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

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.

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-Syn^1-95) with full-length (Syb2) or N-terminally truncated synaptobrevin-2 (Syb2^29-116). 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. 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-Syn^1-95), 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-1^1-264) or synaptobrevin-2 (Syb2^1-96), 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. 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. 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-Syn^1-95) 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-Syn^1-95). 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).