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Science 329 (5999): 1663-1667

Copyright © 2010 by the American Association for the Advancement of Science

{alpha}-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro

Jacqueline Burré1,*, Manu Sharma1,*, Theodoros Tsetsenis1, Vladimir Buchman2, Mark R. Etherton1, and Thomas C. Südhof1,3,{dagger}

1 Department of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University, 1050 Arastradero Road, Palo Alto, CA 94304–5543, USA.
2 School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK.
3 Howard Hughes Medical Institute, Stanford University, 1050 Arastradero Road, Palo Alto, CA 94304–5543, USA.


Figure 1
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Fig. 1. {alpha}-Synuclein directly binds to synaptobrevin-2/vesicle-associated membrane protein 2 (VAMP2) in SNARE complexes. (A to D) {alpha}-Synuclein associates with SNARE complexes immunoprecipitated with antibodies to SNAP-25 from WT and CSP{alpha} KO (CSP–/–) mouse brains containing or lacking transgenic {alpha}-synuclein (tSyn). Shown are (A) representative immunoblots (–Ab, control IP from WT brain without primary antibody; Synt-1, syntaxin-1; Syb2, synaptobrevin-2; {alpha}-Syn, {alpha}-synuclein), (B) quantitations of input levels, (C) levels of immunoprecipitated SNAP-25, and of (D) co-immunoprecipitated proteins, determined by means of quantitative immunoblotting by use of 125I-labeled secondary antibodies (means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 per Student’s t test; n = 3 to 5 mice). (E) Co-immunoprecipitation of {alpha}-synuclein with SNARE complexes reconstituted in transfected HEK293 T cells. Cell lysates were immunoprecipitated with antibodies to (left) {alpha}-synuclein or (right) SNAP-25 and analyzed by means of immunoblotting. (F and G) {alpha}-Synuclein C terminus directly binds to synaptobrevin-2 N terminus. {alpha}-Synuclein was immunoprecipitated from HEK293 T cells co-expressing full-length ({alpha}-Syn) or C-terminally truncated {alpha}-synuclein ({alpha}-Syn1-95) with full-length (Syb2) or N-terminally truncated synaptobrevin-2 (Syb229-116). Immunoprecipitates were analyzed by means of immunoblotting for {alpha}-synuclein and synaptobrevin-2 (figs. S1 and S2). (H) Diagram of the {alpha}-synuclein/synaptobrevin-2 complex on synaptic vesicles (SV). (I to K) Neither CSP{alpha} KO nor transgenic {alpha}-synuclein overexpression detectably alters synaptic strength. Data show sample traces [(I) and (K), top] and summary graphs [(J) and (K), bottom] (means ± SEM) of extracellular recordings from input-output measurements [(I) and (J)] or paired-pulse facilitation experiments (K) in acute hippocampal slices (fig. S3).

 

Figure 2
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Fig. 2. {alpha}-Synuclein directly catalyzes SNARE-complex assembly. (A to C) {alpha}-Synuclein promotes SNARE-complex assembly in a dose-dependent manner. HEK293 T cells were cotransfected with constant amounts of syntaxin-1 (Synt-1), synaptobrevin-2 (Syb2), and SNAP-25 and increasing amounts of {alpha}-synuclein ({alpha}-Syn). Cell lysates were immunoblotted (A) without and (fig. S4) with boiling; SNARE complexes and {alpha}-synuclein were (B) quantified and (C) plotted as a function of each other. (D and E) Full-length but not C-terminally truncated {alpha}-synuclein promotes SNARE-complex assembly. SNARE complexes were immunoprecipitated with antibodies to (left) SNAP-25 or (right) synaptobrevin-2 from HEK293 T cells co-expressing SNARE proteins with emerald (control), full-length {alpha}-synuclein ({alpha}-Syn), or C-terminally truncated {alpha}-synuclein ({alpha}-Syn1-95) and analyzed by means of immunoblotting [(D), representative blots; (E), SNARE-complex quantitation]. (F) Coomassie-stained SDS gel of purified recombinant {alpha}-synuclein ({alpha}-Syn), hemagglutinin (HA)–tagged synaptobrevin-2 (HA-Syb2), SNAP-25, C-terminally truncated syntaxin-1 (Synt-11-264) or synaptobrevin-2 (Syb21-96), and full-length HA-tagged syntaxin-1 (HA-Synt-1). (G) In vitro SNARE-complex assembly assay. Liposomes containing or lacking reconstituted synaptobrevin-2 are mixed with SNAP-25 and C-terminally truncated syntaxin-1, with or without {alpha}-synuclein, and floated by means of density gradient centrifugation. SNARE-complex assembly is measured as coflotation of SNAP-25 and syntaxin-1 with liposomes in the top two fractions. (H and I) Distribution of SNAREs and {alpha}-synuclein in liposome flotation gradients, shown as (H) representative immunoblots and (I) quantitations of liposome-bound proteins. Whereas {alpha}-synuclein ({alpha}-Syn) and synaptobrevin-2 are directly liposome-bound, SNAP-25 and syntaxin-1 (Synt-1) only bind to liposomes via SNARE-complex formation. Data are means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test; n = 3 to 8 independent experiments).

