In this multinuclear NMR study myo-inositol is identified as a glia-specific marker for in vivo NMR studies. The unusually high inositol concentration may participate in the osmoregulatory system in asupcytes. Primary astrocytes also synthesize and export high amounts of hypotaurine, an intermediate of taurine synthesis. Taurine – another osmolyte – is synthesized from cysteine by astrocytes but not by primary neurons. Taurine as well as hypotaurine is accumulated by neurons from the extracellular medium. ¹³C NMR labelling results with 2-¹³C pyruvate indicate a considerable contribution of the anaplerotic pathway in primary neurons from rat. The activity is only half of the activity in primary astrocytes. The ratio of pyruvate carboxy-lase/malic enzyme activity versus pyruvate dehydrogenase activity reflects the degree of maturation. The ¹³C isotopomer ratio of glutamate and glutamine is different for pure astrocyte cultures. Therefore, the different isotopomer ratios of glutamate to glutamine obtained from intact brain studies alone do not prove TCA cycle compartimentation in the brain. Finally, the PCr/ATP ratio in primary astrocytes is 3 times higher than in primary neurons. This has to be considered in case of recovery from ischemic insults.

Oligodendrocytes, the myelin-forming cells of the central nervous system, are in culture characterized by an elaborate process network, terminating in flat membranous sheets that are rich in myelin-specific proteins and lipids, and spirally wrap axons forming a compact insulating layer in vivo. By analogy with other cell types, maintenance and stability of these processes, as well as the formation of the myelin sheath, likely rely on a pronounced cytoskeleton consisting of microtubules and microfilaments. While the specialized process of wrapping and compaction forming the myelin sheath is not well understood, considerably more is known about how cytoskeletal organization is mediated by extracellular and intracellular signals and other interaction partners during oligodendrocyte differentiation and myelination. Here, we review the current state of knowledge on the role of the oligodendrocyte cytoskeleton in differentiation with an emphasis on signal transduction mechanisms and will attempt to draw out implications for its significance in myelination.

Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated ␣-synuclein in oligodendrocytes. p25␣ is an oligodendroglial protein that potently stimulates ␣-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25␣ and ␣-synuclein in the rat oligodendroglial cell line OLN-93 and observed a cellular response characterized by a fast retraction of microtubules from the cellular processes to the perinuclear region followed by a protracted development of apoptosis. This response was dependent on phosphorylation at Ser-129 in ␣-synuclein as demonstrated by site-directed mutagenesis. Treatment of the cells with the kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole that targets kinases like casein kinase 2, and polo-like kinases abrogated the toxicity. The polo-like kinase inhibitor BI 2536 caused apoptosis in the model. Ser-129 phosphorylation was linked to the formation of phosphorylated oligomers detectable by immunoblotting, and their formation was inhibited by 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole. The process of microtubule retraction was also dependent on aggregation as demonstrated by the protective effect of treating the cells with the specific peptide inhibitor of ␣-synuclein aggregation ASI1D and the non-selective inhibitors Congo Red and baicalein. The fast microtubule retraction was followed by the development of the apoptotic markers: activated caspase-3, phosphatidylserine externalization, nuclear condensation, and fragmentation. These markers could all be blocked by the inhibitors of phosphorylation, aggregation, and caspase-3. Hence, the model predicts that both Ser-129 phosphorylation and aggregation control the toxic ␣-syn pathway in oligodendroglial cells and may represent therapeutic intervention points in multiple system atrophy.2 is a soluble protein localized in presynaptic terminals (1). It accumulates as insoluble aggregates in cytoplasmic inclusions in ␣-synucleinopathies, among which Parkinson disease (PD) and dementia with Lewy bodies are the predominant members (2-4). Mutations in the ␣-syn gene leading to amino acid substitutions (A30P, A53T, and E46K) (5-7), and multiplications of the ␣-syn gene (8, 9) are associated with rare familial forms of PD and Lewy bodies.Multiple system atrophy (MSA) represents a special case among the ␣-synucleinopathies. The histological hallmark of MSA is the presence of ␣-syn-containing inclusions in oligodendrocytes, referred to as glial cytoplasmic inclusions (10, 11). Clinically, MSA presents with parkinsonism, ataxia, and autonomic failure, which signify the wide distribution of the degenerative processes (12). The degeneration of oligodendrocytes has been studied using transgenic mice expressing human ␣-syn under the control of oligodendroglial promoters (13-15). These models have demonstrated that expression of ␣-syn in oligodendrocytes results in formation of ␣-syn-containing glial cytoplasmic inclusions, myelin damage, and c...

Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are ␣-synucleinopathies characterized by filamentous ␣-synuclein (␣-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with ␣-syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with ␣-syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble ␣-syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like ␣-syn pathologies. Cell culture studies also revealed that ␣-syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of ␣-syn inclusions in PD and related

Oxidative and nitrative injury is implicated in the pathogenesis of Alzheimer's disease (AD) and Down syndrome (DS), but no direct evidence links this type of injury to the formation of neurofibrillary tau lesions. To address this, we generated a monoclonal antibody (mAb), n847, which recognizes nitrated tau and ␣-synuclein. n847 detected nitrated tau in the insoluble fraction of AD, corticobasal degeneration (CBD), and Pick's disease (PiD) brains by Western blots. Immunohistochemistry (IHC) showed that n847 labeled neurons in the hippocampus and neocortex of AD and DS brains. Double-label immunofluorescence with n847 and an anti-tau antibody revealed partial co-localization of tau and n847 positive tangles, while n847 immmunofluorescence and Thioflavin-S double-staining showed that a subset of n847-labeled neurons were Thioflavin-S-positive. In addition, immuno-electron microscopy revealed that taupositive filaments in tangle-bearing neurons were also labeled by n847 and IHC of other tauopathies showed that some of glial and neuronal tau pathologies in CBD, progressive supranuclear palsy, PiD, and frontotemporal dementia with parkinsonism linked to chromosome 17 also were n847-positive. Finally, nitrated and Thioflavin-S-positive tau aggregates were generated in a oligodendrocytic cell line after treatment with peroxynitrite. Taken together, these findings imply that nitrative injury is directly linked to the formation of filamentous tau inclusions. (Am J