Distinct subcellular changes in proteasome activity and linkage-specific protein polyubiquitination in the amygdala during the consolidation and reconsolidation of a fear memory.

Numerous studies have supported a critical role for the ubiquitin-proteasome system (UPS) in the memory consolidation and reconsolidation processes. The protein targets and functional role of ubiquitin-proteasome activity can vary widely across cellular compartments, however, it is unknown how UPS activity changes within the nuclear, cytoplasmic, and synaptic regions in response to learning or memory retrieval. Additionally, while previous studies have focused on degradation-specific protein polyubiquitination, it is unknown how learning alters other polyubiquitin tags that are not targeted by the proteasome. Using cellular fractionation protocols in combination with linkage-specific polyubiquitin antibodies, we examined subcellular changes in ubiquitin-proteasome activity in the amygdala during memory consolidation and reconsolidation. Following memory acquisition, overall protein ubiquitination and proteasome activity simultaneously increased in the nucleus and decreased in the synaptic and cytoplasmic regions. The nuclear increases were associated with upregulation of degradation-specific (K48) and degradation-independent (K63, M1) polyubiquitin tags, suggesting multiple functions for ubiquitin signaling within this region. Interestingly, retrieval induced a very different pattern of ubiquitin-proteasome activity in the amygdala, consisting of increases in overall protein ubiquitination and proteasome activity and K48-, K63-, and M1-polyubiquitin tags in the synaptic, but not nuclear or cytoplasmic regions. Collectively, learning and memory retrieval dynamically and differentially alter degradation-dependent and degradation-independent ubiquitin-proteasome activity across different cellular compartments, suggesting that the UPS may serve unique functions during memory consolidation and reconsolidation.

Dysregulation of protein degradation in the hippocampus is associated with impaired spatial memory during the development of obesity.

Studies have shown that long-term exposure to high fat and other obesogenic diets results in insulin resistance and altered blood brain barrier permeability, dysregulation of intracellular signaling mechanisms, changes in DNA methylation levels and gene expression, and increased oxidative stress and neuroinflammation in the hippocampus, all of which are associated with impaired spatial memory. The ubiquitin-proteasome system controls the majority of protein degradation in cells and is a critical regulator of synaptic plasticity and memory formation. Yet, whether protein degradation in the hippocampus becomes dysregulated following weight gain and is associated with obesity-induced memory impairments is unknown. Here, we used a high fat diet procedure in combination with behavioral and subcellular fractionation protocols and a variety of biochemical assays to determine if ubiquitin-proteasome activity becomes altered in the hippocampus during obesity development and whether this is associated with impaired spatial memory. We found that only 6 weeks of exposure to a high fat diet was sufficient to impair performance on an object location task in rats and resulted in dynamic dysregulation of ubiquitin-proteasome activity in the nucleus and cytoplasm of cells in the hippocampus. Furthermore, these changes in the protein degradation process extended into cortical regions also involved in spatial memory formation. Collectively, these results indicate that weight gain-induced memory impairments may be due to altered ubiquitin-proteasome signaling that occurs during the early stages of obesity development.

Males and Females Differ in the Subcellular and Brain Region Dependent Regulation of Proteasome Activity by CaMKII and Protein Kinase A.

The ubiquitin-proteasome system (UPS) controls the degradation of ~90% of short-lived proteins in cells and is involved in activity- and learning-dependent synaptic plasticity in the brain. Calcium/calmodulin dependent protein kinase II (CaMKII) and Protein Kinase A (PKA) can regulate activity of the proteasome. However, there have been a number of conflicting reports regarding under what conditions CaMKII and PKA regulate proteasome activity in the brain. Furthermore, this work has been done exclusively in males, leaving questions about whether these kinases also regulate the proteasome in females. Here, using subcellular fractionation protocols in combination with in vitro pharmacology and proteasome activity assays, we investigated the conditions under which CaMKII and PKA regulate proteasome activity in the brains of male and female rats. In males, nuclear proteasome chymotrypsin activity was regulated by PKA in the amygdala but CaMKII in the hippocampus. Conversely, in females CaMKII regulated nuclear chymotrypsin activity in the amygdala, but not hippocampus. Additionally, in males CaMKII and PKA regulated proteasome trypsin activity in the cytoplasm of hippocampal, but not amygdala cells, while in females both CaMKII and PKA could regulate this activity in the nucleus of cells in both regions. Proteasome peptidylglutamyl activity was regulated by CaMKII and PKA activity in the nuclei of amygdala and hippocampus cells in males. However, in females PKA regulated nuclear peptidylglutamyl activity in the amygdala, but not hippocampus. Collectively, these results suggest that CaMKII- and PKA-dependent regulation of proteasome activity in the brain varies significantly across subcellular compartments and between males and females.