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Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells

ABSTRACT: Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission, yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and -generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs and immunoblotting confirmed the presence of endophilins-A1 and -A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+-influx and exocytosis, which we attribute to decreased abundance of presynaptic Ca2+-channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+-influx and promote synaptic vesicle recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV-reformation.

SUBMITTER: Ms. Jana Kroll

PROVIDER: S-SCDT-EMBOJ-2018-100116 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles-LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover.