<p> Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality following cervical spinal cord injury (SCI). Upon SCI, inspiratory signals originating in the rostral ventral respiratory group (rVRG) of the medulla become disrupted from their phrenic motor neuron (PhMN) targets, resulting in diaphragm paralysis. Using the rat model of C2 hemisection SCI, we investigated two mechanisms of axonal plasticity to drive respiratory revovery: promoting regeneration of injured rVRG axons and enhancing contralateral rVRG sprouting. We aimed to stimulate rVRG axon regeneration via induction of the mammalian target of rapamycin (mTOR) pathway, a signaling system that regulates neuronal-intrinsic axon growth potential. Specifically, we targeted two key components of this pathway via: viral vector-mediated expression of a constitutively-active form of ras homolog enriched in brain (Rheb); and systemic treatment with a small-molecule peptide inhibitor of phosphatase and tensin homolog (PTEN). Expression of cRheb selectively in rVRG neurons promoted significant regeneration of ipsilateral rVRG axons. PTEN peptide administration enabled injured rVRG axons to regrow through the lesion and back to the PhMN pool within C3-C5 spinal cord (i.e. several segments from the injury). This robust rVRG axon regrowth coincided with significant restoration of diaphragm activity, as assessed by <i> in vivo</i> electromyography (EMG) recordings. Furthermore, surgical &ldquo;re-lesion&rdquo; through the lesion ablated the functional improvement induced by PTEN inhibition. We also labeled contralateral rVRG axons in a separate cohort; we observed no increases in contralateral rVRG axon input to the PhMN pool ipsilateral to the hemisection, suggesting that increased drive from spared contralateral rVRG is likely not responsible for recovery. To address contralateral sprouting driving diaphragm recovery, we administered a PTP&sigma; inhibitory peptide following C2 hemisection. We found no increase in ipsilateral regeneration following treatment; however, we found robust contralateral rVRG sprouting at all three regions of the spinal cord, C3, C4, and C5. Enhanced sprouting also coincided with diaphragm recovery, which was not ablated upon relesion suggesting that the diaphragm recovery was through contralateral axonal plasticity. Collectively these exciting results demonstrate that targeting both ipsilateral regeneration and contralateral sprouting promotes rVRG-PhMN circuit re-connectivity and recovery of diaphragm function following cervical SCI.</p><p>

<p> Developmental processes, genes and environmental factors interact to produce changes in cognition and behavior over the lifespan of an individual. However, the underlying molecular genetic mechanisms that mediate these changes remain to be fully elucidated. DNA methylation is an epigenetic mechanism that defines cell identity and helps regulate gene expression. DNA methylation is dynamic over development and has been shown to mediate experience-dependent changes, including those resulting from learning and memory and early life adversity. Although methylation mainly occurs at genomic cytosines in the CG dinucleotide context, methylation at non-CG sites was recently found in brain tissue. Non-CG methylation is specifically enriched in neurons and accumulates during the early childhood stages of brain development. The biological impact of non-CG methylation in regulating gene expression and regulating cellular function, if any, remains unclear. A major challenge for addressing this question is the complexity and scale of the DNA methylation landscape, which includes nearly one billion cytosines throughout the genome that are potential sites of modification in every cell. Targeted studies of specific candidate genes and genomic loci do not elucidate the overall configuration of the cellular epigenome. Techniques for comprehensively mapping the genome-wide distribution of DNA methylation are powerful, but they require sophisticated new computational methods of analysis that can reliably distinguish and statistically validate epigenomic differences related to developmental and environmental factors. </p><p> In this thesis we develop new approaches to comprehensively analyze DNA methylation throughout the genome and with single base resolution in order to better characterize the role of CG methylation and elucidate the potential role of CH methylation in mammalian brain cells. First, we consider the impact of enriched early life (peri-pubertal) experience on DNA methylation and gene expression in the dentate gyrus of the hippocampus. In addition to its role in experience-dependent gene regulation, DNA methylation also plays a key role in innate developmental processes, including female X chromosome inactivation. We provide the first allele-specific DNA methylomes from the active and inactive X chromosomes in female brain, and use comprehensive genomic analyses to gain insight into the functional relationship between allele-specific DNA methylation and transcription. These two studies provide new evidence of the dynamic changes in DNA methylation in whole brain tissue caused by environmental and innate developmental factors. However, they do not address the heterogeneity of brain cell types, a hallmark of mammalian brain organization. To address the role of DNA methylation in brain cell diversity, we develop computational methods to analyze data from a new assay that measures single cell methylomes. Using these data, we show that brain cell methylomes can be clustered and used to assess neuronal heterogeneity in the frontal cortex of mouse and human. Upon clustering cells, we are able to gain insight into the role of methylation in the establishment and maintenance of cellular identity in neuronal types. </p><p> Overall, this thesis adds to the increasing evidence that DNA methylation is a cell type-specific, dynamic epigenomic modification of brain cells that is impacted by, and may in turn help to regulate, neuronal development and adaptation. In addition, this thesis provides new computational methods for analyzing large-scale, whole-genome DNA methylation data sets and demonstrates their use in uncovering new insights into the mammalian brain epigenome.</p><p>

