Abstract
α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein-protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1P1R) from the lipid raft fractions. S1P1R regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1P1R became refractory to S1P stimulation required for activating inhibitory G-protein (Gi) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1P1R on internal vesicles, resulting in the reduced amount of CD63 molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn-treated cells. Furthermore, cholesterol-depleting agent-induced S1P1R expulsion from the rafts also resulted in S1P1R uncoupling. Taken together, these results suggest that extracellular α-Syn-induced expulsion of S1P1R from lipid rafts promotes the uncoupling of S1P1R from Gi, thereby blocking subsequent Gi signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.
Highlights
␣-Synuclein (␣-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson’s disease (PD)
These results suggest that extracellular ␣-Syn–induced expulsion of S1P1R from lipid rafts promotes the uncoupling of S1P1R from Gi, thereby blocking subsequent Gi signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes
Because many signaling molecules are concentrated in cholesterol and sphingolipid-rich membrane microdomains known as lipid rafts for their action, the effect of ␣-Syn(A53T), found in hereditary PD, on the distribution of S1P1R in the lipid rafts was assessed by a conventional fractionation analysis
Summary
Because many signaling molecules are concentrated in cholesterol and sphingolipid-rich membrane microdomains known as lipid rafts for their action, the effect of ␣-Syn(A53T), found in hereditary PD, on the distribution of S1P1R in the lipid rafts was assessed by a conventional fractionation analysis. The amount of S1P2R, another subtype of the receptor known to be expressed in this cell line [28], in the raft fractions was not changed by ␣-Syn(A53T) treatment (Fig. 1B, hatched bars in fraction 2) These results suggest that ␣-Syn(A53T) selectively drives S1P1R out of the lipid rafts whereas preserving raft structures. Neuraminidase treatment resulted in the abrogation of the ability of ␣-Syn(A53T) to drive S1P1R out of the raft fractions (Fig. 4, hatched green bars in the raft fractions), indicating gangliosides as a receptor or binding partner of ␣-Syn(A53T) to elicit pathophysiological responses including expulsion of S1P1R from the raft fractions. Upon stimulation by S1P, these subunits dissociate, and the S1P receptor-CFP and G␥-YFP become associated (S1P1R/G␥ ϩ G␣ form high FRET) [25] Under both control and ␣-Syn(A53T)–treated conditions, these fluoroprobe-fused proteins were distributed both in plasma membranes and CD63-positive MVEs (data not shown). Syn(A53T)–treated cells contained reduced CD63-mCherry as compared with untreated cells (Fig. 8), confirming the notion that extracellular ␣-Syn(A53T) causes S1P1R uncoupled from Gi on MVEs and inhibits cargo sorting into ILVs of exosomal MVEs
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