A Novel Target of Proteasomal Degradation Induces Homeostatic Plasticity

Abstract

Chronic manipulation of synaptic activity drives bidirectional alterations in synaptic efficacy. Compensatory tuning of presynaptic neurotransmitter release maintains levels of synaptic activity within an optimal range for efficient information processing. This homeostatic form of plasticity is apparent in cultured hippocampal neurons and appears to involve the ubiquitin-proteasome system (UPS). Examination of presynaptic mechanisms underlying homeostatic plasticity has identified multiple positive regulators of exocytosis subject to proteasomal degradation. However, homeostatic compensation can involve up- or down-regulation of exocytosis and substantially less is known of the negative regulation of vesicle release. Tomosyn, a presynaptically active SNARE protein, is unique in that it is cytosolic and serves to potently inhibit vesicle release at central synapses. Proteomic analysis of tomosyn revealed an interaction with the E3 ubiquitin-ligase HRD1. Here we aim to test the hypothesis that the UPS serves as an activity-dependent mechanism to precisely regulate tomosyn proteostasis, and in turn manipulates exocytosis. Consistent with this hypothesis, endogenous tomosyn levels in cultured rat hippocampal neurons (18-25 DIV) increase following application of the proteasome inhibitors MG132 (50μM, 4h) and lactacystin (10μM, 4h). Moreover, our data indicate that the tomosyn-HRD1 interaction is activity-dependent, as chronic AMPAR blockade (CNQX, 24h) results in an increase in HRD1 co-immunoprecipitation with tomosyn and consequentially a decrease in overall tomosyn protein level. To assess tomosyn's role homeostatic plasticity we use an optical reporter of exocytosis, vGlut1-pHluorin. Results show that chronic activity blockade via CNQX (40μM, 24h) enhances vesicle release in response to a stimulus train (10Hz, 10s) as compared to non-stimulated controls. This effect is dependent upon tomosyn, as shRNA-mediated tomosyn knock-down mitigates the compensatory enhancement of exocytosis. These data strongly implicate tomosyn as a key presynaptic molecular target which is subject to regulation by the UPS and facilitates activity-dependent homeostatic plasticity.