Formation Of Glial Cytoplasmic Inclusions Research Articles

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.

Pharmacological inhibition of FABP7 by MF 6 counteracts cerebellum dysfunction in an experimental multiple system atrophy mouse model.

Multiple system atrophy (MSA) is a rare, fatal neurodegenerative disease characterized by the accumulation of misfolded α-synuclein (αSyn) in glial cells, leading to the formation of glial cytoplasmic inclusions (GCI). We previous found that glial fatty acid-binding protein 7 (FABP7) played a crucial role in alpha-synuclein (αSyn) aggregation and toxicity in oligodendrocytes, inhibition of FABP7 by a specific inhibitor MF 6 reduced αSyn aggregation and enhanced cell viability in cultured cell lines and mouse oligodendrocyte progenitor cells. In this study we investigated whether MF 6 ameliorated αSyn-associated pathological processes in PLP-hαSyn transgenic mice (PLP-αSyn mice), a wildly used MSA mouse model with overexpressing αSyn in oligodendroglia under the proteolipid protein (PLP) promoter. PLP-αSyn mice were orally administered MF6 (0.1, 1 mg ·kg-1 ·d-1) for 32 days starting from the age of 6 months. We showed that oral administration of MF 6 significantly improved motor function assessed in a pole test, and reduced αSyn aggregation levels in both cerebellum and basal ganglia of PLP-αSyn mice. Moreover, MF 6 administration decreased oxidative stress and inflammation levels, and improved myelin levels and Purkinje neuron morphology in the cerebellum. By using mouse brain tissue slices and αSyn aggregates-treated KG-1C cells, we demonstrated that MF 6 reduced αSyn propagation to Purkinje neurons and oligodendrocytes through regulating endocytosis. Overall, these results suggest that MF 6 improves cerebellar functions in MSA by inhibiting αSyn aggregation and propagation. We conclude that MF 6 is a promising compound that warrants further development for the treatment of MSA.

Similar neuronal imprint and no cross-seeded fibrils in \u03b1-synuclein aggregates from MSA and Parkinson\u2019s disease

Aggregated alpha-synuclein (α-syn) is a principal constituent of Lewy bodies (LBs) and glial cytoplasmic inclusions (GCIs) observed respectively inside neurons in Parkinson’s disease (PD) and oligodendrocytes in multiple system atrophy (MSA). Yet, the cellular origin, the pathophysiological role, and the mechanism of formation of these inclusions bodies (IBs) remain to be elucidated. It has recently been proposed that α-syn IBs eventually cause the demise of the host cell by virtue of the cumulative sequestration of partner proteins and organelles. In particular, the hypothesis of a local cross-seeding of other fibrillization-prone proteins like tau or TDP-43 has also been put forward. We submitted sarkosyl-insoluble extracts of post-mortem brain tissue from PD, MSA and control subjects to a comparative proteomic analysis to address these points. Our studies indicate that: (i) α-syn is by far the most enriched protein in PD and MSA extracts compared to controls; (ii) PD and MSA extracts share a striking overlap of their sarkosyl-insoluble proteomes, consisting of a vast majority of mitochondrial and neuronal synaptic proteins, and (iii) other fibrillization-prone protein candidates possibly cross-seeded by α-syn are neither found in PD nor MSA extracts. Thus, our results (i) support the idea that pre-assembled building blocks originating in neurons serve to the formation of GCIs in MSA, (ii) show no sign of amyloid cross-seeding in either synucleinopathy, and (iii) point to the sequestration of mitochondria and of neuronal synaptic components in both LBs and GCIs.

Alpha-Synuclein Inclusion Formation in Human Oligodendrocytes.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by presence of α-synuclein-positive inclusions in the cytoplasm of oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are considered an integral part of the pathogenesis of MSA, leading to demyelination and neuronal demise. What is most puzzling in the research fields of GCIs is the origin of α-synuclein aggregates in GCIs, since adult oligodendrocytes do not express high levels of α-synuclein. The most recent leading hypothesis is that GCIs form via transfer and accumulation of α-synuclein from neurons to oligodendrocytes. However, studies regarding this subject are limited due to the absence of proper human cell models, to demonstrate the entry and accumulation of neuronal α-synuclein in human oligodendrocytes. Here, we generated mature human oligodendrocytes that can take up neuron-derived α-synuclein and form GCI-like inclusions. Mature human oligodendrocytes are derived from neural stem cells via “oligosphere” formation and then into oligodendrocytes, treating the cells with the proper differentiation factors at each step. In the final cell preparations, oligodendrocytes consist of the majority population, while some astrocytes and unidentified stem cell-like cells were present as well. When these cells were exposed to α-synuclein proteins secreted from neuron-like human neuroblastoma cells, oligodendrocytes developed perinuclear inclusion bodies with α-synuclein immunoreactivity, resembling GCIs, while the stem cell-like cells showed α-synuclein-positive, scattered puncta in the cytoplasm. In conclusion, we have established a human oligodendrocyte model for the study of GCI formation, and the characterization and use of this model might pave the way for understanding the pathogenesis of MSA.

