Extracellular α-synuclein drives sphingosine 1-phosphate receptor subtype 1 out of lipid rafts, leading to impaired inhibitory G-protein signaling

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 (G_i) 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 G_i, thereby blocking subsequent G_i signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.

Keywords: ganglioside; lipid raft; signal transduction; sphingosine-1-phosphate (S1P); α-synuclein (α-synuclein).

Figure 1.

Expulsion of S1P₁R from the lipid raft fractions by extracellular α-Syn(A53T). A, SH-SY5Y cells expressing S1P₁R-YFP were incubated without (closed black bars) or with 1 μm α-Syn(A53T) for 18 h (hatched bars) or 0.2 mm MBCD (closed gray bars) for 2 h. Cells were lysed and the lipid raft fractions were separated as described under “Experimental procedures.” The amount of S1P₁R in each fraction was measured using a fluorescence spectrophotometer. Values represent mean ± S.E. of three independent experiments carried out in triplicate. B, SH-SY5Y cells expressing S1P₂R-GFP were incubated with or without 1 μm α-Syn(A53T) for 18 h or 0.2 mm MBCD for 2 h, followed by fractionation and measurement of fluorescence as in (A). Values represent mean ± S.E. of three independent experiments carried out in triplicate. C, SH-SY5Y cells were incubated without (closed black bars) or with 1 μm α-Syn(A53T) (hatched bars) for 18 h or 0.2 mm MBCD (closed gray bars) for 2 h, followed by fractionation. The amount of GM1 in each fraction was measured by dot blot assay using HRP-conjugated CTB (inset) as described under “Experimental procedures.” Values represent mean ± S.E. of three independent experiments carried out in triplicate. Statistical significance was analyzed by Student's t test (*, p < 0.05; **, p < 0.01 versus vehicle control).

Figure 2.

Detection of S1P₁R in the lipid raft fractions by a FRET technique. A, SH-SY5Y cells expressing flotillin 2-YFP were incubated without (vehicle control, closed bars) or with 1 μm α-Syn(A53T) (hatched bars) for 18 h, followed by lipid raft separation and quantification of the fluorescence in each fraction as in Fig. 1A. Values represent mean ± S.E. of three independent experiments carried out in triplicate. B, SH-SY5Y cells expressing both S1P₁R-CFP and flotillin 2-YFP were incubated without (vehicle control) or with either 1 μm α-Syn(A53T) or WT α-Syn for 18 h. Cells were fixed and analyzed for FRET efficiency in the plasma membrane areas using the acceptor photobleaching method. Results are expressed as median on scatter-dot plots (n ≥ 50). Statistical significance was analyzed by Student's t test (**, p < 0.01 versus vehicle control).

Figure 3.

Analysis of extracellular α-Syn–induced expulsion of S1P₁R from the lipid raft fractions as a function of incubation time and α-Syn doses. A, SH-SY5Y cells expressing both S1P₁R-CFP and flotillin 2-YFP were incubated with 1 μm α-Syn(A53T) for various time intervals. FRET efficiency in the plasma membrane areas was measured as described in the legend to Fig. 2B. Values represent mean ± S.E. (n ≥ 50). Statistical significance was analyzed by Student's t test (**, p < 0.01 versus vehicle control). B, cells were incubated with 1 μm α-Syn(A53T) for various time intervals as in A. After removal of the medium, the cells were scraped and disrupted by sonication. After removal of cell debris by low-speed centrifugation, the lysates were subsequently centrifuged at 100,000 × g for 30 min. The clarified medium and the pellets after high-speed centrifugation (cell membranes) were subjected to immunoblot analysis using anti–α-Syn antibody. Note that dimeric forms of the protein increased during incubation. The results are the representative of 3 independent experiments. C, 1 μm α-Syn(A53T) was incubated in Dulbecco's modified Eagle's medium/F-12 medium for 0 h (control) or 24 h at 37 °C (incubated). Samples were then subjected to immunoblot analysis using anti–α-Syn antibody. Note that dimeric and oligomeric forms of the protein increased during incubation. The results are the representative of 3 independent experiments. D, SH-SY5Y cells expressing both S1P₁R-CFP and flotillin 2-YFP were incubated for 40 min without (vehicle control) or with either 1 μm α-Syn(A53T) or preincubated α-Syn(A53T) as described in C. Cells were fixed and analyzed for FRET efficiency in the plasma membrane areas using an acceptor photobleaching method. Results are expressed as median on scatter-dot plots (n ≥ 50). Statistical analysis between nonincubated and preincubated samples showed nonsignificant (Student's t test, p = 0.3 versus nonincubated). E, SH-SY5Y cells expressing both S1P₁R-CFP and flotillin 2-YFP were incubated with various concentrations of either α-Syn(A53T) or WT α-Syn for 18 h. Cells were fixed and analyzed for FRET efficiency in the plasma membrane areas as in Fig. 2B. Values represent mean ± S.E. (n ≥ 50). Statistical significance was analyzed by Student's t test (**, p < 0.01; *, p < 0.05 versus vehicle control).

