Master of Science
Anatomy & Neurobiology
Dr. Peter Hamilton
Dr. William Barton
Dr. Melissa McGinn Greer
Anxiety disorders are the most prevalent mental illness in the United States with many individuals underdiagnosed and undertreated (Anxiety & Depression Association of America, 2021; Bandelow et al., 2017). While stress is a known risk factor for developing anxiety disorders, there is emerging evidence that biological variability in the expression of key proteins in the brain may contribute to regulating an organism’s stress-related behaviors. Earlier work from our laboratory identified a protein, called Regulation of Nuclear Pre-mRNA Domain Containing 2 (RPRD2), that was differentially expressed in the brain of mice stratified in terms of their phenotypic response to chronic stress (Hamilton et al., 2020). RPRD2 protein was elevated in the nucleus accumbens (NAc) of stress resilient animals–mice that display fewer behavioral abnormalities and more successful adaptation to stressors–and is predicted to play a significant role in gene transcription (Hamilton et al., 2020; Sjöstedt et al., 2020). In a cohort of mice exposed to chronic social defeat stress (CSDS), this protein was revealed as differentially expressed in the ventral hippocampus, medial prefrontal cortex, and NAc, of which RPRD2 was the top most affected protein in the NAc (Hamilton et al., 2020). More specifically, previously published reports suggest that RPRD2 functions to modulate RNA polymerase II activity by dephosphorylating serines at position 5 of the carboxyl-terminal domain of RNA polymerase II (Sjöstedt et al., 2020). Due to the correlation between RPRD2 and stress-induced behavioral adaptations in laboratory rodents, I aim to build upon this data and decipher this protein’s influence on stress-related behavior, mechanism of action in the NAc, and any potential impact it may have on medium spiny neuron (MSN) morphology, the major neuron functional unit in the NAc.
In order to expand upon the work conducted by Hamilton et al., I first exposed mice to a period of CSDS and conducted behavioral tests to determine the extent to which elevated levels of RPRD2 in the NAc impacted behavior. Following a period of CSDS, male mice with viral overexpression of RPRD2 spent less time in the open arms of an Elevated Plus Maze (EPM) test, a paradigm that quantifies anxiety-like behavior; however, no difference was observed with a Social Interaction test or thigmotaxis analysis–the tendency of mice to remain close to the walls (Bailey & Crawley, 2009; Kraeuter et al., 2019; Rodgers & Dalvi, 1987). These findings support the conclusion that viral overexpression of RPRD2 specifically impacts anxiety-like behavior, rather than social behavior–another behavioral domain known to be sensitive to chronic stress.
I then began to inquire about not only RPRD2’s behavioral impact, but also RPRD2’s molecular impact in the NAc. It was previously reported that RPRD proteins recruit deacetylases of lysine residues at position 7–specifically, HDAC1–which in turn allows RPRD to decrease phosphorylation of serines at position 5 of the carboxyl-terminus domain of RNA Polymerase II (Ali et al., 2019). Because RNA polymerase II facilitates transcription, I examined RPRD2’s impact on its Phospho Serine 5 levels via Western blots in order to explore its particular role in transcription. Although no significant correlation was found, tissue samples from mice virally overexpressed with RPRD2 revealed a downward trend in RNA Polymerase II Phospho Serine 5 levels (normalized to β-actin). This led me to further inquire about the exact downstream mechanistic impact of this protein and its regulation of transcription within the NAc.
We performed RNA sequencing to determine the gene loci regulated by viral over-expression of RPRD2 in the NAc. RPRD2 overexpression was correlated with upregulated genes canonically enriched in fibroblast cell-types and associated with downregulated genes enriched in ependymal cell-types. Following a gene ontology analysis performed on the significantly regulated differentially expressed genes (DEGs) in the RNA sequencing data, microtubule-based movement was the biological process most significantly associated with RPRD2 downregulated transcripts. Due to this finding, more research is needed regarding the impact of viral overexpression of RPRD2 on downregulating microtubule transport within RPRD2 expressing cells in the NAc.
Furthermore, previous studies have shown decreased strength of NAc synapses of mice expressing depression-like behavior following CSDS (Bagot et al., 2015; LeGates et al., 2018). Thus, enhancing activity in MSNs may result in resilient behavior; contrastingly, diminishing MSNs may result in anxiety-like behavior following CSDS (Francis & Lobo, 2017). To test this, I observed the contribution of viral overexpression of RPRD2 on medium spiny neuron morphology. Based on observation, there appeared to be less dendritic branching in neurons virally overexpressed with RPRD2; however, a Sholl analysis must be conducted in the future in order to solidify this observation.
Overall, I hypothesize that viral overexpression of RPRD2 in the NAc regulates anxiety-like behavior by decreasing RNA polymerase II-mediated transcriptional activity, which consequently impacts medium spiny neuron morphology in the NAc. In synthesizing my research, I will describe my comprehensive knowledge of RPRD2 and its relationship to chronic stress and anxiety, as well as a description of the materials and methods used to demonstrate the effects of RPRD2 in the NAc. I will then describe the results collected and discuss its potential ramifications in understanding the molecular drivers of neuropsychiatric syndromes. The results of this study can help to better understand how RPRD2 serves as a link between chronic stress and behavioral responses, and the mechanistic role of RPRD2 in the NAc. In the future, I hope this data can facilitate studies dedicated to targeting RPRD2 in order to develop effective pharmacotherapies for those with anxiety disorders.
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