Oligomeric α-synuclein inhibits tubulin polymerization

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Abstract

Earlier investigations have demonstrated that tubulin co-localizes with α-synuclein in Lewy bodies and influences the formation of α-synuclein aggregation. However, it is not clear whether aggregated α-synuclein has any effects on the function of tubulin, i.e. tubulin polymerization, a critical mechanism by which neurons maintain their morphology and execute functions. In this study, we evaluated the effects of aggregated α-synuclein on tubulin polymerization in dopaminergic neurons (MES cells), along with mitochondrial function, cell morphology, and viability. The results indicate that MES cells exposed to extracellular oligomeric α-synuclein exhibited decreased tubulin polymerization and mitochondrial function as well as morphological alternation long before cell death. Further investigation showed that internalization of oligomeric α-synuclein by neurons appeared to be critical in the process, although direct interaction between tubulin and intracellular oligomeric α-synuclein was not necessary. Finally, we demonstrated that neurotoxicity induced by oligomeric α-synuclein was largely prevented by overexpressing the neuroprotective protein, DJ-1.

Section snippets

Experimental procedures

Neurotoxicity induced by oligomeric α-synuclein. Oligomeric α-synuclein was produced using a modified protocol published by us recently [13]. Essentially, 100 μL of human recombinant α-synuclein (rPeptide, Athens, GA) was aged in vitro for 12 h with constant agitation using a magnetic stir bar at 37 °C. MES cells were cultured with the methods utilized routinely in our lab [14], and viability was tested with trypan blue (Invitrogen, Carlsbad, CA) viability assay at 1-, 2-, and 3-day intervals.

Toxicity of oligomeric α-synuclein and internalization of α-synuclein

We first investigated the effects of oligomeric α-synuclein on dopaminergic neurons. As shown in Fig. 1A, oligomeric α-synuclein decreased remaining viable neurons significantly over 3 days after treatment. More importantly, the majority of MES cells, though still viable as determined by trypan blue exclusion assay, displayed apparent morphological changes, including shrinkage of cell bodies and dendritic beading after a few hours of treatment. Representative MES cells exposed to oligomeric

Acknowledgments

The study is partially supported by NIH Grants to J.Z. (R01AG025327 and R01ES012703).

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