Objective:  The main purposes of this study were to: 1) investigate the possible effect of extracellular alpha-synuclein (aSYN) on the endocytic rate of dopamine transporter (DAT) and 2) determine whether the endocytic process of DAT could influence on aSYN internalization.

Background: DAT is the transmembrane protein that takes extracellular dopamine back up into dopaminergic neurons. DAT is distributed in both membrane raft and non-raft domains and the number of DATs on cell surface is controlled under the balance between endocytosis and recycling. Flotllin-1 (FLOT1), a caveolae-associated integral membrane protein, is essential for the localization of DAT within membrane microdomains.

Methods: HEK 293 cells stably expressing GFP-DAT were incubated with conditioned medium containing human monomeric aSYN for 30 minutes. Distinct endosomal structures were visualized by co-expression of mStrawberry-tagged Rab GTPases. Time-lapse imaging was performed to explore the spatiotemporal distribution of aSYN and DAT. Lipid rafts were isolated by floatation in a 5-30 % non-linear sucrose density gradient. Co-immunoprecipitation was adopted to demonstrate the molecular interaction between FLOT1 and DAT.

Results: Over-expressed GFP-DAT was localized mainly on plasma membrane, and to a much lesser degree, in the cytoplasm. Surprisingly, in the presence of extracellular aSYN, DAT was actively endocytosed and accumulated in Rab5-positine early endosome together with aSYN. Soon after reaching the early endosome, DAT was re-localized to the plasma membrane via both Rab4- and/or Rab11-mediated recycling pathway, while internalized aSYN was left behind in Rab7-mediated endo-lysosomal compartment. We further demonstrated that after addition of aSYN to the medium, DAT was dissociated from FLOT1 and shifted out of lipid raft compartment.

Conclusions: Extracellular aSYN induces re-distribution of DAT from raft to non-raft domain of cellular membrane, which possibly facilitates DAT endocytosis. Concomitantly, aSYN internalizes into cells by hijacking endocytic trafficking of DAT. These findings may suggest the possible role of DAT on cell-type specific pathological consequences in Synucleiopathies.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/extracellular-alpha-synuclein-internalizes-into-cells-by-hijacking-endocytic-trafficking-of-dopamine-transporter/