Figure 4.

Role of gangliosides in the action of extracellular α-Syn(A53T). A, SH-SY5Y cells expressing S1P₁R-YFP were incubated without (closed black bars) or with 1 μm α-Syn(A53T) (hatched orange bars) or α-Syn(A53T)-AAA (closed blue bars) for 18 h. In some experiments cells were pretreated with 3 milliunits/ml of neuraminidase (neu) for 1 h before treatment of 1 μm α-Syn(A53T) (hatched green bars). Cells were subjected to lipid raft separation and fluorescence intensity was measured in each fraction as described in the legend to Fig. 1A. Values represent mean ± S.E. of three independent experiments carried out in triplicate. Statistical significance was analyzed by Student's t test (**, p < 0.01). B, SH-SY5Y cells expressing both S1P₁R-CFP and flotillin 2-YFP were incubated without (vehicle control, open circles) or with either 1 μm α-Syn(A53T) or α-Syn(A53T)-AAA for 18 h. In some experiments cells were pretreated with 3 milliunits/ml of neuraminidase (neu) for 1 h before treatment of 1 μm α-Syn(A53T). Cells were fixed and analyzed for FRET efficiency in the plasma membrane areas using acceptor photobleaching method. Results are expressed as median on scatter-dot plots (n ≥ 50). Statistical significance was analyzed by Student's t test (*, p < 0.05 versus α-Syn(A53T)).

Figure 5.

Rescue by gangliosides of extracellular α-Syn–induced expulsion of S1P₁R from the lipid raft fractions. SH-SY5Y cells expressing both S1P₁R-CFP and flotillin 2-YFP were incubated without (vehicle control, open circles) or with 1 μm α-Syn(A53T) in the presence of various concentrations of gangliosides for 18 h. Cells were fixed and analyzed for FRET efficiency in the plasma membrane areas as described in the legend to Fig. 2B. Results are expressed as median on scatter-dot plots (n ≥ 20). Statistical significance was analyzed by Student's t test (**, p < 0.01; *, p < 0.05).

Figure 6.

α-Syn(A53T)–induced uncoupling of S1P₁R from G_i both in the plasma membranes and MVEs. A, SH-SY5Y cells expressing S1P₁R-CFP, Gβ, Gγ-YFP, FLAG-Rab5(Q97L), and CD63-mCherry were pretreated without (vehicle control) or with 1 μm α-Syn(A53T) for 18 h and then stimulated with 100 nm S1P for 1 min, fixed, and analyzed for FRET efficiencies in the plasma membrane or MVE areas. Results are expressed as median on scatter-dot plots (n ≥ 20). Statistical significance was analyzed by Student's t test (**, p < 0.01; *, p < 0.05). B, cells expressing S1P₁R-CFP, Gβ, and Gγ-YFP were pretreated without (vehicle control) or with 0.2 mm MBCD for 2 h and then stimulated with 100 nm S1P for 1 min, fixed, and analyzed for FRET efficiencies in the plasma membrane areas. Results are expressed as median on scatter-dot plots (n ≥ 20). Statistical significance was analyzed by Student's t test (**, p < 0.01).

Figure 7.

α-Syn(A53T)–induced inhibition of exosomal cargo sorting into MVEs. A, SH-SY5Y cells expressing both CD63-mCherry and GFP-Rab5(Q79L) were incubated without (vehicle control) or with 1 μm α-Syn(A53T) for 18 h, fixed, and followed by MVE cargo sorting analysis as illustrated in B. Results are expressed as median on scatter-dot plots (n ≥ 33 endosomes; **, p < 0.01 versus control; Student's t test). B, schematic representation of MVE cargo sorting analysis.

Figure 8.

α-Syn(A53T)–induced reduction of cargo content in purified exosomes. A, SH-SY5Y cells expressing CD63-mCherry were incubated without (vehicle control) or with 1 μm α-Syn(A53T) for 18 h, followed by quantification of cargo content in purified exosomes. Exosomes prepared by a sequential centrifugation from cell culture media (5 × 10⁶ cells) were resuspended in equivolume buffer and labeled with DiD and immobilized on streptavidin-functionalized glass surface (see “Experimental procedures”). Images of CD63-mCherry and DiD fluorescence were acquired with a confocal laser scanning microscope. The fluorescence intensity of DiD and CD63-mCherry in DiD-labeled exosomes obtained from the images were plotted on the correlation diagram for each exosome. Note that the number of exosomes in the control and α-Syn(A53T) treatment were 4482 and 4951, respectively, indicating that α-Syn(A53T) treatment has little or no effect on the total number of exosomes. B, an average of cargo (CD63) content per each exosome (coefficient numbers of each red numerical formula in A) is represented. Statistical significance was analyzed by Student's t test (**, p < 0.01).