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.

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.

Relocation of p25α/tubulin polymerization promoting protein from the nucleus to the perinuclear cytoplasm in the oligodendroglia of sporadic and COQ2 mutant multiple system atrophy.

p25α/tubulin polymerization promoting protein (TPPP) is an oligodendroglial protein that plays crucial roles including myelination, and the stabilization of microtubules. In multiple system atrophy (MSA), TPPP is suggested to relocate from the myelin sheath to the oligodendroglial cell body, before the formation of glial cytoplasmic inclusions (GCIs), the pathologic hallmark of MSA. However, much is left unknown about the re-distribution of TPPP in MSA. We generated new antibodies against the N- and C-terminus of TPPP, and analyzed control and MSA brains, including the brain of a familial MSA patient carrying homozygous mutations in the coenzyme Q2 gene (COQ2). In control brain tissues, TPPP was localized not only in the cytoplasmic component of the oligodendroglia including perinuclear cytoplasm and peripheral processes in the white matter, but also in the nucleus of a fraction (62.4%) of oligodendroglial cells. Immunoelectron microscopic analysis showed TPPP in the nucleus and mitochondrial membrane of normal oligodendroglia, while western blot also supported its nuclear and mitochondrial existence. In MSA, the prevalence of nuclear TPPP was 48.6% in the oligodendroglia lacking GCIs, whereas it was further decreased to 19.6% in the oligodendroglia with phosphorylated α-synuclein (pα-syn)-positive GCIs, both showing a significant decrease compared to controls (62.4%). In contrast, TPPP accumulated in the perinuclear cytoplasm where mitochondrial membrane (TOM20 and cytochrome C) and fission (DRP1) proteins were often immunoreactive. We conclude that in MSA-oligodendroglia, TPPP is reduced, not only in the peripheral cytoplasm, but also in the nucleus and relocated to the perinuclear cytoplasm.

Immunohistochemical localization of aggresomal proteins in glial cytoplasmic inclusions in multiple system atrophy

Multiple system atrophy (MSA) is pathologically characterized by the formation of α-synuclein-containing glial cytoplasmic inclusions (GCIs) in oligodendrocytes. However, the mechanisms of GCI formation are not fully understood. Cellular machinery for the formation of aggresomes has been linked to the biogenesis of the Lewy body, a characteristic α-synuclein-containing inclusion of Parkinson's disease and dementia with Lewy bodies. Here, we examined whether GCIs contain the components of aggresomes by immunohistochemistry. Sections from five patients with MSA were stained immunohistochemically with antibodies against aggresome-related proteins and analysed in comparison with sections from five patients with no neurological disease. We evaluated the presence or absence of aggresome-related proteins in GCIs by double immunofluorescence and immunoelectron microscopy. GCIs were clearly immunolabelled with antibodies against aggresome-related proteins, such as γ-tubulin, histone deacetylase 6 (HDAC6) and 20S proteasome subunits. Neuronal cytoplasmic inclusions (NCIs) were also immunopositive for these aggresome-related proteins. Double immunofluorescence staining and quantitative analysis demonstrated that the majority of GCIs contained these proteins, as well as other aggresome-related proteins, such as Hsp70, Hsp90 and 62-kDa protein/sequestosome 1 (p62/SQSTM1). Immunoelectron microscopy demonstrated immunoreactivities for γ-tubulin and HDAC6 along the fibrils comprising GCIs. Our results indicate that GCIs, and probably NCIs, share at least some characteristics with aggresomes in terms of their protein components. Therefore, GCIs and NCIs may be another manifestation of aggresome-related inclusion bodies observed in neurodegenerative diseases.

