| The efficacy of information transfer at synaptic contacts between excitatory central neurons undergoes continual modification in response to neuronal activity and physiological state. This plasticity in synaptic transmission may involve changes in presynaptic release probability, postsynaptic receptor number and sensitivity, and/or synaptic morphology. The molecular mechanisms influencing these distinctive targets are an investigative focus given their importance in learning, memory, and cognitive function. Much attention has focused on transcriptional and translational regulation of the synapse, but post-translational modification and directed turnover of specific protein components is also recognized as critical. Central to targeted protein degradation is the ubiquitin-proteasome system (UPS). While an increasing number of synaptic proteins are known to be susceptible to activity-dependent regulation by the UPS, relatively little has focused on the action of the UPS on known negative regulators of synaptic function. The SNARE protein Tomosyn-1 (Tomo-1) directly inhibits evoked release at central synapses, but it is also present post-synaptically, where no known function has been identified. It was recently discovered that the related Tomosyn-2 protein is subject to ubiquitination and degradation in neuroendocrine pancreatic beta cells, suggesting their secretory activity may be under control of the UPS. The general hypothesis of this dissertation is that a central mechanism underlying modulation of the synapse is the targeted degradation of Tomo-1.;This dissertation made use of a series of complementary biochemical, molecular, and imaging technologies in hippocampal neuronal culture. We demonstrate that Tomo- 1 protein level, independently of its SNARE domain, positively correlates with postsynaptic dendritic spine density in vivo. The data also indicate that the UPS regulates steady-state Tomo-1 level and function. Immunoprecipitated Tomo-1 was ubiquitinated and co-precipitated the E3 ligase HRD1, and both effects dramatically increased upon proteasome inhibition. The interaction was also found in situ, via fixed- cell proximity ligation assay. In vitro reactions indicated direct, HRD1 concentration- dependent Tomo-1 ubiquitination. Furthermore, we demonstrated that neuronal HRD1 knockdown increased Tomo-1 level, and consequently, dendritic spine density. This effect was abrogated by concurrent knockdown of Tomo-1, strongly suggesting a direct HRD1/Tomo-1 effector relationship. We confirmed Tomo-1 is a UPS substrate by identifying 12 lysine residues which are ubiquitinated by HRD1 and generated a non- ubiquitinateable Tomo-1 mutant. Finally, we performed Tomo-1 isoform and homologue comparisons, protein structure modeling, and antibody-based domain targeting of Tomo-1 in neuronal lysates to identify four lysine residues which are highly likely to be ubiquitinated in vivo. In summary, the results of this dissertation indicate that the UPS participates in tuning synaptic efficacy via the precise regulation of neuronal Tomo-1 and spine density. These findings implicate Tomo-1 as a prime target of UPS mediated degradation in the implementation of morphological plasticity in central neurons. |