<p> ABSTRACT The nervous system comprises more diverse and intricately specialized cell types than any other tissue in the body. Understanding the developmental mechanisms that generate cellular diversity in the nervous system is a major challenge in neuroscience. The nematode <i>C. elegans</i> offers the opportunity to study neuronal development at the molecular level with extraordinary resolution.</p><p> My dissertation focuses on the elucidation of genetic mechanisms required for the proper development of the chemosensory BAG neurons, which are specialized for detecting the respiratory gas carbon dioxide (CO₂). Analogs of these neurons play diverse roles in animals from different phyla. CO₂-sensing neurons in the mammalian brainstem are critical regulators of the respiratory motor program, and their dysfunction has been linked to fatal apneas such as Sudden Infant Death Syndrome. In nematodes, CO₂-sensing neurons mediate an avoidance behavior, but their ethological function was not known.</p><p> In my initial studies of BAG neuron development, I demonstrated that a conserved ETS-family transcription factor directly regulates genes required for CO₂-sensing, including the receptor-type guanylate cyclase, GCY-9, which likely functions as a CO₂ receptor. To uncover other genes that function together with <i>ets-5,</i> I carried out a large-scale chemical mutagenesis screen for mutants with improper BAG neuron differentiation. From this screen I identified two new genes required for BAG neuron development: the Pax6 homolog <i>vab-3</i> and the p38 Mitogen-Activated Protein (MAP) kinase <i>pmk-3</i>.</p><p> VAB-3 likely acts during embryonic development to pattern the expression of ETS-5 in head neurons of <i>C. elegans</i>. In loss of function <i> vab-3</i> mutants, ETS-5 protein is misexpressed in hypodermal cells and a motor neuron, in addition to its expression in BAG. VAB-3 likely represses transcription of ETS-5 in some lineages, such as those that give rise to hypodermal cells.</p><p> I next demonstrated that the p38 MAPK PMK-3 functions in a Toll-like receptor (TLR) signaling pathway. This discovery revealed an unexpected role for TLR signaling in neuronal differentiation. Because TLR signaling was known to be required for behavioral responses to microbes, I tested whether BAG neurons were required for pathogen avoidance. I found that this was the case and propose that TLR signaling functions in pathogen avoidance by promoting the development and function of chemosensory neurons that surveil the metabolic activity of environmental microbes.</p><p> Because ETS-5, VAB-3 and TOL-1 are members of gene families that are conserved between nematodes and vertebrates, a similar mechanism might act in the specification and differentiation of CO₂-sensing neurons in other phyla.</p>

<p> Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two debilitating and relatively common early-onset neurodegenerative disorders along a clinicopathologic spectrum. Accounting for ~25% of familial FTD and ~30% of familial ALS, a GGGGCC hexanucleotide repeat expansion in chromosome 9 open reading-frame 72 (C9ORF72) is the most common known genetic cause of both disorders (c9FTD/ALS). While many neuropathologic features of c9FTD/ALS cases mirror archetypal FTD/ALS [i.e., focal neuronal loss, TAR DNA-binding protein 43 (TDP-43) pathology, etc.], a subset of neuronal lesions have been observed which are positive for ubiquitin, but negative for TDP-43, suggesting additional underlying pathology. Our goal of this study was to eliminate the possibility of the involvement other classic neurodegenerative protein (tau and amyloid-&beta;) and identify novel aggregating proteins which could potentially serve as biomarkers and therapeutic targets. Using quantitative neuropathologic assessment, we demonstrated tau and amyloid-&beta; did not consistently co-localize with these ubiquitinated inclusions and was not more common in c9FTD/ALS compared to sporadic FTD and ALS cases (as well as FTD cases with another known mutation, GRN). Following insights from the field of trinucleotide repeat disorders, we discovered novel pathologic dipeptide repeats in c9FTD/ALS generated through the process of repeat-associated non-ATG (RAN) translation. These dipeptide repeat proteins had a very characteristic and consistent morphology, distribution, and pathologic burden amongst c9FTD/ALS cases. These proteins were also highly specific to C9ORF72 repeat expansion carriers, even in clinically and neuropathologically atypical cases. Subsequent research from this initial finding has not only confirmed our results, but also demonstrated the toxicity of these proteins and even their therapeutic potential towards ameliorating disease in individuals harboring the C9ORF72 expansion.</p>

