstrated a significant increase in microglia in the substantia nigra of OPCA cases. There was no difference in the microglial activation state in any region. Cytokine expression in MSA was comparable with controls. Decreased expression of CX3CL1 precursor protein and significantly greater CX3CR1 protein was found in MSA. NanoString analysis revealed the >2-fold greater expression of ARG1, MASP1, NOX4, PTGDR2, and C6 in MSA.

Modeling Multiple System Atrophy Prion Propagation in Astrocytes From Transgenic Mice

Background/AimsThere is a growing body of evidence that astrocytes contain α‐synuclein deposits in Parkinson's disease (PD), multiple system atrophy (MSA), and other α‐synucleinopathies at advanced stages of disease. The native protein isoform of α‐synuclein misfolds into a prion conformation that aggregates and spreads throughout the brain, resulting in neurodegeneration in each of these conditions. We aim to use a new in vitro model of α‐synuclein prion propagation to clarify the role of astrocytes in disease progression of MSA. This system may also prove useful for elucidating the molecular mechanisms of neurotoxicity.MethodsWe isolated primary cell culture astrocytes from the brains of transgenic mice expressing either the wild‐type (wt) form of human α‐synuclein or with the A53T mutation, which is associated with familial PD. Astrocytes were exposed to brain homogenates from MSA patients, mouse‐passaged MSA, recombinant wt α‐synuclein fibrils, and cell lysate from MSA‐infected astrocyte cultures. Following exposure, cells were thoroughly washed and cultured in fresh medium. We used confocal microscopy and high content analysis to examine α‐synuclein intracellular morphology and phosphorylation at multiple time points.ResultsWhile exogenously added α‐synuclein fibrils persisted unphosphorylated, they efficiently induced aggregation and phosphorylation of endogenously expressed α‐synuclein in exposed astrocytes. We observed rapid, progressive, and dose‐dependent α‐synuclein aggregate formation in astrocytes expressing either wt or A53T human α‐synuclein exposed to fibrils or MSA homogenates. Moreover, confocal microscopy revealed two morphologically distinct types of α‐synuclein assemblies – filamentous and granular. Both types progressively formed glial cytoplasmic inclusions, were hyperphosphorylated, thioflavin S positive, ubiquitinated, and co‐localized with the p62 marker, as is also seen in the brains of human MSA patients.ConclusionsOur studies investigate the formation of α‐synuclein aggregates in astrocytes and report these cells as potential candidates in which to examine various pathological forms of α‐synuclein prions. This cell culture model may allow studies on mechanisms of neurotoxicity and might provide a novel approach for drug discovery against MSA and other α‐synucleinopathies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiple System Atrophy: An Oligodendroglioneural Synucleinopathy1.

Multiple system atrophy (MSA) is an orphan, fatal, adult-onset neurodegenerative disorder of uncertain etiology that is clinically characterized by various combinations of parkinsonism, cerebellar, autonomic, and motor dysfunction. MSA is an α-synucleinopathy with specific glioneuronal degeneration involving striatonigral, olivopontocerebellar, and autonomic nervous systems but also other parts of the central and peripheral nervous systems. The major clinical variants correlate with the morphologic phenotypes of striatonigral degeneration (MSA-P) and olivopontocerebellar atrophy (MSA-C). While our knowledge of the molecular pathogenesis of this devastating disease is still incomplete, updated consensus criteria and combined fluid and imaging biomarkers have increased its diagnostic accuracy. The neuropathologic hallmark of this unique proteinopathy is the deposition of aberrant α-synuclein in both glia (mainly oligodendroglia) and neurons forming glial and neuronal cytoplasmic inclusions that cause cell dysfunction and demise. In addition, there is widespread demyelination, the pathogenesis of which is not fully understood. The pathogenesis of MSA is characterized by propagation of misfolded α-synuclein from neurons to oligodendroglia and cell-to-cell spreading in a “prion-like” manner, oxidative stress, proteasomal and mitochondrial dysfunction, dysregulation of myelin lipids, decreased neurotrophic factors, neuroinflammation, and energy failure. The combination of these mechanisms finally results in a system-specific pattern of neurodegeneration and a multisystem involvement that are specific for MSA. Despite several pharmacological approaches in MSA models, addressing these pathogenic mechanisms, no effective neuroprotective nor disease-modifying therapeutic strategies are currently available. Multidisciplinary research to elucidate the genetic and molecular background of the deleterious cycle of noxious processes, to develop reliable biomarkers and targets for effective treatment of this hitherto incurable disorder is urgently needed.