Role of TPPP/p25 on α-synuclein-mediated oligodendroglial degeneration and the protective effect of SIRT2 inhibition in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder presenting variable combinations of parkinsonism, cerebellar ataxia, corticospinal and autonomic dysfunction. Alpha-synuclein (α-SYN)-immunopositive glial cytoplasmic inclusions (GCIs) represent the neuropathological hallmark of MSA, and tubulin polymerization promoting protein (TPPP)/p25 in oligodendroglia has been known as a potent stimulator of α-SYN aggregation. To gain insight into the molecular pathomechanisms of GCI formation and subsequent oligodendroglial degeneration, we ectopically expressed α-SYN and TPPP in HEK293T and oligodendroglial KG1C cell lines. Here we showed that TPPP specifically accelerated α-SYN oligomer formation and co-immunoprecipitation analysis revealed the specific interaction of TPPP and α-SYN. Moreover, phosphorylation of α-SYN at Ser-129 facilitated the TPPP-mediated α-SYN oligomerization. TPPP facilitated α-SYN-positive cytoplasmic perinuclear inclusions mimicking GCI in both cell lines; however, apoptotic cell death was only observed in KG1C cells. This apoptotic cell death was partly rescued by sirtuin 2 (SIRT2) inhibition. Together, our results provide further insight into the molecular pathogenesis of MSA and potential therapeutic approaches.

Selective Nuclear Shrinkage of Oligodendrocytes Lacking Glial Cytoplasmic Inclusions in Multiple System Atrophy: A 3-Dimensional Volumetric Study

Glial cytoplasmic inclusions (GCIs) are a pathologic hallmark of multiple system atrophy (MSA), but their pathogenetic roles need to be clarified. To determine possible roles of GCIs in individual cells, serial optical sections obtained by confocal microscopy were reconstructed to yield 3-dimensional (3D) images of the nuclei to quantify nuclear volume. Oligodendroglial nuclear volumes were determined in the pons of 6 MSA and 7 control patients. The nuclear volumes were significantly smaller in the MSA group as a whole (135.81 +/- 60.83 microm, mean +/- SD; n = 404) than in the control group (188.05 +/- 55.71 microm; n = 308; p < 0.001). This difference was due to a significantly smaller nuclear volume of oligodendrocytes without GCIs (GCI group, 91.26 +/- 23.77 microm; n = 210) compared with the control group (p < 0.001) and compared with the oligodendrocytes with GCIs (GCI group, 184.03 +/- 51.18 microm; n = 194; p < 0.001); the difference between the latter GCI and control groups was not significant (p > 0.05). This selective decrease in nuclear volume restricted to the GCI group cannot be explained if nuclear shrinkage was accelerated in the presence of GCIs. Conversely, GCI formation might be linked, either directly or indirectly, to a mechanism that counteracts rather than accelerates nuclear shrinkage. This novel 3-dimensional strategy provides pivotal data that link GCI formation and degeneration in MSA.

LRRK2 and parkin immunoreactivity in multiple system atrophy inclusions

Certain genetic defects in LRRK2 and parkin are pathogenic for Parkinson's disease (PD) and both proteins deposit in the characteristic Lewy bodies. LRRK2 is thought to be involved in the early initiation of Lewy bodies. The involvement of LRRK2 and parkin in the similar cellular deposition of fibrillar alpha-synuclein in glial cytoplasmic inclusions (GCI) in multiple system atrophy (MSA) has not yet been assessed. To determine whether LRRK2 and parkin may be similarly associated with the abnormal deposition of alpha-synuclein in MSA GCI, paraffin-embedded sections from the basal ganglia of 12 patients with MSA, 4 with PD and 4 controls were immunostained for LRRK2, parkin, alpha-synuclein and oligodendroglial proteins using triple labelling procedures. The severity of neuronal loss was graded and the proportion of abnormally enlarged oligodendroglia containing different combinations of proteins assessed in 80-100 cells per case. Parkin immunoreactivity was observed in only a small proportion of GCI. In contrast, LRRK2 was found in most of the enlarged oligodendroglia in MSA and colocalised with the majority of alpha-synuclein-immunopositive GCI. Degrading myelin sheaths containing LRRK2-immunoreactivity were also observed, showing an association with one of the earliest oligodendroglial abnormalities observed in MSA. The proportion of LRRK2-immunopositive GCI was negatively associated with an increase in neuronal loss and alpha-synuclein-immunopositive dystrophic axons. Our results indicate that an increase in LRRK2 expression occurs early in association with myelin degradation and GCI formation, and that a reduction in LRRK2 expression in oligodendroglia is associated with increased neuronal loss in MSA.