<p> The membrane-bounded lysosome is a dynamic organelle responsible for macromolecule degradation, receptor down regulation, stress survival and pH balance. Intact lysosomal function is critical for proper cell function, as defects in lysosomal trafficking have been associated with neurodegenerative diseases such as Alzheimer&rsquo;s disease. <i>Saccharomyces cerevisiae </i>, budding yeast, is an ideal model organism for studying lysosomal protein trafficking because its vacuole is functionally analogous to the mammalian lysosome and both share conserved trafficking machinery components. </p><p> A previous yeast genomic deletion strain screen in our lab uncovered fourteen deletion strains with h&barbelow;ypersensitivity to <u>hy </u>gromycin B (<i>hhy</i> mutants), all of which exhibited defective vacuolar trafficking, morphology and/or function. Additionally, recent microscopic findings in our lab support that the late-endosome dependent pathway is the affected pathway and that <i>hhy</i>s have compromised T&barbelow;arget of R&barbelow;apamycin C&barbelow;omplex 1&barbelow; signaling. TORC1 regulates cell growth and cell proliferation and its hyperactivity is associated with cancer and metabolic diseases. As with lysosomes, the vacuole also serves as the platform for TORC1 signaling. </p><p> To further assess if the late-endosome dependent pathway is affected by hygromycin B treatment in <i>hhy</i> mutants, vacuolar delivery of two vacuolar markers was assessed. Alkaline phosphatase (ALP) delivery was assessed as a late-endosome independent pathway marker and carboxypeptidase Y (CPY) delivery was assessed as a late-endosome dependent pathway marker. To date, we have observed that hygromycin B treatment disrupts CPY trafficking via the late-endosome dependent pathway while ALP trafficking via the late-endosome independent pathway is unaffected by hygromycin B treatment. </p><p> Rapamycin is the direct inhibitor of TORC1. Rapamycin and its analogs (rapalogs) are currently administered in the treatment of TORC1 hyperactivity. However, therapeutic inhibition of TORC1 signaling by rapamycin is associated with severe toxicities. Since our recent results implicate compromised vacuolar trafficking of Tor1 kinase in <i>hhy</i> mutants, we tested vesicular trafficking inhibiting drugs in combination with rapamycin in order to explore a cumulative effect on TORC1 inhibition assessed by growth of wild type cells. Additionally, we explored the effects of combining two vesicular trafficking drugs on wild type cell growth. </p><p> We established a cumulative effect on wild type growth upon using low concentrations of rapamycin in combination with vesicular trafficking inhibitory drugs. Thus, rapamycin and vesicular trafficking inhibitory drugs have potential for drug combination therapy against TORC1 hyperactivity at lower drug concentrations. Drug combination treatment may be a new and effective way to regulate TORC1 function at sub-toxic levels of rapamycin.</p>

<p> Na⁺/Ca²⁺ exchangers are low affinity/high capacity transporters that mediate Ca²⁺ extrusion by coupling Ca²⁺ efflux to the influx of Na⁺ ions. Their primary function is to maintain Ca²⁺ homeostasis in cells of all organisms and they play a particularly important role in excitable cells that experience transient Ca²⁺ fluxes. While their functions have been studied extensively in muscle cells, much is still unknown about their contributions to the nervous system. Data suggests that Na⁺/Ca²⁺ exchangers play a key role in neuronal processes such as memory formation, learning, oligodendrocyte differentiation and axon guidance. They are also implicated in pathologies such as Alzheimer&rsquo;s Disease, Parkinson&rsquo;s Disease, Multiple Sclerosis and Epilepsy. While they are implicated in critical neuronal processes, a clear understanding of their mechanism remains unknown. This dissertation examines the role of Na⁺/Ca²⁺ exchangers in the invertebrate model organism <i>Caenorhabditis elegans </i>. There are ten identified Na⁺/Ca²⁺ exchanger genes in <i>C. elegans</i> (labeled <i>ncx-1</i> to <i>ncx</i>-10). Data presented here is the first comprehensive description of their genetics and function in <i>C. elegans</i>. The expression pattern of all 10 Na⁺/Ca²⁺ exchanger genes is described and their phylogeny is examined comparatively across humans and flies. Analysis of <i>ncx-2</i> and <i>ncx-8</i> mutants shows important roles for Na⁺/Ca²⁺ exchanger genes in egg-laying, lipid storage and longevity, suggesting a role in diverse biological functions for Na⁺/Ca²⁺ exchangers in <i>C. elegans</i>. The function of an NCLX type Na⁺/Ca²⁺ exchanger NCX-9 is also detailed comprehensively. Analysis of <i> ncx-9</i> mutants shows that NCX-9 is required for asymmetrical axon guidance choices made by the DD and VD GABAergic motor neuron circuit. Pathway analysis shows that NCX-9 regulates asymmetric circuit patterning through RAC-dependent UNC-6/Netrin signaling and LON-2/Glypican Heparan Sulfate signaling. <i> In vitro</i> analysis of NCX-9 physiology in HEK cells shows that NCX-9 is a mitochondrial Na⁺/Ca²⁺ exchanger, similar to NCLX, which is its homolog in humans.</p>

