Decreases in K63 Polyubiquitination in the Hippocampus Promote the Formation of Contextual Fear Memories in Both Males and Females

ABSTRACT Over 90% of protein degradation in eukaryotic cells occurs through the ubiquitin‐proteasome system (UPS). In this system, the ubiquitin protein can bind to a substrate on its own or it can form a chain with multiple ubiquitin molecules in a process called polyubiquitination. There are 8 different sites on ubiquitin at which polyubiquitin chains can be formed, the second most abundant of which, lysine‐63 (K63), is independent of the degradation process, though this mark has rarely been studied in the brain or during learning‐dependent synaptic plasticity. Recently, we found that knockdown of K63 polyubiquitination in the amygdala selectively impaired contextual fear memory formation in female, but not male, rats. It is unknown, however, whether the sex‐specific requirement of K63 polyubiquitination occurs in other brain regions that are required for contextual fear memory formation, including the hippocampus. Here, we found that CRISPR‐dCas13‐mediated knockdown of K63 polyubiquitination in the hippocampus significantly enhanced contextual fear memory in both male and female rats, a result that is in striking contrast to what we observed in the amygdala for both sex‐specificity and directionality. Using unbiased proteomics, we found that following fear conditioning K63 polyubiquitination was primarily decreased at target proteins in the hippocampus of both males and females. Importantly, the target proteins and downstream functional pathways influenced by K63 polyubiquitination changes diverged significantly by sex. Together, these data suggest that unlike what we previously reported in the amygdala, decreases in K63 polyubiquitination in the hippocampus are a critical regulator of memory formation in the hippocampus of both males and females.