<p> The human brain functions within a narrow range of temperatures and variations outside of this range incur cellular damage and death and, ultimately, death of the organism. Other organisms, like the poikilotherm <i>Drosophila melanogaster</i>, have adapted mechanisms to maintain brain function over wide ranges in temperature and, if exposed to high temperatures where brain function is no longer supported, these animals enter a protective coma to promote survival of the organism once the acute temperature stress is alleviated. </p><p> This research characterized the role of different neuronal cell types, including glia, in the protection of brain function during acute hyperthermia, specifically looking at two protective pathways: the heat shock protein (HSP) pathway and the cGMP-dependent protein kinase G (PKG) pathway. Whole animal behavioral assays were used in combination with tissue-specific genetic manipulation of protective pathways to determine the specific cell types sufficient to confer protection of neuronal function during acute hyperthermia. Using the neuromuscular junction (NMJ) preparation, calcium imaging techniques were combined with pharmacological and genetic manipulations to test the hypothesis that alterations in ion channel conductance via endogenous mechanisms regulating the cellular response to high temperature stress alter neuronal function. </p><p> Expression of <i>foraging</i> RNAi to inhibit PKG expression in neurons or glia demonstrated protection of function during acute hyperthermia measured behaviorally through the extension of locomotor function. This extension of function with the tissue-specific inhibition of PKG was also confirmed at the cellular level using the genetically encoded calcium indicator (GECI), GCaMP3, to image calcium dynamics at the NMJ, where preparations expressing <i> foraging</i> RNAi could continue to elicit changes in calcium dynamics in response to stimulation. Over the course of this study, the mechanism underlying a novel glial calcium wave in the peripheral nervous system was characterized in order to elucidate glia&rsquo;s role in the protection of neuronal function during acute hyperthermia.</p><p>

<p> Behavior in higher eukaryotes is a complex process which integrates signals in the environment, the genetic makeup of the organism, and connectivity in the nervous system to produce extremely diverse adaptations to the phenomenon of existence. Unraveling the subcellular components that contribute to behavioral output is important for both understanding how behavior occurs in an unperturbed state, as well as understanding how behavior changes when the underlying systems that generate it are altered. Of the numerous molecular species that make up a cell, the regulation of messenger RNAs (mRNAs), the coding template of all proteins, is of key importance to the proper maintenance and functioning of cells of the brain, and thus the synaptic signals and information integration which underlie behavior. RNA binding proteins, a class of regulatory molecules, associate with mRNAs and facilitate their maturation from pre-spliced nascent transcripts, their stabilization and degradation ensuring appropriate levels are maintained, as well as their translation and subcellular compartmentalization, which ensures that proteins are translated at the appropriate level and in the places where they are required to fulfill their cellular functions. Our laboratory identified polymorphisms in the gene coding for the CUGBP and ELAV-like Factor 6 (CELF6) RNA binding protein to be associated with Autism Spectrum Disorder risk in humans. ASD is a spectrum of disorders of early neurodevelopment which present with lowered sociability and communication skills as well as restricted patterns of interests. When expression of the <i>Celf6</i> gene was ablated in mice, we found that they exhibited reductions to early communication as well as altered aspects of their exploratory behavior. In this dissertation, I explore the communication changes in young mouse pups with loss of CELF6 protein and identify that despite being able to produce vocalization patterns similar to their wild-type littermates, they nevertheless exhibit reduced response to maternal separation. Despite a history of literature on other CELF family proteins, the functions of the CELF6 protein in the brain have not been previously described. I provide characterization of the mRNA binding targets of CELF6 in the brain, and show that they share common UGU-containing sequence motifs which has been noted for other CELF proteins, and that CELF6 binding occurs primarily in the 3' untranslated regions (3' UTR) of mRNA. I hypothesized that this mode of interaction would result in regulation of mRNA degradation or translation efficiency as 3' UTR regions are known for providing binding sites for numerous regulators of such processes. In order to answer this question, I cloned sequence elements from the 3' UTRs of target mRNAs into a massively parallel reporter assay which has enabled me to test the effect of CELF6 expression on hundreds of binding targets simultaneously. When expressed in vitro, I found that CELF6 induced reduction to reporter library levels but exhibited few effects on translation efficiency, and I was able to rescue effects to reporter abundance mutation of binding motifs. Intriguingly, like CELF6, CELF3, CELF4, and CELF5 were all able to produce the same effect. CELF5 and CELF6 both showed similar, intermediate repression of reporter library mRNAs, while CELF3 and CELF4 exerted the strongest levels of repression. The level of repression under these conditions was somewhat predicted by number of motifs present per element, however a large amount of the variance in reporter levels is still unexplained and a mechanism for CELF6's action is unknown. Nevertheless, the work I present in this dissertation shows that CELF6 and other members of its family are key regulators of mRNA abundance levels which has direct implications to downstream consequence in the cell. As several of CELF6 binding target mRNAs are known regulators of neuronal signaling and synaptic function, the information I present is crucial for future experimentation. This work well help lead us to understand how behavior is altered when this protein is absent, along the way uncovering important mechanistic steps connecting the molecular landscape of cells to the behavior of organisms.</p><p>