In vitro models of multiple system atrophy from primary cells to induced pluripotent stem cells.

Multiple system atrophy (MSA) is a rare neurodegenerative disease with a fatal outcome. Nowadays, only symptomatic treatment is available for MSA patients. The hallmarks of the disease are glial cytoplasmic inclusions (GCIs), proteinaceous aggregates mainly composed of alpha‐synuclein, which accumulate in oligodendrocytes. However, despite the extensive research efforts, little is known about the pathogenesis of MSA. Early myelin dysfunction and alpha‐synuclein deposition are thought to play a major role, but the origin of the aggregates and the causes of misfolding are obscure. One of the reasons for this is the lack of a reliable model of the disease. Recently, the development of induced pluripotent stem cell (iPSC) technology opened up the possibility of elucidating disease mechanisms in neurodegenerative diseases including MSA. Patient specific iPSC can be differentiated in glia and neurons, the cells involved in MSA, providing a useful human disease model. Here, we firstly review the progress made in MSA modelling with primary cell cultures. Subsequently, we focus on the first iPSC‐based model of MSA, which showed that alpha‐synuclein is expressed in oligodendrocyte progenitors, whereas its production decreases in mature oligodendrocytes. We then highlight the opportunities offered by iPSC in studying disease mechanisms and providing innovative models for testing therapeutic strategies, and we discuss the challenges connected with this technique.

The Relevance of Iron in the Pathogenesis of Multiple System Atrophy: A Viewpoint.

Iron is essential for cellular development and maintenance of multiple physiological processes in the central nervous system. The disturbance of its homeostasis leads to abnormal iron deposition in the brain and causes neurotoxicity via generation of free radicals and oxidative stress. Iron toxicity has been established in the pathogenesis of Parkinson’s disease; however, its contribution to multiple system atrophy (MSA) remains elusive. MSA is characterized by cytoplasmic inclusions of misfolded α-synuclein (α-SYN) in oligodendrocytes referred to as glial cytoplasmic inclusions (GCIs). Remarkably, the oligodendrocytes possess high amounts of iron, which together with GCI pathology make a contribution toward MSA pathogenesis likely. Consistent with this observation, the GCI density is associated with neurodegeneration in central autonomic networks as well as olivopontocerebellar and striatonigral pathways. Iron converts native α-SYN into a β-sheet conformation and promotes its aggregation either directly or via increasing levels of oxidative stress. Interestingly, α-SYN possesses ferrireductase activity and α-SYN expression underlies iron mediated translational control via RNA stem loop structures. Despite a correlation between progressive putaminal atrophy and iron accumulation as well as clinical decline, it remains unclear whether pathologic iron accumulation in MSA is a secondary event in the cascade of neuronal degeneration rather than a primary cause. This review summarizes the current knowledge of iron in MSA and gives evidence for perturbed iron homeostasis as a potential pathogenic factor in MSA-associated neurodegeneration.

Pathological Endogenous α-Synuclein Accumulation in Oligodendrocyte Precursor Cells Potentially Induces Inclusions in Multiple System Atrophy.

SummaryGlial cytoplasmic inclusions (GCIs), commonly observed as α-synuclein (α-syn)-positive aggregates within oligodendrocytes, are the pathological hallmark of multiple system atrophy. The origin of α-syn in GCIs is uncertain; there is little evidence of endogenous α-syn expression in oligodendrocyte lineage cells, oligodendrocyte precursor cells (OPCs), and mature oligodendrocytes (OLGs). Here, based on in vitro analysis using primary rat cell cultures, we elucidated that preformed fibrils (PFFs) generated from recombinant human α-syn trigger multimerization and an upsurge of endogenous α-syn in OPCs, which is attributable to insufficient autophagic proteolysis. RNA-seq analysis of OPCs revealed that α-syn PFFs interfered with the expression of proteins associated with neuromodulation and myelination. Furthermore, we detected cytoplasmic α-syn inclusions in OLGs through differentiation of OPCs pre-incubated with PFFs. Overall, our findings suggest the possibility of endogenous α-syn accumulation in OPCs that contributes to GCI formation and perturbation of neuronal/glial support in multiple system atrophy brains.