Synaptotagmin 7 is enriched at the plasma membrane through γ-secretase processing to promote vesicle docking and control synaptic plasticity in mouse hippocampal neurons

Abstract

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. 'Zap-and-freeze' electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via γ-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release

“…Syt7 KO neurons had no apparent phenotype (Figure 1B-E; 66% synchronous and 34% asynchronous, p = 0.39). The 2% reduction in AR in the KO did not reach statistical significance here, but we note that this trend is consistent with earlier measurements using iGluSnFR (Figure 1B, C; p = 0.39, against WT) (Vevea et al, 2021). By contrast, the asynchronous component was markedly reduced in Doc2α KO neurons (Figure 1B-E, C; 84% synchronous and 16% asynchronous).…”

“…In contrast, syt7 KO neurons displayed depression instead of facilitation of synchronous release after the second pulse, as well as faster and more severe synaptic depression as the train progressed (Figure 3D). This result is in line with a large body of work showing that syt7 is essential for both facilitation and resistance to depression (Chen et al, 2017; Jackman et al, 2016; Liu et al, 2014; Vevea et al, 2021). As in single-stimulus measurements (Figure 1), AR was decreased in Doc2α KOs, but not syt7 KOs, after the first pulse of the stimulus train.…”

“…These findings indicate that Doc2α is required for the majority of AR. The peak average fluorescence intensity in both mutants was similar to WT (Figure 1D), demonstrating that synchronous release is not affected in these mutants (Vevea et al, 2021; Yao et al, 2011).…”

“…We stimulated cultured hippocampal neurons 50 times at 10 and 20 Hz and measured iGluSnFR responses (Figure 3A). To resolve the response from each pulse during the train, we used the faster, low-affinity version of iGluSnFR (S72A; Marvin et al, 2018; Vevea et al, 2021). At 20 Hz, WT neurons exhibited facilitation of synchronous release in response to the second pulse, and thereafter depressed as the train went on, eventually reaching a steady state (Figure 3B, D).…”

Allosteric stabilization of calcium and phosphoinositide dual binding engages three synaptotagmins in fast exocytosis

“…Differences in the distance these bridges may span, the binding rates and affinities for Ca 2+ and membranes, which determine the Ca 2+ sensitivity, duration, speed and sequence of molecular interaction, might explain their diverse roles in SV docking, priming and fusion and versatile effects on spontaneous-, synchronous-and delayed neurotransmission (Augustin et al, 1999;Bacaj et al, 2013;Basu et al, 2007;Liu et al, 2014;Luo and Sudhof, 2017;Quade et al, 2019;Varoqueaux et al, 2002;Vevea et al, 2021;Wen et al, 2010;Xu et al, 2017). Further research will be necessary to gain understanding how these processes shape the energetic landscape of neurotransmitter release.…”