Disruption of the interaction of alpha-synuclein with microtubules enhances cell surface recruitment of the dopamine transporter.
Mutations in alpha-synuclein have been implicated in the genesis of Parkinson's disease. A probable normative function of alpha-synuclein is the maintenance of dopamine homeostasis, partly through a negative modulation of dopamine transporter (DAT) activity, by reducing its level at the cell surface. To study the possible involvement of the microtubular network in the alpha-synuclein-dependent trafficking of DAT, we treated cotransfected cells and primary mesencephalic neurons with either colchicine, vinblastine, or nocodazole, each of which disrupts microtubules or affects microtubule dynamics. Treatment of both types of cells with vinblastine, colchicine, or nocodazole reversed alpha-synuclein-mediated inhibition of DAT activity, resulting in an increased rate of dopamine uptake and and increased level of extracellular dopamine-induced oxidative stress, with accelerated cell death. Treatment with these agents also reversed the alpha-synuclein-induced decrease in levels of cell surface-associated DAT. This effect of colchicine, vinblastine, or nocodazole was not linked to a disruption of formation of the alpha-synuclein-DAT complex but paradoxically caused an increased level of interaction between these proteins. Both alpha-synuclein and DAT co-immunoprecipitated with both alpha- and beta-tubulins, in both transfected cells and rat primary mesencephalic dopaminergic neurons, suggesting heteromeric complex formation between these various proteins. Treatment with the microtubule depolymerizing agents disrupted the heteromeric protein complex between either alpha-synuclein or the DAT, and alpha- or beta-tubulins. These results indicate a previously unappreciated role of microtubules in the modulation of DAT trafficking, and provide insight into a novel mechanism by which alpha-synuclein regulates DAT activity, by tethering the transporter to the microtubular network.
Wersinger
Christophe
C
Department of Pediatrics, Georgetown University, Washington, DC 20007, USA.
Sidhu
Anita
A
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D051381:Rats; D013378:Substantia Nigra / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D014747:Vinblastine / Q000494:pharmacology; D051844:alpha-Synuclein / Q000187:drug effects / Q000378:metabolism*
D050483:Dopamine Plasma Membrane Transport Proteins; D014404:Tubulin; D051844:alpha-Synuclein; D014747:Vinblastine; D015739:Nocodazole; D003078:Colchicine; D004298:Dopamine