The 'α-synucleinopathy syndicate': multiple system atrophy and Parkinson's disease.

Multiple System Atrophy (MSA) and Parkinson's diseases (PD) are elite members of the α-synucleinopathy organization. Aberrant accumulations of the protein α-synuclein characterize them. A plethora of evidence indicates the involvement of these rogue inclusions in a cascade of events that disturb cellular homeostasis resulting in neuronal dysfunction. These two neurodegenerative diseases share many features both clinically and pathologically. Cytotoxic processes commonly induced by reactive free radical species have been associated with oxidative stress and neuroinflammation, frequently reported in both diseases. However, it appears they have characteristic and distinct α-synuclein inclusions. It is glial cytoplasmic inclusions in the case of MSA while Lewy bodies manifest in PD. This is probably related to the etiology of the illness. At present, precise mechanism(s) underlying the characteristic configuration of neurodegeneration are unclear. Furthermore, the "prion-like" transmission from cell to cell prompts the suggestion that perhaps these α-synucleinopathies are prion-like diseases. The possibility of some underlying genetic foul play remains controversial. But as major culprits of pathological processes or even single triggers of PD and MSA are the same-like oxidative stress, iron-induced pathology, mitochondriopathy, loss of respiratory activity, loss of proteasomal function, microglial activation, neuroinflammation-it is not farfetched to assume that in sporadic PD and also in MSA a variety of combinations of susceptibility genes contribute to the regional specificity of pathological onset. These players of pathology, as mentioned above, in a synergistic combination, are responsible for driving the progression of PD, MSA and other neurodegenerative disorders. Elucidating the triggers and progression factors is vital for advocating disease modification or halting its progression in both, MSA and PD.

MOBP and HIP1 in multiple system atrophy: New α-synuclein partners in glial cytoplasmic inclusions implicated in the disease pathogenesis.

AIMS: Multiple system atrophy (MSA) is a fatal neurodegenerative disease. Similar to Parkinson's disease (PD), MSA is an α-synucleinopathy, and its pathological hallmark consists of glial cytoplasmic inclusions (GCIs) containing α-synuclein (SNCA) in oligodendrocytes. We previously identified consistent changes in myelin-associated oligodendrocyte basic protein (MOBP) and huntingtin interacting protein 1 (HIP1) DNA methylation status in MSA. We hypothesized that if differential DNA methylation at these loci is mechanistically relevant for MSA, it should have downstream consequences on gene regulation. METHODS: We investigated the relationship between MOBP and HIP1 DNA methylation and mRNA levels in cerebellar white matter from MSA and healthy controls. Additionally, we analysed protein expression using western blotting, immunohistochemistry and proximity ligation assays. RESULTS: We found decreased MOBP mRNA levels significantly correlated with increased DNA methylation in MSA. For HIP1, we found a distinct relationship between DNA methylation and gene expression levels in MSA compared to healthy controls, suggesting this locus may be subjected to epigenetic remodelling in MSA. Although soluble protein levels for MOBP and HIP1 in cerebellar white matter were not significantly different between MSA cases and controls, we found striking differences between MSA and other neurodegenerative diseases, including PD and Huntington's disease. We also found that MOBP and HIP1 are mislocalized into the GCIs in MSA, where they appear to interact with SNCA. CONCLUSIONS: This study supports a role for DNA methylation in downregulation of MOBP mRNA in MSA. Most importantly, the identification of MOBP and HIP1 as new constituents of GCIs emphasizes the relevance of these two loci to the pathogenesis of MSA.

Neuropathology of multiple system atrophy: Kurt Jellinger`s legacy.

Multiple System Atrophy (MSA) is a rare, fatal neurodegenerative disorder. Its etiology and exact pathogenesis still remain poorly understood and currently no disease-modifying therapy is available to halt or slow down this detrimental neurodegenerative process. Hallmarks of the disease are α-synuclein rich glial cytoplasmic inclusions (GCIs). Neuropathologically, various degrees of striatonigral degeneration (SND) and olivopontocerebellar atrophy (OPCA) can be observed. Since the original descriptions of this multifaceted disorder, several steps forward have been made to clarify its neuropathological hallmarks and key pathophysiological mechanisms. The Austrian neuropathologist Kurt Jellinger substantially contributed to the understanding of the underlying neuropathology of this disease, to its standardized assessment and to a broad systematical clinic-pathological correlation. On the occasion of his 90th birthday, we reviewed the current state of the art in the field of MSA neuropathology, highlighting Prof. Jellinger's substantial contribution.

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

BACKGROUND: The 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. RESULTS: We 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. CONCLUSION: In 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.

Synucleinopathies: common features and hippocampal manifestations.

