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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In the development of the central nervous system, thousands of neuronal subtypes must be generated, each with their own unique molecular properties. This process is governed by selector transcription factors, which specify cell-identities by binding to cell-type specific genomic regulatory elements. These regulatory elements, dispersed across extragenic regions of the genome, establish precise long-distance interactions with target gene promoters to regulate their expression. While prior studies have emphasized the roles of distally bound selector transcription factors in cell-type specification, the involvement of gene promoters in the regulation of gene expression remains underexplored. In this dissertation, I analyze the role of promoter elements in regulating neuronal gene expression programs using a comprehensive approach that combines high-throughput genomics and targeted experimental manipulations. In Chapter 2, I reveal a highly flexible regulatory system utilized in the nervous system: neuronal promoters are universal and can thus be activated by any enhancer found within their regulatory neighborhood. This model of promiscuous neuronal promoters raises two important questions: Is promoter promiscuity a universal phenomena? What are the promoter elements that facilitate universality? To address these questions in Chapter 3, I first find that promoters of genes associated with pluripotency exhibit incompatibility with neuronal enhancers. Then, to test what promoter elements encode for this incompatibility, and also which elements endow neuronal promoters with their promiscuity, I developed a novel promoter-screening strategy. Through this work, I discovered novel aspects of enhancer-promoter communication. First, core promoters are universal and can be induced by non-cell identity matched distal enhancers. Second, promoter-proximal regions serve to modulate expression from universal core promoters by either dampening or potentiating their responsiveness to distal enhancers. This work suggests that in addition to distal regulatory elements, promoter-proximal regions also play an active role in fine-tuning cell-type specific gene expression programs by either modulating induction or repressing ectopic expression. Finally, in Chapter 4, I explore another aspect of the regulation of cell-identity during development, shifting my focus away from selector transcription factors and instead on “secondary” transcription factors induced during differentiation. Here, I utilize a multi-omic approach to characterize the role of Mnx1 in motor neuron development. Analysis of its effects on gene expression, distal genomic binding patterns, and influence on the overall regulatory landscape reveals that Mnx1 plays a role in maintaining the motor neuron cell-identity by ensuring robust expression of motor neuron genes and preventing ectopic expression of genes normally restricted to alternate neuronal subtypes. This suggests that “secondary” transcription factors play a role in refining cellular identities established by selector transcription factors. Integrating these findings with prior research in central nervous system development underscores that while neuronal gene expression programs are primarily established through the actions of selector transcription factor-bound distal regulatory elements, promoters and secondary transcription factors contribute to the fine-tuning of transcription and, consequently, cell identity.

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Neurovascular injuries are leading causes of disability implicated in neurological dysfunction. Much of the Central Nervous System (CNS) homeostasis depends on concerted signaling between neurons, glial cells, and vasculature–the neurovascular unit (NVU). Neurovascular injuries disrupt the NVU causing hypoxia, ischemia, neuroinflammation, and neuronal death. Much of the neuroinflammatory responses associated with neurovascular injuries have been characterized, but the contribution of specific signaling pathways from the injured endothelium to inflammatory response remains to be established. To understand vascular-glial communication in the context of vascular injury, the Troy lab has used a mouse model of retinal vascular injury, retinal vein occlusion (RVO). The retina is a CNS enclosed tissue that allows live visualization of vascular and neuronal condition upon injury, genotype, and/or treatment. Previous studies in the laboratory determined that non-apoptotic expression of endothelial caspase-9 (EC Casp9) was key for the development of retinal edema, capillary ischemia, and neuronal death. Caspases are known for their role in mediating cell death, but how and if glial cells orchestrated outcomes remain unknown. This thesis work aimed to investigate the role of caspase-9 signaling in vascular-glial communication and its contribution to pro-inflammatory cytokine levels and neurodegeneration in neurovascular injury. To answer this, we first optimized the mouse model of RVO and profiled the levels of caspases in RVO retinas treated or untreated with a caspase-9 inhibitor using immunohistochemistry. Then, we used tamoxifen inducible endothelial and astroglial caspase-9 KO lines, subjected them to RVO and measured glial changes, cytokine levels, capillary ischemia, retinal edema, neuronal death, and vision dysfunction. We first found that RVO induces a range of cell-specific levels of caspases and that inhibition of caspase-9 specifically modulated the levels of endothelial caspase-9 and 8, neuronal caspase-9, 7, and 6, astroglial caspase-6, and leukocytic caspase-9 and 7. Our studies also suggest that endothelial caspase-9 induces a decrease in reactive microglia, inflammatory cytokines, cleaved- caspase-6 and GFAP cleavage in astrocytes. EC Casp9 deletion also altered changes in GFAP, nestin and AQP4 levels in Müller glia. Through investigating an astroglial caspase-9 KO, we discovered that astroglial caspase-9 could be upstream of astroglia caspase-6. Additionally, we found that astroglial caspase-9 loss protected hypoxic retinas from capillary ischemia but not from retinal edema nor neuronal death. Lastly, we used an optokinetic test to study the potential role of endothelial and astroglial caspase-9 in RVO-induced vision disfunction. Our results indicate that removing caspase-9 from endothelial cells or astrocytes protected contrast sensitivity damage in visual function one day post-RVO. In sum, the present thesis work demonstrates that endothelial and astroglial caspase-9 signaling can lead to inflammation and worsening of visual function in neurovascular injury.