Multiple system atrophy: A primary oligodendrogliopathy

To this day, the cause of multiple system atrophy (MSA) remains stubbornly enigmatic. A growing body of observations regarding the clinical, morphological, and biochemical phenotypes of MSA has been published, but the interested student is still left without a clue as to its underlying cause. MSA has long been considered a rare cousin of Parkinson's disease and cerebellar degeneration; it is rich in acronyms but poor in genetic and environmental leads. Because of the worldwide research efforts conducted over the last two decades and the discovery of the alpha-synuclein-encoding SNCA gene as a cause of rare familial Parkinson's disease, the MSA field has seen advances on three fronts: the identification of its principal cellular target, that is, oligodendrocytes; the characterization of alpha-synuclein-rich glial cytoplasmic inclusions as a suitable marker at autopsy; and improved diagnostic accuracy in living patients resulting from detailed clinicopathological studies. The working model of MSA as a primary glial disorder was recently strengthened by the finding of dysregulation in the metabolism of myelin basic protein and p25alpha, a central nervous system-specific phosphoprotein (also called tubulin polymerization promoting protein, TPPP). Intriguingly, in early cases of MSA, the oligodendrocytic changes in myelin basic protein and p25alpha processing were recorded even before formation of glial cytoplasmic inclusions became detectable. Here, we review the evolving concept that MSA may not just be related to Parkinson's disease but also share traits with the family of demyelinating disorders. Although these syndromes vary in their respective cause of oligodendrogliopathy, they have in common myelin disruption that is often followed by axonal dysfunction.

Transient overexpression of alpha-Synuclein in OLN-93 oligodendroglial cells is not cytotoxic and not affected by HSP70

Alpha-Synuclein (alpha-syn) accumulates in neurons and glia in a number of neurodegenerative disorders. The cytotoxicity and solubility of alpha-syn can be modified by the molecular chaperone HSP70. In multiple system atrophy (MSA), alpha-syn-positive glial cytoplasmic inclusions (GCIs) originating in oligodendrocytes are specifically prominent. Alpha-Synuclein is not detectable in mature oligodendrocytes in the normal brain, but we have shown previously that it is present in cultured rat brain oligodendrocytes [1] and down-regulated as the cultures mature. Hence, GCI formation might be causally related to a specific upregulation of the protein during pathological situations.

Analysis of alpha-synuclein aggregation and associated pathologies in a cellular model of multiple system atrophy

Intracellular aggregation of alpha-synuclein is a neuropathological hallmark of a variety of neurodegenerative diseases, including Parkinson's disease and multiple system atrophy (MSA). The deposition of the normally neuronal alpha-synuclein protein within (oligodendro)glial cytoplasmic inclusions (GCIs) and its relationship with oligodendrocyte death in MSA have not been elucidated. We developed a cellular model that recapitulates features of the degenerative process that occurs in MSA. Ectopic human alpha-synuclein expression in primary oligodendrocytes was achieved by deriving cultures from transgenic mice, which express human alpha-synuclein under the control of the myelin proteolipid protein promoter. High level of alpha-synuclein expression in oligodendrocytes induced alpha-synuclein accumulation, proteasome dysfunction and alters their sensitivity to proteasome inhibition. The large perinuclear inclusions formed, displayed biochemical and histological features of GCIs including the presence of phosphorylated and oxidized forms of alpha-synuclein as well as components of the ubiquitin-proteasome system and molecular chaperones. The fibrillar (amyloid) nature of alpha-synuclein inclusions was indicated by thioflavin S staining and proven by immunoelectron microscopy. Under these conditions, alpha-synuclein accumulation was accompanied by oligodendrocyte apoptosis, as evidenced by caspase-3 activation and chromatin condensation. These effects were not due to secondary effects of proteasome inhibitors, because high-level over-expression by lentiviral delivery of alpha-synuclein alone, but not beta-synuclein, caused protein aggregation and cell death. Moreover, lenti-beta-synuclein prevented aggregation in alpha-synuclein transgenic oligodendrocytes challenged with proteasome inhibitor. Thus, induction of alpha-synuclein and pathological impairment of the ubiquitin-proteasome system may synergistically contribute to GCI formation and ultimately cause oligodendrocyte dysfunction and cell death in MSA.