Parkinson's disease (PD), dementia with Lewy Bodies (DLB), and multiple system atrophy (MSA) are three major synucleinopathies characterized by α-synuclein-containing inclusions in the brains of patients. Because the cell types and brain structures that are affected vary markedly between the disorders, the patients have different clinical manifestations in addition to some overlapping symptoms, which are the basis for differential diagnosis. Cognitive impairment and depression associated with hippocampal dysfunction are frequently observed in these disorders. While various α-synuclein-containing inclusions are found in the hippocampal formation, increasing evidence supports that small α-synuclein aggregates or oligomers may be the real culprit, causing deficits in neurotransmission and neurogenesis in the hippocampus and related brain regions, which constitute the major mechanism for the hippocampal dysfunctions and associated neuropsychiatric manifestations in synucleinopathies.

Glia and alpha-synuclein in neurodegeneration: A complex interaction.

α-Synucleinopathies (ASP) comprise adult-onset, progressive neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) that are characterized by α-synuclein (AS) aggregates in neurons or glia. PD and DLB feature neuronal AS-positive inclusions termed Lewy bodies (LB) whereas glial cytoplasmic inclusions (GCIs, Papp-Lantos bodies) are recognized as the defining hallmark of MSA. Furthermore, AS-positive cytoplasmic aggregates may also be seen in astroglial cells of PD/DLB and MSA brains. The glial AS-inclusions appear to trigger reduced trophic support resulting in neuronal loss. Moreover, microgliosis and astrogliosis can be found throughout the neurodegenerative brain and both are key players in the initiation and progression of ASP. In this review, we will highlight AS-dependent alterations of glial function and their impact on neuronal vulnerability thereby providing a detailed summary on the multifaceted role of glia in ASP.

Mitochondrial impairment and oxidative stress compromise autophagosomal degradation of α-synuclein in oligodendroglial cells.

α-Synuclein (α-syn)-containing glial cytoplasmic inclusions originating in oligodendrocytes are characteristically observed in multiple system atrophy. The mechanisms of glial cytoplasmic inclusion formation remain rather elusive. α-Syn over-expression, uptake from the environment, oxidative stress or impairment of the proteolytic degradation systems have been discussed. Here, we investigated whether in oligodendrocytes autophagy plays a major role in the degradation and aggregation of endogenously expressed α-syn and of α-syn taken up from the extracellular environment. Furthermore, we studied whether in cells with impaired mitochondria the accumulation and aggregation of exogenously added α-syn is promoted. Using primary cultures of rat brain oligodendrocytes and an oligodendroglial cell line, genetically engineered to express green fluorescent protein-microtubule-associated light chain 3 with or without α-syn to monitor the autophagic flux, we demonstrate that both exogenously applied α-syn and α-syn stably expressed endogenously are effectively degraded by autophagy and do not affect the autophagic flux per se. Mitochondrial impairment with the protonophore carbonyl cyanide 3-chlorophenylhydrazone or 3-nitropropionic acid disturbs the autophagic pathway and leads to the accumulation of exogenously applied α-syn and enhances its propensity to form aggregates intracellularly. Thus, mitochondrial dysfunction and oxidative stress, which occur over time and are significant pathological features in synucleinopathies, have an impact on the autophagic pathway and participate in pathogenesis. Glial cytoplasmic inclusions are characteristically observed in multiple system atrophy, their occurrence might be related to failure in protein degradation systems. Here, we show that in oligodendrocytes autophagy is the major route of α-synuclein degradation which is either endogenously expressed or added exogenously (1, 2). Mitochondrial impairment (3) disturbs the autophagic flux and leads to the accumulation of exogenously applied α-synuclein, and enhances its propensity to form aggregates intracellulary (4).

Alpha-synuclein mRNA expression in oligodendrocytes in MSA.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α-Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser-capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P < 0.0001). At the cellular level, MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls.

Immunohistochemical localization of spatacsin in α-synucleinopathies.

Spatacsin (SPG11) is a major mutated gene in autosomal recessive spastic paraplegia with thin corpus callosum (ARHSP-TCC) and is responsible for juvenile Parkinsonism. To elucidate the role of spatacsin in the pathogenesis of α-synucleinopathies, an immunohistochemical investigation was performed on the brain of patients with Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) using anti-spatacsin antibody. In PD, Lewy bodies (LBs) in the brain stem were positive for spatacsin. These LBs showed intense staining in their peripheral portions and occasionally in the central cores. Lewy neurites were also spatacsin-positive. In DLB, cortical LBs were immunolabeled by spatacsin. In MSA, glial cytoplasmic inclusions (GCI) and a small fraction of neuronal cytoplasmic inclusions (NCI) were positive for spatacsin. The widespread accumulation of spatacsin observed in pathologic α-synuclein-containing inclusions suggests that spatacsin may be involved in the pathogenesis of α-synucleinopathies.

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.

