Endophilin A and B Join Forces With Clathrin to Mediate Synaptic Vesicle Recycling in Caenorhabditis elegans

Abstract

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.

Synapsin is required for dense core vesicle capture and cAMP-dependent neuropeptide release

“…To assess the role of synapsin in synaptic transmission, and specifically in neuropeptide release, we turned to ultrastructural analysis. High-pressure-freeze electron microscopy (HPF-EM; Kittelmann et al, 2013; Yu et al, 2018) can be used to analyze the content and distribution of synaptic organelles in the terminals of cholinergic motor neurons, including SVs, docked SVs, DCVs and large vesicles (LVs), which are endosomes formed after activity-induced SV release ( Fig. 4a, b ).…”

“…Primed SVs (forming the readily releasable pool - RRP) fuse with the PM following Ca 2+ influx into the synaptic terminal. SVs are recycled after endocytosis from endosomal compartments (Haucke et al, 2011; Hua et al, 2013; Jahne et al, 2015; Kittelmann et al, 2013; Kononenko and Haucke, 2015; Rizzoli, 2014; Steuer Costa et al, 2017; Watanabe et al, 2013a; Watanabe et al, 2013b; Watanabe et al, 2014; Yu et al, 2018).…”

Time course and temperature dependence of synaptic vesicle endocytosis

“…CME was reported to act at the plasma membrane to retrieve synaptic vesicle components only at room temperature. At physiological temperatures, other clathrin‐independent mechanisms that involve endosomal intermediates maintain synaptic vesicle recycling .…”

“…This pathway operates at a faster pace than CME (a few seconds compared to 30 s to a minute) and it has been termed fast endophilinmediated endocytosis (FEME, [54,64]) in non-neuronal mammalian cells. Endophilins, and possibly FEME, also regulate the number of synaptic vesicles and synaptic vesicle endocytosis at peripheral (neuromuscular junction) and central (hippocampal) synapses [53,57,65]. During UFE, endophilin works together with synaptojanin to accelerate the speed of endocytosis via regulation of neck formation in membrane invaginations and subsequently they regulate clathrin uncoating after vesicle reformation for synaptic endosomes [53] (also see [57,65]).…”

“…Endophilins, and possibly FEME, also regulate the number of synaptic vesicles and synaptic vesicle endocytosis at peripheral (neuromuscular junction) and central (hippocampal) synapses [53,57,65]. During UFE, endophilin works together with synaptojanin to accelerate the speed of endocytosis via regulation of neck formation in membrane invaginations and subsequently they regulate clathrin uncoating after vesicle reformation for synaptic endosomes [53] (also see [57,65]). Through their roles in synaptic vesicle endocytosis, synaptojanin and endophilin help sustain the efficiency of neurotransmission during prolonged high frequency stimulation at the neuromuscular junction, even at room temperature [66].…”