Fear memory formation requires a coordination of molecular events, including protein synthesis, protein degradation, and epigenetic regulation of gene expression, throughout a circuit of brain regions. One mechanism highly studied for its involvement in protein degradation during fear memory is the ubiquitin-proteasome system (UPS), which utilizes the small protein ubiquitin to label proteins. Ubiquitin contains eight linkage sites that each lead to a unique outcome for the protein being labeled and a protein can gain one (monoubiquitination) or multiple (polyubiquitination) ubiquitins. The 26S proteasome is the catalytic component of the UPS and is comprised of a 20S catalytic core surrounded by two 19S regulatory caps. Phosphorylation of 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Interestingly, sex differences have been observed in proteasome-mediated protein degradation in the amygdala and hippocampus during fear memory formation. However, female subjects have only recently been regularly included in rodent behavioral studies so the majority of data on mechanisms of fear memory apply solely to the male brain. Considering post-traumatic stress disorder (PTSD) is two to three times more prevalent in females compared to males, understanding the mechanisms involved in fear memory in both sexes is important for understanding sex-specific development of fear-based disorders, such as PTSD.
Importantly, the UPS also has non-proteolytic functions independent of proteasome-mediated protein degradation. For example, monoubiquitination and some forms of polyubiquitination do not lead to protein degradation. Additionally, 19S cap regulatory subunit RPT6 has been found to function independently of its role in the proteasome, where it has a transcription-like role in the hippocampus of male rats during fear memory formation. Unfortunately, proteasome-independent functions of the UPS have not been extensively studied in terms of different forms of ubiquitination. Additionally, it is unclear whether phosphorylation of RPT6 is necessary for its non-proteolytic roles in memory formation and the role of proteasome-independent RPT6 in general has not been investigated in female subjects.
Here, we address these gaps in knowledge by 1) investigating sex-differences in the role of lysine 63 (K63-) polyubiquitination, a proteasome-independent ubiquitin linkage, in the amygdala during fear memory formation, 2) studying the role of proteasome-independent RPT6 in the hippocampus of female rats during fear memory formation, and 3) identifying proteasome-independent RPT6 target genes as well as the role of phosphorylation status of RPT6 at Serine-120 for its transcriptional activity during memory formation in the hippocampus of male rats. We first found that levels of K63-polyubiquitination targeting in the amygdala were increased in female, but not male, rats during fear memory formation. Interestingly, K63-polyubiquitination targeted proteins involved in ATP synthesis and proteasome functions in the amygdala of females and genetic manipulation of the K63 codon in the ubiquitin coding gene led to decreased ATP levels and proteasome activity. Additionally, this manipulation only led to impaired fear memory in females, suggesting that K63-polyubiquitination has a sex-selective role in the amygdala, where it regulates fear memory in females, but not males.
We then investigated the role of proteasome-independent RPT6 in the hippocampus of females and males during fear memory formation. In females, we found RPT6 did not bind to DNA regions in the c-fos gene, a previously identified RPT6 target gene in males. However, RPT6 did bind to monoubiquitination of histone H2B at lysine-120 (H2BubiK120), an epigenetic modification identified as an RPT6 binding partner in males, suggesting a potential role for proteasome-independent RPT6 in transcriptional regulation in the hippocampus of female rats. In males, we identified RPT6 targets genes during memory formation, found that RPT6 DNA binding alone altered gene expression, and lastly observed that pRPT6-S120 was necessary for RPT6 to bind DNA and regulate transcription during memory formation. Collectively, these data reveal sex-differences in proteasome-independent roles of the UPS through ubiquitination and proteasomal subunits in both the amygdala and hippocampus during fear memory formation. Considering males and females have differences in PTSD prevalence, understanding proteasome-independent roles of the UPS in both sexes may lead to a better understanding of PTSD development as well as potential therapeutic targets in each sex. / Doctor of Philosophy / In order to remember a fear-provoking memory, or any memory, it must be stored in the brain after the event. To store a fear memory, cells in specific areas of the brain have to destroy some proteins and activate or shut off certain genes using epigenetic mechanisms. Although the DNA itself never changes, epigenetic mechanisms recruit proteins to sit on the DNA to make it more (activate) or less (shut off) accessible. For protein destruction, it has been shown that brain cells use a mechanism called the ubiquitin-proteasome system (UPS) during fear memory storage. The UPS uses a small protein called ubiquitin to label proteins in the cell. Ubiquitin is versatile in its ability to label proteins due to it having eight different binding sites that can be used as a label. Some proteins only gain one ubiquitin (monoubiquitination), while other proteins can gain multiple ubiquitin proteins (polyubiquitination) and both the number of ubiquitins and the label used determines what happens to the protein. When a protein is labeled to be destroyed, the UPS uses a large complex of proteins called the 26S proteasome, which contains a section called the 20S catalytic core and two 19S regulatory caps that sit above and below the 20S core. It has been shown that when one of the proteins in the 19S regulatory cap called RPT6 gains a phosphate molecule at the 120th amino acid, which is a serine amino acid, it increases the number of proteins destroyed by the 26S proteasome. Interestingly, the UPS does not destroy proteins in the same way in male and female brains during storage of a fear memory. This is important because females are 2-3 times more likely to develop post-traumatic stress disorder (PTSD) than males, but it is unclear why, making the study of mechanisms involved in fear memory storage in both males and females important.
The UPS also functions in ways that do not involve destroying proteins. For example, proteins with a monoubiquitination label are often not destroyed. Additionally, it has been observed that RPT6, a protein in the 19S regulatory cap, can work outside of the 19S regulatory cap of the 26S proteasome to activate genes in brain cells of male rats. Sadly, the ubiquitin labels that do not cause protein destruction have not been well studied. It is also unclear whether RPT6 must gain a phosphate group to activate genes and if it activates genes in both male and female brains during fear memory storage.
In the present study, we investigated the role of a common ubiquitin linkage that does not cause protein destruction called lysine 63- (K63-) polyubiquitination during fear memory storage in the amygdala, the emotional control center of the brain, in male and female rats. We found that K63-polyubiquitination is increased in female, but not male, rats during fear memory storage. In females, K63-polyubiquitination was involved in making new ATP as well as controlling the number of proteins destroyed by the 26S proteasome. Lastly, we found that female, but not male, rats required K63-polyubiquitination in the amygdala for fear memory storage, suggesting a female-specific use of this ubiquitin label.
In this study, we also studied the role of RPT6 in the hippocampus, another key area of the brain for memory storage, of male and female rats. In females, we found RPT6 did not activate the same gene we previously identified in male rats, but it did bind with a monoubiquitination label on a protein that DNA is wrapped around, known as a histone. Due to this finding, it appears that RPT6 may act freely of the 19S regulatory cap to alter accessibility of genes in the hippocampus of females. On the other hand, in males we found that RPT6 activates some genes and shuts off other genes and can do so by sitting on DNA by itself. We also found that the addition of phosphate to RPT6 was necessary for it to sit on a gene and activate it during fear memory storage. These results show differences in UPS mechanisms during fear memory storage between males and females, which will be important for understanding why females are more likely to develop PTSD than males and for identifying potential treatment options.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/116506 |
| Date | 18 October 2023 |
| Creators | Farrell, Kayla Brianne |
| Contributors | Animal and Poultry Sciences, Jarome, Timothy J., Gilbert, Elizabeth Ruth, Lee, Kiho, Campbell, Susan |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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