Abstract
Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated alpha-synuclein in oligodendrocytes. p25alpha is an oligodendroglial protein that potently stimulates alpha-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25alpha and alpha-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 alpha-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 alpha-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 alpha-syn pathway in oligodendroglial cells and may represent therapeutic intervention points in multiple system atrophy.
Highlights
␣-Synuclein (␣-syn)2 is a soluble protein localized in presynaptic terminals [1]
Coexpression of ␣-Synuclein and p25␣ Causes Microtubule Relocalization to the Perinuclear Region—To model the cellular effects of ␣-syn-dependent degeneration in Multiple system atrophy (MSA), we studied the effect of coexpressing ␣-syn and p25␣ in rat oligodendroglial OLN-93 cells
Phenotypes obtained with respect to ␣-syn aggregation and cell death have only been marginally successful, and many cell lines tolerate transgenic expression of human wt and mutant ␣-syn [46]
Summary
␣-Synuclein (␣-syn) 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 [2,3,4]. The degeneration of oligodendrocytes has been studied using transgenic mice expressing human ␣-syn under the control of oligodendroglial promoters [13,14,15]. These models have demonstrated that expression of ␣-syn in oligodendrocytes results in formation of ␣-syn-containing glial cytoplasmic inclusions, myelin damage, and cellular degeneration. Our results suggest that specific signaling pathways amenable for treatment at several levels may be involved in ␣-syn-dependent degeneration in MSA
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