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

Molecular Psychiatry volume 28, pages 2594–2605 (2023)Cite this article

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Abstract

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.

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Fig. 1: Sex-selective changes in K63-polyubiquitin protein targets in the amygdala of male and female rats during fear memory formation.
Fig. 2: CRISPR-dCas13b-ADAR2DD RNA editing system can specifically and efficiently edit different polyubiquitin linkage sites.
Fig. 3: CRISPR-dCas13b-ADAR2DD mediated knockdown of K63-polyubiquitination in the BLA impairs contextual fear memory in female, but not male, rats.
Fig. 4: CRISPR-dCas13b-ADAR2DD mediated knockdown of K63-polyubiquitination in the BLA does not broadly impact anxiety or auditory fear memory in females.
Fig. 5: CRISPR-dCas13b-ADAR2DD mediated knockdown of K63-polyubiquitination in the BLA alters total ATP levels but not proteasome activity in female rats 1 h after contextual fear conditioning and alters proteasome activity but not ATP levels in female rats 4 h after contextual fear conditioning.

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Acknowledgements

This work was supported by National Institutes of Health grants MH122414, MH123742, MH120498, MH120569, MH131587, AG071523 and AG079292 to T.J.J.

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Authors and Affiliations

  1. School of Animal Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

    Kayla Farrell & Timothy J. Jarome

  2. School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

    Madeline Musaus & Timothy J. Jarome

  3. Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

    Aubrey Auerbach

  4. Translational Biology, Medicine and Health Graduate Program, Virginia Polytechnic Institute and State University, Roanoke, VA, USA

    Shaghayegh Navabpour & Timothy J. Jarome

  5. Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

    W. Keith Ray & Richard F. Helm

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Contributions

KF, MM, and TJJ designed the experiments. KF, MM, and AA performed the experiments. KF and TJJ analyzed data. WKR and RH performed mass spectrometry experiments. KF and SN analyzed proteomic data. KF and TJJ wrote the manuscript.

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Correspondence to Timothy J. Jarome.

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Farrell, K., Musaus, M., Auerbach, A. et al. Proteasome-independent K63 polyubiquitination selectively regulates ATP levels and proteasome activity during fear memory formation in the female amygdala. Mol Psychiatry 28, 2594–2605 (2023). https://doi.org/10.1038/s41380-023-02112-0

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