Sugeno N, Hasegawa T, Tanaka N, Fukuda M, Wakabayashi K, Oshima R, Konno M, Miura E, Kikuchi A, Baba T, Anan T, Nakao M, Geisler S, Aoki M, Takeda A

Alpha-synuclein (aS) is a major constituent of Lewy bodies, which are not only a pathological marker for Parkinson's disease (PD) but also a trigger for neurodegeneration. Cumulative evidence suggests that aS spreads from cell to cell and thereby propagates neurodegeneration to neighboring cells. Recently, Nedd4-1 (neural precursor cell expressed developmentally ...

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down-regulated protein 4-1), an E3 ubiquitin ligase, was shown to catalyze the K63-linked poly-ubiquitination of intracellular aS and thereby facilitate aS degradation by the endo-lysosomal pathway. Because Nedd4-1 exerts its activity in close proximity to the inner leaflet of the plasma membrane, we speculate that after the internalization of aS, membrane-resident aS is preferentially ubiquitinated by Nedd4-1. To clarify the role of Nedd4-1 in aS internalization and endo-lysosomal sequestration, we generated aS mutants, including ΔPR1 (1-119, 129-140), ΔC (1-119), and ΔPR2 (1-119, 134-140), that lack the PR (proline rich) sequence, a putative Nedd4-1 recognition site. We show that wild type (wt) aS, but not ΔPR1, ΔPR2, or ΔC aS, is modified by Nedd4-1 in vitro, acquiring a K63-linked ubiquitin chain. Compared with the mutants lacking the PR sequence, wt-aS is preferentially internalized and translocated to endosomes. The over-expression of Nedd4-1 increased aS in endosomes, whereas RNAi-mediated silencing of Nedd4-1 decreased endosomal aS. Although aS freely passes though plasma membranes within minutes, a pulse-chase experiment revealed that the over-expression of Nedd4-1 markedly decreased the re-secretion of internalized aS. Together, these findings demonstrate that Nedd4-1-linked K63 ubiquitination specifies the fate of extrinsic and de novo synthesized aS by facilitating their targeting to endosomes.

Date: May. 15, 2014

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