Functional cooperation of α-synuclein and VAMP2 in synaptic vesicle recycling

Abstract

The function of α-synuclein (α-syn) has been long debated, and two seemingly divergent views have emerged. In one, α-syn binds to VAMP2, acting as a SNARE chaperone—but with no effect on neurotransmission—while another posits that α-syn attenuates neurotransmitter release by restricting synaptic vesicle mobilization and recycling. Here, we show that α-syn–VAMP2 interactions are necessary for α-syn–induced synaptic attenuation. Our data connect divergent views and suggest a unified model of α-syn function.

The normal function of the small presynaptic protein α-synuclein (α-syn) is of exceptional interest, not only in the context of neurodegeneration, but also as a cytosolic regulator of neurotransmission (1). Over the years, two seemingly divergent views have emerged. In one, α-syn binds to VAMP2 (synaptobrevin-2) and chaperones SNARE complexes, but with no effect on neurotransmission (2). Alternatively, we and others have advocated the concept that α-syn is a physiologic attenuator of neurotransmitter release, based on evidence that modest α-syn overexpression attenuates synaptic vesicle (SV) recycling and exocytosis (3–7). Furthermore, we proposed a model where α-syn helps in physiologic clustering of SV pools, restricting their egress to the presynaptic plasma membrane, thus attenuating SV recycling (6). Here, we asked if the two seemingly divergent views can be reconciled.

Results and Discussion

Although previous studies have documented α-syn–VAMP2 binding, some have reportedly failed to detect such associations (8). Interaction at synapses is also unclear. In support of ref. 2, our coimmunoprecipitation (co-IP) experiments show that full-length α-syn [amino acids 1 to 140 (α-syn 1–140)] binds VAMP2, whereas a deletion lacking the reported VAMP2-binding region (α-syn 1–95, see ref. 2) does not (Fig. 1 A and B). To validate α-syn–VAMP2 interaction at synapses, we used the bimolecular fluorescence complementation (BiFC) assay, where candidate interacting proteins are tagged to N- and C-terminal Venus fragments (VN and VC) that are reconstituted upon interaction of the protein partners (6). Indeed, there was robust complementation of VAMP2:VN and α-syn 1–140:VC (but not VAMP2:VN and α-syn 1–95:VC) in HEK cells and presynaptic boutons (Fig. 1 C–E)—the latter indicating synaptic α-syn–VAMP2 interactions.

Fig. 1.

α-Syn sequence lacking the VAMP2-binding domain fails to attenuate SV recycling. (A and B) α-Syn sequence (A) and co-IP of α-syn–VAMP2 (B). Neuro2a cells were cotransfected with myc-tagged α-syn and VAMP2 and then immunoprecipitated with an anti-myc antibody. Note that α-syn 96–140 is the VAMP2-binding region (repeated twice). (C) Principle of our BiFC assay. (D) HEK cells were transfected with VN:VAMP2 and α-syn:VC (α-syn 1–140 or 1–95). Note punctate fluorescence with VN:VAMP2 + α-syn(1–140):VC, which was greatly attenuated with α-syn 1–95 sequence lacking VAMP2-binding domain; quantification of data shown Right (mean ± SEM; α-syn 1–140, 1 ± 0.06499, n = 38 from 3 independent experiments; α-syn 1–95, 0.3074 ± 0.01676, n = 39; ****P < 0.0001). (E) Cultured hippocampal neurons were cotransfected with the constructs listed; note α-syn–VAMP2 Venus complementation at boutons with WT α-syn; quantification shown Right (mean ± SEM from 2 independent experiments; α-syn 1–140, 1 ± 0.03949, n = 280; α-syn 1–95, 0.1691 ± 0.007379, n = 312; ****P < 0.0001). (F) α-Syn 1–110 contains the VAMP2-binding site that starts at amino acid 96. (G) Principle of pHluorin assay (Top) with representative images (Below). (H and I) Experiments in cultured hippocampal neurons (pHluorin). (H) While α-syn 1–110 attenuates SV recycling (Left), α-syn 1–95 has no effect (Right) (note that some error bars are too small to see). (I) Quantification of data in H (mean ± SEM from at least 3 independent experiments); control, 0.4703 ± 0.03215, n = 16; α-syn 1–140, 0.3165 ± 0.02914, n = 13; α-syn 1–110, 0.3464 ± 0.05906, n = 8; α-syn 1–95, 0.4763 ± 0.0192, n = 17; **P = 0.0025, *P = 0.0459 (one-way ANOVA followed by Dunnett’s post hoc test).

A previous study showed that overexpression of an α-syn sequence lacking the C terminus (α-syn 1–110) also attenuated SV recycling (4), leading to the notion that VAMP2 binding may not be critical for synaptic function. However, we noticed that the α-syn 1–110 sequence used in ref. 4 has a 15-aa region (α-syn 96–110) that overlaps with the reported VAMP2-binding site that starts at amino acid 96 (see Fig. 1F). Accordingly, we first asked if α-syn 1–95—a sequence that definitively lacks the VAMP2-binding site—can attenuate SV recycling in pHluorin assays (6) that report exo/endocytic cycles as fluctuations of SV luminal fluorescence (see Fig. 1G). Interestingly, while α-syn 1–110 attenuated SV recycling, α-syn 1–95 had no effect (Fig. 1 H and I).