<p> Rod and cone photoreceptors in the retina are polarized sensory neurons that possess uniquely modified primary cilium, called the outer segment, to capture photons. Circadian-mediated shedding and renewal of outer segment membrane discs requires extensive vesicular transport of protein cargo from the endoplasmic reticulum and Golgi to the base of the cilium. Endocytosis is vesicle transport process of capturing and/or recycling extrinsic components and is shown to occur in retina of early vertebrates, such as <i>Xenopus</i> laevis. In this thesis, I have explored the hypothesis that a critical endocytosis-associated protein Rabgef1 is critical for the genesis of photoreceptor outer segments in the mammalian retina. After demonstrating high expression of Rabgef1 concordant with photoreceptor maturation, I characterized morphology and function of retina from <i>Rabgef1</i>-loss of function (<i>Rabgef1</i>^{&ndash;/&ndash;}) mice. Though no gross defect was observed by histology and immunohistochemistry before eye opening (postnatal day 14), transmission electron microscopy demonstrated ultrastructural defects in photoreceptor outer segments by P8. Progressive, yet rapid, photoreceptor degeneration and near-complete ablation of the visual response were evident at and after P15. I show that the outer segment defect noted in <i>Rabgef1</i>^{&ndash;/&ndash;} mice was not due to defective ciliogenesis or trafficking of cargo proteins to the cilium. In concordance with other systems, Rabgef1 was enriched in purified endocytic vesicles from the retina and interacted with Rabaptin5, confirming its role in Rab5-mediated endocytosis. Curiously, <i>Rabgef1</i>^{&ndash;/&ndash;} photoreceptors accumulated enlarged vesicular/endosomal structures within the inner segment, similar to loss of function mutations in the yeast orthologue of Rabgef1, Vps9p. My studies provide the first evidence of an essential role of Rabgef1-mediated fusion and recycling of endocytic vesicles in the formation and/or renewal of outer segment membrane discs in the mammalian retina. Rabgef1 and other components of the endocytic pathway should therefore be considered as candidates for human retinopathies. </p><p>