Progressive accumulation of ubiquitin and disappearance of α-synuclein epitope in multiple system atrophy-associated glial cytoplasmic inclusions: triple fluorescence study combined with Gallyas-Braak method

Alpha-synuclein (alphaS) and ubiquitin (Ub) are shared constituents of glial cytoplasmic inclusions (GCIs) and Lewy bodies (LBs), both composed of fibrillary structures. Staining profiles of GCIs were investigated with triple immunofluorescence involving immunostaining for alphaS and Ub, both amplified with catalyzed reporter deposition, and a fluorochrome, thiazin red (TR) that has an affinity to fibrillary structures. After observation for the triple-fluorescent images, the sections were subsequently stained with the Gallyas-Braak method. Sections of putamen, cerebellar white matter and motor cortex from patients suffering from multiple system atrophy (MSA) with varying duration of the disease (4-15 years) were quantified for these staining profiles of Gallyas-positive GCIs. Although most of GCIs were positive for Ub and variably positive for alphaS, they were consistently negative for TR. The result was opposite in LBs in Lewy body disease with variable affinity to TR, suggesting that the construction of GCIs is different from that of LBs. These four staining features (alphaS, Ub, TR and Gallyas) alone failed to exhibit apparent correlation with disease duration, lesion site or severity of degeneration as reported previously. The fraction of alphaS-negative and Ub-positive GCIs, however, linearly increased along the disease progression, while that of alphaS-positive and Ub-negative GCIs decreased in contrast. This reciprocal change suggests that alphaS immunoreactivity in GCIs is being replaced by Ub immunoreactivity during the disease progression, which resulted in the ultimate predominance of alphaS-negative and Ub-positive GCIs in the most advanced case. Interestingly, this predominance of alphaS-negative and Ub-positive GCIs was a feature of motor cortex, where degeneration usually remains mild in spite of robust appearance of Gallyas-positive GCIs. Another fraction, alphaS-positive and Ub-positive GCIs were frequent in cerebellar white matter, suggesting that GCI evolution is heterogeneous and dependent also on area examined. Progressive accumulation of Ub with concomitant disappearance of alphaS epitope and their colocalization, partly shared with LBs, may represent a process of GCI formation, possibly linked to an aspect of degeneration in MSA.

P39 immunoreactivity in glial cytoplasmic inclusions in brains with multiple system atrophy.

Glial cytoplasmic inclusions (GCIs) characteristically occur in the oligodendrocytes of patients with multiple system atrophy (MSA). Cyclin-dependent kinase 5 (Cdk5) regulates cytoskeletal dynamism through phosphorylation of cytoskeletal proteins such as neurofilament proteins, tau and microtubule-associated protein 2. We examined the immunohistochemical localization of p39, a Cdk5 activator, in human brain specimens obtained post mortem from controls and patients with MSA. Among control specimens, p39 immunoreactivity was found in some neuronal somata and axons. Similar immunoreactivity was detected in MSA neuronal cell bodies and axons, but a number of inclusions were heavily labeled. These results suggest that p39 and Cdk5 are up-regulated in oligodendrocytes and may be involved in the formation of GCIs through phosphorylation of cytoskeletal proteins in oligodendrocytes in MSA.

Cyclin-dependent kinase 5 and mitogen-activated protein kinase in glial cytoplasmic inclusions in multiple system atrophy.

Glial cytoplasmic inclusions (GCI) characteristically occur in the oligodendrocytes of patients with multiple system atrophy (MSA). However, the molecular mechanisms underlying GCI formation are unknown. To investigate whether these inclusions are related to proline-directed protein kinases that have been associated with neuronal inclusion bodies in some other neurodegenerative diseases, we immunohistochemically probed tissue samples from MSA brains with a panel of antibodies against cyclin-dependent kinases and mitogen-activated protein kinase. We unexpectedly detected cyclin-dependent kinase 5- (cdk5) and mitogen-activated protein kinase- (MAPK) immunoreactivities in GCI. We also found TAU1 immunoreactivity in GCI, and a strong expression of microtubule-associated protein (MAP) 2 immunoreactivity in oligodendrocytes of MSA brains. This immunoreactivity was not observed in the normal or neurological controls. The accumulated evidence suggest a close association between GCI and the microtubular cytoskeleton. Cdk5 phosphorylates tau and MAP2, and MAPK is capable of phosphorylating MAP2. The present results suggest that the aberrant or ectopic expression of cdk5 and MAPK causes abnormal phosphorylation of microtubular cytoskeletal proteins, thus leading to GCI formation in affected oligodendrocytes.