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. 2010 Sep 24;329(5999):1663-7.
doi: 10.1126/science.1195227. Epub 2010 Aug 26.

Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro

Jacqueline Burré  1 , Manu Sharma, Theodoros Tsetsenis, Vladimir Buchman, Mark R Etherton, Thomas C Südhof
Affiliations

Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro

Jacqueline Burré et al. Science. .

Abstract

Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we show that maintenance of continuous presynaptic SNARE-complex assembly required a nonclassical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/vesicle-associated membrane protein 2 (VAMP2) and promoted SNARE-complex assembly. Moreover, triple-knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and died prematurely. Thus, synucleins may function to sustain normal SNARE-complex assembly in a presynaptic terminal during aging.

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Figures

Figure 1
Figure 1. α-Synuclein directly binds to synaptobrevin-2/VAMP2 in SNARE complexes
(A-D) α-Synuclein associates with SNARE-complexes immunoprecipitated with SNAP-25 antibodies from wild-type (WT) and CSPα KO (CSP-/-) mouse brains containing or lacking transgenic α-synuclein (tSyn). Panels show representative immunoblots (A; -Ab = control IP from WT brain without primary antibody; Synt-1 = syntaxin-1; Syb2 = synaptobrevin-2; α-Syn = α-synuclein), and quantitations of input levels (B), immunoprecipitated SNAP-25 (C), and co-immunoprecipitated proteins (D), performed by quantitative immunoblotting using 125I-labeled secondary antibodies (means ± SEMs, *p<0.05; **p<0.01; ***p<0.001 per Student's t-test; n=3-5). (E) Co-immunoprecipitation of α-synuclein with SNARE-complexes reconstituted in transfected HEK293T cells. Cell lysates were immunoprecipitated with antibodies to α-synuclein (left panel) or SNAP-25 (right panel), and analyzed by immunoblotting. (F & G) α-Synuclein C-terminus directly binds to synaptobrevin-2 N-terminus. α-Synuclein was immunoprecipitated from HEK293T cells co-expressing full-length (α-Syn) or C-terminally truncated α-synuclein (α-Syn1-95) with full-length (Syb2) or N-terminally truncated synaptobrevin-2 (Syb229-116). Immunoprecipitates were analyzed by immunoblotting for α-synuclein and synaptobrevin-2 (see also Figs. S1 and S2). (H) Diagram of the α-synuclein/synaptobrevin-2 complex on synaptic vesicles (SV). (I-K) Neither CSPα KO nor transgenic α-synuclein overexpression detectably alters synaptic strength. Data show sample traces (I and K top) and summary graphs (J and K bottom; means ± SEMs) of extracellular recordings from input-output measurements (I and J) or paired-pulse facilitation experiments (K) in acute hippocampal slices (see also Fig. S3).
Figure 2
Figure 2. α-Synuclein directly catalyzes SNARE-complex assembly
(A-C) α-Synuclein promotes SNARE-complex assembly in a dose-dependent manner. HEK293T cells were co-transfected with constant amounts of syntaxin-1 (Synt-1), synaptobrevin-2 (Syb2), and SNAP-25, and increasing amounts of α-synuclein (α-Syn). Cell lysates were immunoblotted without (A) and with boiling (Fig. S4); SNARE-complexes and α-synuclein were quantified (B), and plotted as a function of each other (C). (D & E) Full-length but not C-terminally truncated α-synuclein promotes SNARE-complex assembly. SNARE complexes were immunoprecipitated with antibodies to SNAP-25 (left) or synaptobrevin-2 (right) from HEK293T cells co-expressing SNARE proteins with emerald (control), full-length α-synuclein (α-Syn), or C-terminally truncated α-synuclein (α-Syn1-95), and analyzed by immunoblotting (D, representative blots; E, SNARE-complex quantitation). (F) Coomassie-stained SDS gel of purified recombinant α-synuclein (α-Syn), H A-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 α-synuclein, and floated by density gradient centrifugation. SNARE-complex assembly is measured as co-flotation of SNAP-25 and syntaxin-1 with liposomes in the top two fractions. (H & I) Distribution of SNAREs and α-synuclein in liposome flotation gradients, shown as representative immunoblots (H) and quantitations of liposome-bound proteins (I). Note that whereas α-synuclein (α-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 ± SEMs (*p<0.05; **p<0.01; ***p<0.001 by Student's t-test; n=3-8).
Figure 3
Figure 3. αβγ-Synuclein TKO mice exhibit impaired survival and decreased SNARE-complex assembly
(A & B) Neurological impairments in αβγ-synuclein TKO mice (at P300). Data show (A) latency to fall off an inverted wire grid (grid-hanging time; WT, n=6; TKO, n=7), forelimb grip strength (measured using a digital grip strength meter; WT, n=9; TKO, n=11), and footslips during beamwalking (WT, n=9; TKO, n=11), and (B) the latency to fall off an accelerating rotarod (5 min trials; WT, n=9; TKO, n=11). (C) Hind-limb clasping of WT and TKO mice as a function of age (left, representative images; right, clasping incidence; WT, n=41; TKO, n=57). (D) Age-dependent mortality of WT and TKO mice (WT, n=41; TKO, n=57). (E) Age-dependent changes in protein levels in TKO mice (left, representative blots; right, quantitations). (F) SNARE-complex assembly analyzed by co-immunoprecipitation of SNAREs in brain lysates from WT and TKO mice at P40 (top) and P200 (bottom). Left panels show representative blots, right panels quantitations from multiple independent experiments (n=3-5; Syb2 = synaptobrevin-2; Synt-1 = syntaxin-1). See Figs. S6-S9 for further data. Data are means ± SEMs, *p<0.05; **p<0.01; ***p<0.001 by Student's t-test (A, E and F), two-way ANOVA (B), or Mantel-Cox test (C).
Figure 4
Figure 4. α-Synuclein boosts SNARE-complex assembly during synaptic activity
(A) Linear relationship between α-synuclein levels and SNARE-complex assembly in αβγ-synuclein TKO neurons infected at DIV4 with increasing amounts of lentivirus expressing α-synuclein, and analyzed by immunoblotting of non-boiled samples at DIV17 (n=3-6). (B) Full-length (α-Syn) but not truncated α-synuclein (α-Syn1-95) enhances SNARE-complex assembly in TKO neurons, as measured by co-immunoprecipitation of syntaxin-1 (Synt-1) and SNAP-25 with synaptobrevin-2 (Syb2; control = plain virus; left, representative blots; right, quantitations; n=3-6). (C) Activity-dependence of SNARE-complex assembly in cultured WT and TKO neurons. Neurons were incubated at DIV10 for 36 hrs in control medium, 0.5 μM tetrodotoxin (TTX), or 4 mM Ca2+ (Ca2+). SNARE-complex assembly was measured by co-immunoprecipitation of SNARE-proteins with SNAP-25 and synaptobrevin-2. Representative blots are shown on top (Pre-Imm = pre-immune control), and quantitations from multiple independent experiments on the bottom (n=3-6). (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 (α-Syn) or C-terminally truncated α-synuclein (α-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 by immunoblotting for SNAP-25 in unboiled samples (n=3). See Figures S10 and S11 for further data. Data are means ± SEMs, *p<0.05; **p<0.01; ***p<0.001 by Student's t-test (A-C) or one-way ANOVA (D).
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