Altering the expression of Tomosyn-1 (Tomo-1), a soluble, R-SNARE domaincontaining protein, significantly affects behavior in mice, Drosophila, and Caenorhabditis elegans. Yet, the mechanisms that modulate Tomo-1 expression and its regulatory activity remain poorly defined. Here, we found that Tomo-1 expression levels influence postsynaptic spine density. Tomo-1 overexpression increased dendritic spine density, while Tomo-1 knockdown (KD) decreased spine density. These findings identified a novel action of Tomo-1 on dendritic spines, which is unique because it occurs independently of Tomo-1's C-terminal R-SNARE domain. We also demonstrate that the ubiquitinproteasome system (UPS), which is known to influence synaptic strength, dynamically regulates Tomo-1 protein levels. Immunoprecipitated and affinity-purified Tomo-1 from cultured rat hippocampal neurons was ubiquitinated, and the levels of ubiquitinated Tomo-1 dramatically increased upon pharmacological proteasome blockade. Moreover, Tomo-1 ubiquitination appeared to be mediated through an interaction with the E3 ubiquitin ligase HRD1, as immunoprecipitation of Tomo-1 from neurons coprecipitated HRD1, and this interaction increases upon proteasome inhibition. Further, in vitro reactions indicated direct, HRD1 concentrationdependent Tomo-1 ubiquitination. We also noted that the UPS regulates both Tomo-1 expression and functional output, as HRD1 KD in hippocampal neurons increased Tomo-1 protein level and dendritic spine density. Notably, the effect of HRD1 KD on spine density was mitigated by additional KD of Tomo-1, indicating a direct HRD1/Tomo-1 effector relationship. In summary, our results indicate that the UPS is likely to participate in tuning synaptic efficacy and spine dynamics by precise regulation of neuronal Tomo-1 levels.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.