Females, but not males, require protein degradation in the hippocampus for contextual fear memory formation

Abstract

Strong evidence supports a role for protein degradation in fear memory formation. However, these data have been largely done in only male animals. Here, we found that following contextual fear conditioning, females, but not males, had increased levels of proteasome activity and K48 polyubiquitin protein targeting in the dorsal hippocampus, the latter of which occurred at chaperones or RNA processing proteins. In vivo CRISPR-dCas9-mediated repression of protein degradation in the dorsal hippocampus impaired contextual fear memory in females, but not males. These results suggest a sex-specific role for protein degradation in the hippocampus during the consolidation of a contextual fear memory.

Figure 1.

Females, but not males, have increased proteasome activity in the dorsal hippocampus following contextual fear conditioning. (A) Experimental design showing that the dorsal CA1 region of the hippocampus was taken and dissected 1 h after contextual fear conditioning. Male and female rats were trained at separate times. (B) Behavioral performance of male and female rats during the training session. (C–E) Female rats that underwent contextual fear conditioning (n = 10) had increased proteasome activity in the nuclear (C), but not cytosolic (D) or synaptic (E), regions of cells in the hippocampus when compared with their naïve counterparts (n = 9 nuclear, 10 cytosolic/synaptic). (F–H) Male rats that underwent contextual fear conditioning did not have altered proteasome activity in the nucleus (n = 7; F), cytosolic (n = 10; G) or synaptic (n = 10; H) compartments relative to naïve controls (n = 8 nuclear/synaptic, 9 cytosolic). (I) Female rats that underwent an immediate shock procedure (n = 8) had increased proteasome activity in the nuclear compartment of cells in the hippocampus when compared with their naïve counterparts (n = 7). (*) P < 0.05 from naïve.

Figure 2.

Proteomic analysis of degradation-specific K48 polyubiquitination targets in the dorsal hippocampus of male and female rats following contextual conditioning. Male and female rats were trained to a contextual fear conditioning task and the dorsal CA1 region of the hippocampus collected 1 h later for K48-specific tandem ubiquitin binding entity (K48-TUBE) liquid chromatography-mass spectrometry (MC/MS) analysis. Males and females were trained at separate times (n = 5 per group) and compared with same sex naïve animals (n = 5 per group). (A) Identification of fear conditioning-induced K48 polyubiquitination targets for both females (orange bars) and males (green bar). Proteins are identified on the Y-axis and log change in K48 polyubiquitination (trained/naïve) is on the X-axis. Negative log value indicates a loss of K48 polyubiquitination with fear conditioning. (B,C) Ingenuity pathway analysis (IPA) was performed on female hippocampus samples that underwent mass spectrometry analysis. (B) IPAs of downstream targets of ribosomal RNA processing 12 protein (RRP12). Green indicates a decrease in function due to K48-mediated degradation, and the dotted gray arrow denotes indirect decreased function of ribonucleotide reductase (RNR). (C) IPAs of downstream targets of heat shock protein 40 (HSP40). Green indicates a decrease in function due to K48-mediated degradation, and the solid gray arrow denotes direct inactivation of the tyrosine kinase fibroblast growth factor receptor 1 (FGFR1) and sorting nexin 1 (SNX1).

Figure 3.

CRISPR–dCas9-mediated down-regulation of protein degradation in the dorsal hippocampus impairs memory for a contextual fear conditioning task in females but not males. (A) Experimental design showing that the fear conditioning took place 2 wk after the dorsal hippocampus CRISPR–dCas9 injection, with testing occurring 24 h after training. Synthetic guide RNAs (gRNAs) targeted ubiquitin (Uba52) and proteasome subunit (Psmd14) coding genes and were combined with a transcriptionally repressive dCas9-KRAB-MECP2 fusion. Male and female cohorts were run at separate times. (B,C) In females, injection of the CRISPR–dCas9 constructs did not impact performance during training (B) but significantly impaired retention during testing (C) relative to controls (n = 6 per group). (D,E) In males, injection of the CRISPR–dCas9 constructs did not impact performance during training (D) or retention during testing (E) relative to controls (n = 8 per group). (*) P < 0.05 from Uba52 + Psmd14-gRNA.