<p> The study of how genotypes encode phenotypes is germane if not central to every area of genetics research. This thesis focuses on the application of targeted and Whole Exome Sequencing (WES) in the discovery, identification and understanding of Mendelian disorders primarily with Spinocerebellar Ataxia(s) (SCA) phenotypes. Analogous to Mendelian disorders as a whole, SCA are a complex group of disorders with both shared and unique clinical symptoms. Currently, 28 genetically unique forms of autosomal dominant SCA have been identified. This thesis begins by exploring the potential role of targeted Next Generation Sequencing (NGS) as a clinical and diagnostic tool. The benefits of using targeted sequencing in a clinical setting are two-fold. First, it presents the opportunity to rapidly screen symptomatic individuals for all known genetic variants associated with ataxia phenotypes, thereby greatly increasing the likelihood and accuracy of a diagnosis. Second, symptomatic patients who test negative for all known variants may harbor a novel genetic variant. The identification of novel forms of SCA is of great importance both clinically and in basic research. As a case in point, this thesis uses WES to identify the genetic basis of a rare autosomal dominant SCA affecting a multi-generational kindred of Canadian European descent. Four affected and two unaffected individuals spanning three generations have been sequenced to identify the causal variant. The ability of WES to identify the pathogenic variant of a Mendelian disorder from only four affected individuals is a significant benchmark in genomics research. It is both feasible and probable that as reference databases become more extensive, the genetic diagnosis of human disease from a single affected individual will be common practice. This thesis concludes by examining Mendelian disease variants from a broader perspective. Large exome variant cohorts of asymptomatic individuals are examined for the presence of known pathogenic Mendelian variants. The presence of such variants in a reference database is empirical evidence that pathogenic variants, while necessary, are not sufficient to cause many Mendelian disorders. Specifically, we demonstrate that variable penetrance and expressivity are pervasive factors in Mendelian genetics that have yet to be fully appreciated.</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> Similar to the pattern observed in people with substance abuse disorders, laboratory animals will exhibit escalation of cocaine intake when the drug is readily available and will exhibit increased drug-seeking behaviors after long periods of abstinence. Additionally, there are long term changes in neuron structure, receptor function, and neurotransmission associated with abstinence from cocaine in humans and animals. DNA methylation is an epigenetic modification to the DNA structure that mediates mRNA expression to confer different cell types, but has recently been implicated in learning and memory mechanisms. The long-term control that DNA methylation has over gene expression in animals makes it a prime candidate for controlling gene expression over the course of abstinence in animals with previous drug experience. Therefore, here, I investigated the contribution of behavioral contingency of cocaine administration on escalation of cocaine intake and re-exposure to cocaine cues as well as DNA methylation and gene expression within the dorsal medial prefrontal cortex (dmPFC) in adult male Sprague-Dawley rats. I exposed rats to daily training for saline (1 h/ day) or cocaine (0.25 mg/kg/inf) in limited- (1 h access per day), prolonged- (6 h access per day), or limited + yoked-access (1 h contingent + 5 h non-contingent access per day) for 15 days. Rats were then put through forced abstinence for 1, 14, or 60 days, and then the dmPFC was dissected out. Saline- and prolonged-access rats were additionally separated into cue- and no cue- conditions after 60 days of abstinence, where cue rats were re-exposed to the operant chamber without cocaine delivery for 2 h. These studies led to 4 main findings. 1) cocaine contingency affects mRNA expression for glutamatergic genes, 2) DNA methylation changes dynamically throughout abstinence, 3) re-exposure to cocaine cues rapidly alters DNA methylation and mRNA expression, and 4) DNA methylation, hydroxymethylation, and transcription factor binding all contribute to altered mRNA expression.</p><p>

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.