<p> Parkinson&rsquo;s disease (PD) is an adult onset neurodegenerative disease that is characterized by deficiencies in movement, cognition, and Lewy body neuropathology within the brain. Motor and cognitive deficiencies progressively worsen through the course of disease concurrent with increasing neuropathology and neurodegeneration. Approximately 10&ndash;15% of PD patients have a family history of PD with a confirmed genetic cause. Presently PD pathogenesis is incompletely understood and there are no treatments capable of halting or reversing this disease. The extended disease-course and age-dependent nature of PD, especially in genetic cases where a mutation is present from birth, affirm that aging itself is the most important risk factor for disease. We hypothesize that specific cellular changes that occur during the normal process of aging confer susceptibility to disease-causing mutations which, while tolerated at younger ages, contribute to disease with age. Accurate animal models of PD and aging provide the ability to elucidate disease mechanisms and explore novel strategies targeting the aging process. To test the role of aging in PD we utilize the nematode <i>Caenorhabditis elegans</i> because this animal has been used extensively to study animal aging at a cellular level. We confirm that disease phenotypes in genetic <i>C. elegans</i> models of PD such as neurodegeneration, protein aggregation, and mitochondrial deficits are proportional to this organism&rsquo;s brief lifespan. This indicates that PD progresses according to biological age and not merely to chronological time. As a proof-of-principle we also show that delaying aging by mutation of the gene encoding the insulin-IGF receptor, <i>daf-2</i>, can rescue multiple deficits present in nematode models of PD. Overall we demonstrate that biological aging is a crucial for the development of various PD associated phenotypes and that delaying aging is sufficient to delay these phenotypes. Therefore targeting aging itself may be a sound strategy for the halting or the prevention of PD.</p><p>

<p> Despite being comprised of networks with extensive positive feedback, the brain is able to prevent runaway activity. Neural networks are remarkably good at maintaining an activity setpoint while still permitting learning-related or developmental plasticity. To accomplish the delicate balance between change and stability, neural networks employ a group of homeostatic negative feedback mechanisms. This suite of homeostatic mechanisms sense and adjust neuronal excitability to keep firing rates within some target range. To date, the most well described manner in which neurons homeostatically regulate their excitability is through adjustment of excitatory or inhibitory synaptic weights, or by modulating their intrinsic excitability. It is perplexing why the neuron should have several means to accomplish the same outcome. Experiments demonstrating the collaborative or solo induction of homeostatic mechanisms have provided only limited insight into how homeostatic signaling pathways are organized to generate and maintain firing rate set-points (FRSP).</p><p> In order for neurons to maintain a FRSP, deviations from this value must modulate an internal signal that subsequently triggers homeostatic mechanisms to restore excitability to its set-point. The CaMKIV pathway is a calcium-dependent signaling element that plays a crucial role in regulating excitatory synaptic strength. The CaMKIV cascade is highly sensitive to activity and can modulate transcription, making it an ideal candidate to integrate incoming activity and modulate the excitability of neurons. Therefore, the major aim of this thesis was to characterize the role of CaMKIV in inducing multiple forms of homeostatic plasticity in tandem. Here we leverage our expertise in measuring homeostasis in neocortical neurons <i>in vitro</i> to determine how manipulating the activation state of nuclear CaMKIV affects neuronal excitability. </p><p> We found that excitatory synaptic scaling and intrinsic plasticity were bidirectionally induced by manipulating CaMKIV activity even without any perturbations to network activity. In contrast, CaMKIV had no impact on inhibitory synaptic weights. Additionally, we found that CaMKIV activity bidirectionally regulated spontaneous firing rates. Taken together, our data suggests that CaMKIV activity is used by the neuron to monitor the firing set point and gate homeostatic mechanisms to correct for drift from this target. The data presented in this thesis contribute that excitatory synaptic scaling and intrinsic excitability are tightly coordinated through bidirectional changes in the same signaling pathway, while inhibitory synaptic scaling is sensed and regulated through an independent signaling mechanism. This body of work contributes to a better understanding of neuronal homeostasis and will hopefully help us determine how malfunctions in homeostatic plasticity contributes to neurological and neurodevelopmental disorders.</p><p>