In co-IP experiments, α-syn 1–140 and 1–110 bound VAMP2 with equal affinity (Fig. 2A), suggesting that α-syn 96–110 might be the VAMP2-binding domain. Indeed, scrambling the α-syn 96–110 amino acids abrogated α-syn–VAMP2 interaction (Fig. 2B). To narrow down the amino acid region required for binding, we did alanine scanning of the α-syn 96–110 region, sequentially mutating amino acids starting at α-syn 96 to alanine—an inert methyl functional group mimicking secondary structures of other amino acids (Fig. 2C). As shown in Fig. 2D, the first ∼9 amino acids starting at α-syn 96 seem critical for VAMP2 binding in this setting. However, we note that other C-terminal sequences may also be important, particularly if such interactions do not affect SV recycling. Finally, these α-syn–VAMP2 deletions and subtle mutations also abrogated α-syn effects on SV recycling (Fig. 2 E and F).

Fig. 2.

Mapping of the α-syn–VAMP2 binding domain and requirement of α-syn–VAMP2 interactions for α-syn–mediated SV attenuation. (A and B) Both α-syn 1–140 and 1–110 bind VAMP2 with equal affinity, and scrambling of amino acid sequences in α-syn 96–110 attenuates α-syn–VAMP2 binding (co-IP in neuro2a, repeated twice). (C and D) Sequential amino acids (from α-syn 96) were mutated to alanine, and association of these mutants (myc-tagged) with VAMP2 was evaluated (co-IP in neuro2a cells). Note that mutations in α-syn 96–104 show the greatest disruption (repeated twice). (E) Scrambled and KKD mutations in the α-syn 96–110 sequence abrogated α-syn–mediated synaptic attenuation, as determined by pHluorin assays in hippocampal neurons. (F) Quantification of data in E (mean ± SEM from at least 3 independent experiments); control, 0.423 ± 0.029, n = 6; α-syn 1–140, 0.178 ± 0.032, n = 6; KKD, 0.453 ± 0.070, n = 5; Scr-1, 0.464 ± 0.041, n = 6; **P = 0.0017 (one-way ANOVA followed by Dunnett’s post hoc test). (G–I) Optical single vesicle clustering experiments were carried out as described in ref. 10. Briefly, VAMP2-containing synaptic-like vesicles were first immobilized on a glass slide assembled in a microfluidic chamber, and then WT or mutant α-syn protein was added. After extensive washing (to remove unbound α-syn), DiI-labeled VAMP2-containing vesicles were added to the chamber, and clustering of the labeled vesicles was visualized by prism-type total internal reflection fluorescence microscopy (after extensive washing to remove unbound vesicles). As shown in representative images (G) and quantitative data (H), α-syn induced vesicle clustering, and deletions or subtle mutations in the VAMP2-binding site markedly abrogated the number of vesicle clusters. Mean ± SEM from 4 independent experiments where observer was blinded to the conditions; α-syn 1–140, 100%; α-syn 1–110, 83.15% ± 6.439%; no α-syn, 21.83% ± 7.437%; α-syn 1–95, 29.94% ± 8.332%; KKD, 33.89% ± 3.465%; Scr-1, 30.49% ± 8.138%; NS, nonsignificant; ****P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc test). (I) Scatter plots showing number of vesicle clusters (on y axis) and fluorescence intensities (on x axis) of all Dil-labeled clusters, along with a smoothened curve through the data points.

Previously, we found that α-syn multimers appear to cluster SVs (6), resembling phenotypes in yeast where α-syn induced vesicle clusters (9). Here, we used a single-vesicle optical microscopy system to directly visualize α-syn–induced clustering of small synaptic-like vesicles in vitro (Fig. 2G; see ref. 10 for details of methods). As shown in Fig. 2 H and I, α-syn 1–140 induced vesicle clustering, whereas any perturbation in the α-syn–VAMP2 binding region abrogated this effect. Synaptic targeting of α-syn deletions/mutations—determined by a quantitative ratiometric paradigm (11)—was comparable to wild-type (WT) α-syn (targeting of α-syn 1–110, α-syn 1–95, KKD, and Scr-1 was ∼93%, 86%, 98%, and 95% of the WT protein, respectively; changes were statistically nonsignificant).

Taken together, the data indicate that α-syn–VAMP2 binding is essential for α-syn function and advocate an “interlocking model” where α-syn multimers on the SV surface interact with VAMP2 on adjacent SVs, helping to maintain physiologic SV clustering. An understanding of normal α-syn function is likely critical to appreciate pathologic triggers in disease (12, 13). Degradation of α-syn by chaperone-mediated autophagy (CMA) is strongly implicated in synucleinopathies, and interestingly, a pentapeptide region consistent with a CMA recognition motif—α-syn 95–99 (see ref. 14)—also lies within the VAMP2-binding site. Thus, this small region in the C terminus of α-syn may be a key “hub” in pathophysiologic transition. Our findings link divergent views in the field and offer a unified model of α-syn function that provides a new platform for future studies probing the pathobiology of this enigmatic protein. Along with the paper in PNAS by Atias et al. (15), the collective evidence points to a scenario where VAMP2 and synapsin (another cytosolic presynaptic protein with known roles in SV clustering) cooperate to help cluster SVs and regulate SV recycling. Whereas VAMP2 and α-syn directly induce SV clustering, synapsin assists in clustering by enhancing α-syn targeting to SVs, perhaps by facilitating the axonal transport of α-syn (see ref. 15 for details).