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Stress plays a significant role in neural development and brain function. To better understand the mechanisms underlying the impact of stress on brain development and neuroendocrine function, this study focuses on the phenylethanolamine-N-methyltransferase (Pnmt) enzyme as a key mediator of stress hormone signaling. Pnmt is activated as part of a positive feedback mechanism during stress to convert norepinephrine to epinephrine and amplify the sympathetic response. Most of our knowledge about Pnmt is derived from its role in the systemic production of epinephrine from adrenal chromaffin cells, but it is also known to be expressed in the central nervous system, including the brainstem, retina, hypothalamus, and cerebellum. Given the importance of the central nervous system in modulating stress responses, this project sought to investigate cellular Pnmt expression in the central nervous system using a genetic-marking strategy with a Pnmt-Cre-recombinase knock-in driver strain (Pnmt+/Cre) and a β-galactosidase (βGal) reporter strain (R26R+/βGal) in parallel with Pnmt-specific immunofluorescent histochemical staining to identify Pnmt+ cells in the adult mouse cerebellum, hypothalamus, and cerebral cortex. The results show extensive patterns of active and historical Pnmt protein expression throughout the cerebellum and hypothalamus, with significant neuropeptide Y co-expression in the hypothalamus and considerable historical Pnmt expression throughout the cerebral cortex. To quantify baseline Pnmt mRNA levels across embryonic and postnatal neural development and elucidate differential Pnmt isoform expression through tissue-specific regulation in the developing brain, quantitative polymerase chain reaction (qPCR) was performed in the brainstem, cerebellum, and cerebral cortex with isoform-specific primers. Initial results show a developmental, tissue-specific Pnmt isoform shift between embryonic and postnatal neural development by an intron-retention alternative splicing mechanism. Ultimately, these findings provide an anatomical "blueprint" for investigating the role of central nervous system Pnmt expression in health and disease, and emphasize the role of Pnmt in early neural development, illustrating how stress impacts the formation of neural connections during formative periods of brain maturation.

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<p>Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin protein. The huntingtin protein has many roles in vesicular and endocytic trafficking, which can be modified in HD cells. When mutant huntingtin is expressed in HD, protein levels of the huntingtin interacting protein, Huntingtin-associated protein of 40kDa (HAP40) are increased. This increase in HAP40 protein levels causes the formation of a complex between carboxyl terminal huntingtin, HAP40 and active Rab5 on the early endosome. This complex induces a switch from early endosomal movements on microtubules to movements on actin filaments, greatly reducing both the speed and distance of movement. The main objective of this research is to determine where the interaction occurs between huntingtin and HAP40 on the huntingtin protein. Here we show that HAP40 interacts with the amino terminus of huntingtin, specifically the first 17 amino acids (N17). In co-localization studies, we found that HAP40 co-localizes with the carboxyl terminal fragment of huntingtin corresponding to amino acids 2570-2634; however, GFP trap immunoprecipitation analysis revealed no interaction between the carboxyl terminal fragments of huntingtin and HAP40. An interaction was discovered between HAP40 and N17, which was then confirmed by far western blot. These results demonstrate that HAP40 interacts with N17. By identifying the interaction site between HAP40 and huntingtin, modifications can be explored to prevent the interaction of HAP40 with huntingtin. This would restore motility on microtubules reinstating fast, long-range movements of early endosomes.</p> / Master of Science (MSc)

Digitized by Kansas Correctional Industries

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Medical Sciences

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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