<p> Neural stem progenitor cells (NSPCs) in the mammalian brain contribute to life-long neurogenesis and brain health. Adult mammalian neurogenesis primarily occurs in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus. Epigenetic repression is a crucial regulator of cell fate specification during adult neurogenesis. How epigenetic repression impacts adult neurogenesis and how epigenetic dysregulation may impact neoplasia or tumorigenesis remains poorly understood. Examination of epigenetic regulation in the adult mammalian brain is complicated by the heterogeneous nature of neurogenic niches and by the highly orchestrated fate specification processes within neural stem progenitor cells involving myriad intrinsic and extrinsic factors. To overcome these challenges, we utilized a cross-species approach. To model histone modifications as they exist <i>in vivo</i> for epigenetic profiling, we isolated neural stem progenitor cells from the adult SVZ and SGZ of non-human primate baboon brains. To determine cellular and molecular changes within the adult SVZ and SGZ following loss of epigenetic repression, we utilized multiple mouse models, including conditional <i> Ezh2</i> and <i>Suv4-20h1</i> knockouts. To model the non-cell type specific effects common to small molecule screening and brain chemotherapeutic agents, induction of conditional knockout utilized a recombinant Cre protein. Finally, to model epigenetic mechanisms during SVZ-associated glioblastoma (GBM) tumorigenesis, we conducted comparative analysis between healthy NSPCs and GBM specimens from humans. The convergence of baboon, mouse and human models of adult neurogenesis revealed that epigenetic repression is a critical mechanism regulating proper neural cell fate and that epigenetic dysregulation may be a driver of GBM.</p><p>

The anteroventral periventricular nucleus (AVPV) of the preoptic area mediates the positive feedback effects of estradiol (E2) on LH surge in rats. Consistent with their role in female reproduction, the neurons in this region are more numerous in adult females than males. This sex difference is established due to E2-mediated cell death in the developing male AVPV. Loss of neurons in the AVPV permanently abolishes the ability of E2 to trigger LH surge release. However, the identity of the neurons lost during AVPV masculinization and the mechanism underlying E2-triggered cell death have not been clearly defined to date. This dissertation shows that, developmental exposure to E2 permanently reduces the number of dual-phenotype GABAergic/Glutamatergic (GABA/Glu) neurons, supporting the role of these neurons in female-specific LH surge release. My results identified a key role for TNFα-activated NFκB-mediated cell survival in establishing sex differences in the GABA/Glu population in the AVPV. GABAergic neurons in males had higher levels of TRAF Interacting Protein (TRIP), an inhibitor of the NFκB pathway. Thus, the male AVPV had lower levels of nuclear NFκB and its downstream target, pro-survival Bcl-2 mRNA than females. I also showed that E2 produces these sex differences by upregulating TRIP gene expression and thus reduces the number of GABA/Glu neurons in the male AVPV. Using the N42 GABA/Glu cell line as an in vitro model for the AVPV, I verified that E2 reversed TNFα-mediated effects on NFκB activation, Bcl-2 mRNA, and caspase activity. Moreover, E2 could directly upregulate TRIP mRNA levels, only in the presence of TNFα. To understand the nature of this cooperativity, I cloned the proximal TRIP promoter, identified an ERE half-site and a novel TNFRE in a region 1000 base pairs upstream of the transcription start site. Mutation of either of these sites abolished the stimulatory effect of E2 on promoter activity, suggesting that cooperative action of E2 and TNFα is required for TRIP promoter activation. To summarize, these studies provide a novel mechanism for sexual differentiation of the AVPV in which E2 acts cooperatively with TNFα to inhibit a neuroprotective pathway in GABA/Glu neurons of the male AVPV.

Progesterone regulates female reproduction in many ways, yet it is still unclear how signals are conveyed through nuclear and extranuclear receptors. The traditional notion was that progesterone binds classical progesterone receptors to alter gene transcription. This view has been challenged by the discovery of additional progesterone signaling molecules important for progesterone actions in non-neural cells. In granulosa cells, the progesterone receptor membrane component 1 (Pgrmc1) mediates progesterone effects by forming a receptor complex with binding partner, Serpine mRNA binding protein 1, but it is unknown whether these molecules function similarly in the brain. To begin to address these issues, I investigated the neural role of Pgrmc1 in female mouse brain, rat brain and in neural cells. By examining the neuroanatomical localization, hormonal regulation, and colocalization of Pgrmc1 within key neurons in the neural control of ovulation, Pgrmc1 emerged as a candidate signaling molecule likely to mediate progesterone functions. Furthermore, Pgrmc1 levels regulate the expression of several diverse genes and signaling pathways in neural cells. Taken together, these results demonstrate that Pgrmc1 function is likely to impact diverse neural functions.

