Regulation of STIM1 and SOCE by the Ubiquitin-Proteasome System (UPS)

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca2+ entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3′s), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca2+ homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function

Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and Overexpressing Mutant Mice

SCRAPPER, an F-box protein coded by FBXL20, is a subunit of SCF type E3 ubiquitin ligase. SCRAPPER localizes synapses and directly binds to Rab3-interacting molecule 1 (RIM1), an essential factor for synaptic vesicle release, thus it regulates neural transmission via RIM1 degradation. A defect in SCRAPPER leads to neurotransmission abnormalities, which could subsequently result in neurodegenerative phenotypes. Because it is likely that the alteration of neural transmission in Scrapper mutant mice affect their systemic condition, we have analyzed the behavioral phenotypes of mice with decreased or increased the amount of SCRAPPER. We carried out a series of behavioral test batteries for Scrapper mutant mice. Scrapper transgenic mice overexpressing SCRAPPER in the hippocampus did not show any significant difference in every test argued in this manuscript by comparison with wild-type mice. On the other hand, heterozygotes of Scrapper knockout [SCR (+/−)] mice showed significant difference in the contextual but not cued fear conditioning test. In addition, SCR (+/−) mice altered in some tests reflecting anxiety, which implies the loss of functions of SCRAPPER in the hippocampus. The behavioral phenotypes of Scrapper mutant mice suggest that molecular degradation conferred by SCRAPPER play important roles in hippocampal-dependent fear memory formation

The ataxia (axJ) mutation causes abnormal GABAA receptor turnover in mice

Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax(J) gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. Ax(J) mouse mutants, characterized by cerebellar ataxia, display both increased GABA(A) receptor (GABA(A)R) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA(A)R alpha1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(A)R turnover at cerebellar synapses contributes to ax(J)-mediated behavioural impairment

APCCdh1 mediates EphA4-dependent downregulation of AMPA receptors in homeostatic plasticity

Homeostatic plasticity is crucial for maintaining neuronal output by counteracting unrestrained changes in synaptic strength. Chronic elevation of synaptic activity by bicuculline reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs), but the underlying mechanisms of this effect remain unclear. We found that activation of EphA4 resulted in a decrease in synaptic and surface GluR1 and attenuated mEPSC amplitude through a degradation pathway that requires the ubiquitin proteasome system (UPS). Elevated synaptic activity resulted in increased tyrosine phosphorylation of EphA4, which associated with the ubiquitin ligase anaphase-promoting complex (APC) and its activator Cdh1 in neurons in a ligand-dependent manner. APCCdh1 interacted with and targeted GluR1 for proteasomal degradation in vitro, whereas depletion of Cdh1 in neurons abolished the EphA4-dependent downregulation of GluR1. Knockdown of EphA4 or Cdh1 prevented the reduction in mEPSC amplitude in neurons that was a result of chronic elevated activity. Our results define a mechanism by which EphA4 regulates homeostatic plasticity through an APC Cdh1-dependent degradation pathway. © 2011 Nature America, Inc. All rights reserved.link_to_subscribed_fulltex