Dysfunction in mechanisms that regulate the development, maintenance, and plasticity of synaptic connections have been linked to many neuropsychiatric and neurodevelopmental disorders, and thus a deep molecular understanding of these processes will be crucial in relieving the severe health burden these disorders impose. One aspect of synaptic communication that is critical for information processing in the brain is the maintenance of precise functional alignment between presynaptic neurotransmitter release and postsynaptic function at individual synapses, but the local signals that control this aspect of “synaptic homeostasis” are poorly understood. This project will focus on a recently discovered homeostatic pathway in hippocampal neurons that functions to tune presynaptic neurotransmitter release when postsynaptic receptor activation is deficient. My laboratory has recently described a unique homeostatic signaling pathway that couples synaptic inactivity to mTOR complex 1 (mTORC1) signaling in postsynaptic dendrites. mTORC1 activation, in turn, drives the local dendritic translation and release of brain-derived neurotrophic factor (BDNF), which elicits compensatory increases in neurotransmitter release from apposed presynaptic terminals but only if those terminals have been recently active. This state-dependent gating of synaptic homeostasis by local presynaptic activity ensures coupling of mTORC1 trans-synaptic signaling to spike-driven neurotransmitter release. During the previous period of support, we discovered that BDNF elicits presynaptic compensation via the proteasome-dependent degradation of the synaptic regulator tomosyn1 (Tomo1), which is catalyzed by the E3 ubiquitin ligase HRD1. During these investigations, we uncovered an unexpected and critical role for activity-dependent recruitment of the proteasome to axonal boutons. We now propose to test the central hypothesis that activity-dependent sequestration of proteasomes in presynaptic terminals confers state-dependent gating of synaptic homeostasis driven by postsynaptic mTORC1 signaling. Our investigations will examine how regulated phosphorylation of the 19S proteasome subunit Rpt6 dynamically regulates proteasome distribution in axons (Aim 1), define the core signaling pathway in axon terminals that links neural activity (and specifically, P/Q/N voltage-gated Ca2+ channel activity) with posttranslational modifications of the proteasome important for synaptic targeting (Aim 2), and define the molecular mechanisms that control proteasome sequestration in axon terminals (Aim 3). In each of these aims, the relationship of the mechanisms uncovered with mTORC1 trans-synaptic homeostatic signaling will be rigorously tested. The proposed experiments employ state of the art optical imaging of synaptic vesicle cycling and release, genetic models targeting mTORC1 signaling and proteasome phosphorylation, rigorous electrophysiological measurements, and are focused on a fundamentally new area...