Removal of protein activity by genetic mutation or pharmacological inhibition has been used extensively to understand the normal function of a protein. However, null mutations eliminate gene function in all cells and pharmacological agents can diffuse through tissues to have similar global effects that can obscure the physiological function of a protein. This is a particular problem when studying proteins that function in many cell types or that have different cell-specific activities. The most direct strategy to study the function of a protein is to reduce or eliminate its activity only in specific cell types, rather than in all cells of an organism. The idea of targeting gene knockdown to specific cell types or to individual cells is not new and many strategies aim to do just this. However, these strategies result in variable knockdown efficiencies and can have silencing effects in neighboring cells and therefore knockdown is never cell-specific. We developed a novel method to knock down the expression of any gene and to restrict this knockdown to specific cell types in C. elegans. In this method we replaced endogenous genes with single copy integrated transgenes containing an engineered sequence tag that introduces premature stop codons (PTCs) into transgene mRNA. This tag causes the natural stop codon to be recognized as a PTC by the host's nonsense-mediated decay (NMD) machinery and does not disrupt gene function. In NMD-competent animals, a PTC-containing transgene is degraded and in NMD-defective animals, a PTC-containing transgene is expressed. Therefore, the expression of PTC-containing transgenes can be controlled by cell-specific activation of NMD. Using this technique, we replaced two endogenous genes with PTC-containing transgenes and directed degradation of their mRNA to specific cell types by restoring NMD activity in these cells. The single copy transgenes were expressed at levels comparable to the endogenous genes and were knocked down to ∼10% of endogenous by NMD, resulting in both global and cell-specific null-like phenotypes. This knockdown strategy can be used to cell-specifically knock down essentially any gene in the C. elegans genome and should provide new insights into understanding protein function.

<p> Mitochondrial metabolism of reactive oxygen species (ROS) is tightly regulated during brain development. Imbalance has been correlated to neuropsychiatric disorders. Nevertheless, the contribution of ROS accumulation to aberrant cortical circuit organization and function remains unknown. Individuals with 22q11 deletion syndrome (22q11DS) are highly susceptible to psychiatric disorders; therefore, 22q11DS has been suggested as a model for studying the neurodevelopmental origins of these disorders. Six genes &ndash;<i>Mrpl40, Tango2, Prodh, Zdhhc8, Txnrd2</i> and <i>Scl25a1</i>&ndash; located in the 22q11DS commonly deleted region encode proteins that localize to mitochondria. This project aimed to characterize the effects of altered mitochondrial function, due to diminished dosage of these genes, on cortical projection neuron development, using the <i>LgDel</i> mouse model of 22q11DS. I found growth deficits in <i>LgDel</i> neurons that are due to increased mitochondrial ROS and are <i>Txnrd2</i>-dependent. Antioxidant treatment, by n-acetyl cysteine (NAC), rescues neuronal morphogenesis in <i>LgDel</i> and <i> Txnrd2</i>-depleted neurons <i>in vitro</i> and <i>in vivo.</i> Electroporation of <i>Txnrd2</i> restores ROS levels and normal dendritic and axonal growth. <i>Txnrd2</i>-dependent redox regulation underlies a key aspect of cortical circuit differentiation in a mouse model of 22q11DS. These studies define the effects of mitochondrial accumulation of ROS on neuronal integrity, and establish the role of altered pyramidal neuron differentiation in the formation of circuits in 22q11DS. These data provide novel insight into the role of redox imbalance in aberrant development of cortical circuits.</p><p>