Synapsins regulate α-synuclein functions

Abstract

The normal function of α-synuclein (α-syn) remains elusive. Although recent studies suggest α-syn as a physiologic attenuator of synaptic vesicle (SV) recycling, mechanisms are unclear. Here, we show that synapsin-a cytosolic protein with known roles in SV mobilization and clustering-is required for presynaptic functions of α-syn. Our data offer a critical missing link and advocate a model where α-syn and synapsin cooperate to cluster SVs and attenuate recycling.

Keywords: alpha synuclein; neurotransmission; synapsin.

Fig. 1.

Synapsins are required for α-syn–mediated synaptic attenuation. (A) Experimental design: cultured hippocampal neurons were transduced (AAV1/2) at 5 d in vitro (DIV); sypHy/FM1-43 were imaged at 12 to 14 DIV. (B) SypHy in WT neurons. Modest overexpression of hα-syn-mCherry (184% ± 10%) attenuated sypHy responses in WT neurons (Control: mCherry); (Inset) similar effect of untagged hα-syn (stimulation: 10 V/cm, 20 Hz; n = 9 to 13, >30 synapses per coverslip, ≥3 cultures; one-way ANOVA, Tukey’s post hoc analysis). (C) SypHy in synapsin TKO neurons. Hα-syn failed to attenuate synaptic responses in TKO neurons (Left); reintroduction of tag blue fluorescent protein (mTagBFP)-synapsin Ia (Right) reinstated hα-syn–induced synaptic attenuation. (D) Quantification of data in C (n = 6 to 15; t test). (E) Evaluation of exocytosis. Bafilomycin A (Baf) blocked SV reacidification after endocytosis. Hα-syn reduced exocytosis in WT neurons (Left), but not in TKO neurons (Right). Dashed line indicates total SV pool revealed by NH₄Cl. (F) Quantification of data in E (n = 6 to 17; t test). (G) FM-dye release. Neurons loaded with FM1-43 were stimulated to evaluate exocytosis. Hα-syn reduced FM-dye release in WT neurons, but not in TKO neurons. (H) Quantification of data in G (n = 5 to 9; t test). ns, not significant, *P < 0.05, ***P < 0.001.

Fig. 2.

Interaction of α-syn and synapsin in neuronal cell lines and synapses. (A) Coimmunoprecipitation (co-IP) of synapsin and α-syn. Neuro2a cells were cotransfected with enhanced green fluorescent protein (EGPF)-synapsin Ia (or its deletions) and myc-hα-syn, and then immunoprecipitated with an anti-EGFP antibody (Millipore). Full-length EGFP-synapsin I (but not EGFP alone) interacted with α-syn (lanes 1 and 2). Synapsin C/D domains (lanes 5 and 6) are critical for this interaction [Bottom: inputs; anti-myc (Abcam); repeated twice]. (B) FRET combinations. Donor: hα-syn-cerulean (α-syn-cer); acceptor: soluble Venus (ven), Venus-synapsin Ia (ven-SynIa), or the SV protein synaptophysin I-Venus (SypI-ven). (C) FRET data. Note FRET in synapses between hα-syn-cerulean and Venus-synapsin Ia, but not soluble Venus (control). No FRET was seen with synaptophysin I-Venus (n = 8 to 26; one-way ANOVA, Tukey’s post hoc analysis). (D) FRET between hα-syn and synapsin Ia was reduced by stimulation, recovering during rest (n = 101 synapses in 3 experiments; Friedman’s ANOVA, post hoc analysis: Wilcoxon’s test/Bonferroni’s correction). (E) FM-FRAP schematic. SVs are loaded with FM1-43 and a single bouton is bleached. Hα-syn inhibits intersynaptic SV traffic and FM recovery. (F) FM-FRAP experiments. Hα-syn dampens recovery in WT neurons (Left), but not in TKO neurons (Right). (G) Quantification of data in F (n = 10 to 17 experiments, 3 synapses per experiment; t test). (H, Top) Synaptic enrichment. Hα-syn-mCherry and soluble EGFP (volume filler) in WT and TKO neurons. (H, Bottom) Ratio images (scale to left) and intensity curves; red indicates α-syn, green indicates EGFP. (I) Quantification of data in H. Hα-syn-mCherry (vs. soluble mCherry) is enriched in WT, but not in TKO boutons. Reintroduction of synapsin Ia restored α-syn synaptic enrichment (n = 15 to 18; one-way ANOVA, Tukey’s post hoc analysis). (J) Synaptic enrichment of endogenous vGlut1 (normalized by WT) is similar in WT and TKO neurons overexpressing hα-syn (n = 14 to 15; t test). (K) Synapsins help α-syn associate with SVs, thereby facilitating SV clustering [α-syn also binds VAMP2 to help clustering (21), not shown]. Loss of synapsins decreases α-syn targeting and disrupts clustering of SVs. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.