The ubiquitin-proteasome system functionally links neuronal Tomosyn-1 to dendritic morphology

Abstract

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.

STXBP5/tomosyn regulates the small RhoA GTPase to control the dendritic stability of neurons and the surface expression of AMPA receptors

“…Together, this evidence suggests that tomosyn may interact with the small Rho GTPase RhoA via 14 the WD40 domain. In the current study, FRET analysis revealed an increase of RhoA activity in tomosyn knockdown neurons.…”

“…One study has shown that tomosyn regulates neurite outgrowth in immature neurons by strongly binding to Rho-associated serine/threonine kinase (ROCK)-phosphorylated syntaxin-1 (13). Tomosyn has also been shown to regulate dendritic morphology via the ubiquitin-proteasome system (14). Here, we provide evidence showing that tomosyn regulates RhoA signaling and the trafficking of glutamate receptors to maintain the structural stability of neurons.…”

Synaptotagmin-9 and Tomosyn-1 molecular complex regulates Stx1A SNAREs to inhibit insulin secretion from pancreatic β-cells

“…We show that the loss of Syt9 by post-transcriptional mechanisms decreases Tomosyn-1 inhibitory function, thereby increasing Stx1A-SNARE-mediated insulin secretion. Post-translational regulation by Hrd1 E3-polyubiquitin ligase 80,81 and protein kinase A 47 in neurons, and mono-ubiquitination in β-cells 72 , has been shown to affect Tomosyn-1 function. Thus, we propose that Syt9 stabilizes Tomosyn-1 in the Syt9-Tomosyn-1-Stx1A non-fusogenic complex to prevent PM fusion of insulin granules.…”

Homeostatic Roles of the Proteostasis Network in Dendrites

“…Other work in rat hippocampal cultures has indicated that inhibiting lysosomes causes loss of excitatory synapses (Goo et al, 2017). Furthermore, hippocampal spine density increased after knockdown of E3 ubiquitin ligase HRD1 in rat neuronal cell cultures (Saldate et al, 2018). Though perhaps paradoxical on first glance, it is likely that UPS facilitates both LTD and LTP through complex ubiquitin signals (Mabb and Ehlers, 2018;Yun et al, 2018).…”