Multiple system atrophy: Clinical, evolutive and histopathological characteristics of a series of cases.

BACKGROUND AND OBJECTIVE: Multiple system atrophy is a rare and fatal neurodegenerative disorder, characterized by autonomic dysfunction in association with either parkinsonism or cerebellar signs. The pathologic hallmark is the presence of alpha-synuclein aggregates in oligodendrocytes, forming glial cytoplasmic inclusions. Clinically, it may be difficult to distinguish form other parkinsonisms or ataxias, particularly in the early stages of the disease. In this case series we aim to describe in detail the features of MSA patients. MATERIAL AND METHODS: Unified MSA Rating Scale (UMSARS) score, structural and functional imaging and cardiovascular autonomic testing, are summarized since early stages of the disease. RESULTS: UMSARS proved to be useful to perform a follow-up being longitudinal examination essential to stratify risk of poor outcome. Neuropathological diagnosis showed an overlap between parkinsonian and cerebellar subtypes, with some peculiarities that could help to distinguish from other subtypes. CONCLUSION: A better description of MSA features with standardized test confirmed by means of neuropathological studies could help to increase sensitivity.

Pathological Relevance of Post-Translationally Modified Alpha-Synuclein (pSer87, pSer129, nTyr39) in Idiopathic Parkinson's Disease and Multiple System Atrophy.

Aggregated alpha-synuclein (α-synuclein) is the main component of Lewy bodies (LBs), Lewy neurites (LNs), and glial cytoplasmic inclusions (GCIs), which are pathological hallmarks of idiopathic Parkinson's disease (IPD) and multiple system atrophy (MSA). Initiating factors that culminate in forming LBs/LNs/GCIs remain elusive. Several species of α-synuclein exist, including phosphorylated and nitrated forms. It is unclear which α-synuclein post-translational modifications (PTMs) appear within aggregates throughout disease pathology. Herein we aimed to establish the predominant α-synuclein PTMs in postmortem IPD and MSA pathology using immunohistochemistry. We examined the patterns of three α-synuclein PTMs (pS87, pS129, nY39) simultaneously in pathology-affected regions of 15 IPD cases, 5 MSA cases, and 6 neurologically normal controls. All antibodies recognized LBs, LNs, and GCIs, albeit to a variable extent. pS129 α-synuclein antibody was particularly immunopositive for LNs and synaptic dot-like structures, followed by nY39 α-synuclein antibody. GCIs, neuronal inclusions, and small threads were positive for nY39 α-synuclein in MSA. Quantification of the LB scores revealed that pS129 α-synuclein was the dominant and earliest α-synuclein PTM, followed by nY39 α-synuclein, while lower amounts of pSer87 α-synuclein appeared later in disease progression in PD. These results may have implications for novel biomarker and therapeutic developments.

Multiple system atrophy: Clinical, evolutive and histopathological characteristics of a series of cases.

BACKGROUND AND OBJECTIVE: Multiple system atrophy is a rare and fatal neurodegenerative disorder, characterized by autonomic dysfunction in association with either parkinsonism or cerebellar signs. The pathologic hallmark is the presence of alpha-synuclein aggregates in oligodendrocytes, forming glial cytoplasmic inclusions. Clinically, it may be difficult to distinguish form other parkinsonisms or ataxias, particularly in the early stages of the disease. In this case series we aim to describe in detail the features of MSA patients. MATERIAL AND METHODS: Unified MSA Rating Scale (UMSARS) score, structural and functional imaging and cardiovascular autonomic testing, are summarized since early stages of the disease. RESULTS: UMSARS proved to be useful to perform a follow-up being longitudinal examination essential to stratify risk of poor outcome. Neuropathological diagnosis showed an overlap between parkinsonian and cerebellar subtypes, with some peculiarities that could help to distinguish from other subtypes. CONCLUSION: A better description of MSA features with standardized test confirmed by means of neuropathological studies could help to increase sensitivity.

BCAS1-positive immature oligodendrocytes are affected by the α-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.

Physiological and pathological role of alpha-synuclein in Parkinson's disease through iron mediated oxidative stress; the role of a putative iron-responsive element.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer's disease (AD) and represents a large health burden to society. Genetic and oxidative risk factors have been proposed as possible causes, but their relative contribution remains unclear. Dysfunction of alpha-synuclein (alpha-syn) has been associated with PD due to its increased presence, together with iron, in Lewy bodies. Brain oxidative damage caused by iron may be partly mediated by alpha-syn oligomerization during PD pathology. Also, alpha-syn gene dosage can cause familial PD and inhibition of its gene expression by blocking translation via a newly identified Iron Responsive Element-like RNA sequence in its 5'-untranslated region may provide a new PD drug target.

The molecular tweezer CLR01 reduces aggregated, pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy.

Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development.

