Proteasome-independent K63 polyubiquitination selectively regulates ATP levels and proteasome activity during fear memory formation in the female amygdala

Females are more likely than males to develop post-traumatic stress disorder (PTSD). However, the neurobiological mechanisms responsible for these sex differences remain elusive. The ubiquitin proteasome system (UPS) is involved in fear memory formation and implicated in PTSD development. Despite this, proteasome-independent functions of the UPS have rarely been studied in the brain. Here, using a combination of molecular, biochemical, proteomic, behavioral, and novel genetic approaches, we investigated the role of proteasome-independent lysine-63 (K63)-polyubiquitination, the second most abundant ubiquitin modification in cells, in the amygdala during fear memory formation in male and female rats. Only females had increased levels of K63-polyubiquitination targeting in the amygdala following fear conditioning, which targeted proteins involved in ATP synthesis and proteasome function. CRISPR-dCas13b-mediated knockdown of K63-polyubiquitination in the amygdala via editing of the K63 codon in the major ubiquitin gene, Ubc , impaired fear memory in females, but not males, and caused a reduction in learning-related increases in ATP levels and proteasome activity in the female amygdala. These results suggest that proteasome-independent K63-polyubiquitination is selectively involved in fear memory formation in the female amygdala, where it is involved in the regulation of ATP synthesis and proteasome activity following learning. This indicates the first link between proteasome-independent and proteasome-dependent UPS functions in the brain during fear memory formation. Importantly, these data are congruent with reported sex differences in PTSD development and may contribute to our understanding of why females are more likely to develop PTSD than males.