Functional cooperation of α-synuclein and VAMP2 in synaptic vesicle recycling - PubMed (original) (raw)

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

Jichao Sun et al. Proc Natl Acad Sci U S A. 2019.

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.

Keywords: Parkinson’s disease; alpha synuclein; synaptic transmission.

Copyright © 2019 the Author(s). Published by PNAS.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

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).

Fig. 2.

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. . 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.

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