LRRK2 p.Ile1371Val Mutation in a Case with Neuropathologically Confirmed Multi-System Atrophy.

BACKGROUND: Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are among the most common genetic causes of Lewy body Parkinson's disease (PD). However, LRRK2 mutations can also lead to a variety of pathological phenotypes other than typical PD, including relatively pure nigrostriatal cell loss without alpha-synuclein-positive Lewy bodies or Lewy neurites, progressive supranuclear palsy (PSP), and multiple system atrophy (MSA). The mechanisms behind this remarkable pleomorphic pathology are currently unclear. OBJECTIVE: To genetically and pathologically characterize a case with a LRRK2, p.Ile1371Val rare variant and pathologically proven MSA. METHODS: From the brain donation program at the Parkinson's Institute and Clinical Center, we selected 26 brains with family history and a with clinicopathological diagnosis of PD (n = 20), MSA (n = 4), or PSP (n = 2). We performed neuropathological evaluation, including alpha-synuclein and tau immunohistochemistry and sequenced 188 genes that have been reported as causative for or associated with neurodegenerative diseases. RESULTS: We identified a known LRRK2, p.Ile1371Val genetic variant in a case with clinically diagnosed and pathologically proven MSA. Neuropathology revealed that the olivopontocerebellar system was more affected than the striatonigral system. CONCLUSIONS: Our data suggest that genetic variants in the LRRK2 gene can present clinically and neuropathologically as MSA. One other LRRK2 genetic variant (LRRK2, p.Ile2020Thr) has been reported with a neuropathological diagnosis of MSA. Interestingly, the LRRK2 variant (LRRK2, p.Ile1371Val) identified here has been reported previously in a postmortem case with Lewy body PD.Future studies are critical to discover the mechanisms leading to different neurodegenerative trajectories both in neuronal and glial cell populations.

Activated caspase-9 immunoreactivity in glial and neuronal cytoplasmic inclusions in multiple system atrophy.

The mitochondria play an important role in apoptotic cell death, and the released cytochrome c from the mitochondria promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9, resulting in the activation of caspase-9 and the promotion of the apoptotic cascade. To investigate the role of mitochondria-dependent apoptotic cell death in patients with multiple system atrophy (MSA), we performed immunohistochemical studies on apoptosome-related proteins in formalin-fixed, paraffin-embedded sections from 8 normal subjects and 10 patients with MSA. We then performed double-labeling immunohistochemistry for activated caspase-9 and α-synuclein in some sections from 10 patients with MSA. In the brains with MSA, glial cytoplasmic inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs) were intensely immunoreactive for cytochrome c, Apaf-1 and caspase-9. Activated caspase-9 immunoreactivities were also confirmed to be densely localized to both GCIs and NCIs using two types of anti-cleaved caspase-9 antibodies. The semiquantitative analyses using the upper pontine sections double-immunostained with cleaved caspase-9 and α-synuclein demonstrated that approximately 80% of GCIs and NCIs were immunopositive for cleaved caspase-9. Our results suggest that the formation of the apoptosome accompanied by the activation of caspase-9 may occur in brains affected by MSA, and that a mitochondria-dependent apoptotic pathway may be partially associated with the pathogenesis of MSA.

DOPAL is transmissible to and oligomerizes alpha-synuclein in human glial cells.

INTRODUCTION: Glial cytoplasmic inclusions (GCIs) containing alpha-synuclein (AS) are a neuropathologic hallmark of multiple system atrophy (MSA). Oligomerized AS is thought to be the pathogenic form of the protein. Glial cells normally express little AS, but they can take up AS from the extracellular fluid. 3,4-Dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in the intra-neuronal metabolism of dopamine (DA), potently oligomerizes AS. In this study we tested whether DOPAL is taken up by human glial cells and augments intracellular oligomerization of AS. METHODS: DOPAL (exogenous or endogenous from co-incubation with PC12 cells) and AS (native or A53T mutant form) were added to the incubation medium of glial cells (glioblastoma or MO3.13 oligodendrocytes). Glial cellular contents of DOPAL and its intracellular metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at up to 180 min of incubation. Glial cellular AS oligomers were quantified by Western blotting. RESULTS: Neither glioblastoma nor MO3.13 cells contained endogenous catecholamines or AS. Co-incubation of the cells with DA-producing PC12 cells produced time-related increases in DOPAL and DOPAC contents. Similarly, glial cellular DOPAL and DOPAC contents increased rapidly after addition of DOPAL to the medium. After addition of native or A53T-AS, intracellular AS also increased. Incubation of glial cells with both DOPAL and AS enhanced the intracellular oligomerization of native and A53T-AS. CONCLUSIONS: DOPAL is transmissible to glial cells and enhances intracellular oligomerization of AS. An interaction of DOPAL with AS might help explain the formation of CGIs in MSA.