 

Figure 3
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Fig. 3. {alpha}β{gamma}-Synuclein TKO mice exhibit impaired survival and decreased SNARE-complex assembly. (A and B) Neurological impairments in {alpha}β{gamma}-synuclein TKO mice (at P300). Data show (A) latency to fall off an inverted wire grid (grid-hanging time; WT, n = 6 mice; TKO, n = 7 mice), forelimb grip strength (measured using a digital grip strength meter; WT, n = 9 mice; TKO, n = 11 mice), and footslips during beamwalking (WT, n = 9 mice; TKO, n = 11 mice) and (B) the latency to fall off an accelerating rotarod (5-min trials; WT, n = 9 mice; TKO, n = 11 mice). (C) Hind-limb clasping of WT and TKO mice as a function of age (left, representative images; right, clasping incidence; WT, n = 41 mice; TKO, n = 57 mice). (D) Age-dependent mortality of WT and TKO mice (WT, n = 41 mice; TKO, n = 57 mice). (E) Age-dependent changes in protein levels in TKO mice (left, representative blots; right, quantitations). (F) SNARE-complex assembly analyzed by means of co-immunoprecipitation of SNAREs in brain lysates from WT and TKO mice at P40 (top) and P200 (bottom). (Left) Representative blots. (Right) Quantitations from multiple independent experiments (n = 3 to 5 mice; Syb2, synaptobrevin-2; Synt-1, syntaxin-1). See figs. S6 to S9 for further data. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 by [(A), (E), and (F)] Student's t test, (B) two-way analysis of variance (ANOVA), or (C) Mantel-Cox test.

 

Figure 4
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Fig. 4. {alpha}-Synuclein boosts SNARE-complex assembly during synaptic activity. (A) Linear relationship between {alpha}-synuclein levels and SNARE-complex assembly in {alpha}β{gamma}-synuclein TKO neurons infected at DIV4 with increasing amounts of lentivirus expressing {alpha}-synuclein and analyzed by means of immunoblotting of nonboiled samples at DIV17 (n = 3 to 6 cultures). (B) Full-length ({alpha}-Syn) but not truncated {alpha}-synuclein ({alpha}-Syn1-95) enhances SNARE-complex assembly in TKO neurons, as measured by means of co-immunoprecipitation of syntaxin-1 (Synt-1) and SNAP-25 with synaptobrevin-2 (Syb2) (control is plain virus; left, representative blots; right, quantitations; n = 3 to 6 cultures). (C) Activity-dependence of SNARE-complex assembly in cultured WT and TKO neurons. Neurons were incubated at DIV10 for 36 hours in control medium, 0.5 µM tetrodotoxin (TTX), or 4 mM Ca2+ (Ca2+). SNARE-complex assembly was measured with co-immunoprecipitation of SNARE proteins with SNAP-25 and synaptobrevin-2. Representative blots are shown at top (Pre-Imm, pre-immune control), and quantitations from multiple independent experiments are shown at bottom (n = 3 to 6 cultures). (D) Time course of activity-induced changes in SNARE-complex assembly in WT and TKO neurons and in TKO neurons infected with lentiviruses expressing full-length ({alpha}-Syn) or C-terminally truncated {alpha}-synuclein ({alpha}-Syn1-95). Neurons were incubated in control medium, 0.5 µM TTX, or 4 mM Ca2+ for the indicated times, and SNARE-complex assembly was measured with immunoblotting for SNAP-25 in unboiled samples (n = 3 cultures). See figs. S10 and S11 for further data. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 by [(A) to (C)] Student’s t test or (D) one-way ANOVA.

 


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