Neuroprogenitor cells are an important resource because of their potential to replace damaged cells in the brain caused by trauma and disease. It is of great importance to better understand which factors influence the differentiation and migration of these cells. Previously it has been reported that neuroprogenitor cells undergoing apoptotic stress have increased levels of Amyloid precursor protein (APP) and increased APP expression results in glial differentiation. APP activity was also shown to be required for staurosporine induced glial differentiation of neuroprogenitor cells. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is expressed during inflammatory. The binding of MCP-1 to its chemokine receptor induces expression of novel transcription factor MCP-1 induced protein (MCPIP). MCPIP expression subsequently leads to cell death. Previous studies have shown that pro apoptotic factors have the ability to induce neural differentiation. Therefore, we investigated if MCPIP expression leads to differentiation of NT2 neuroprogenitor cells. Results showed that MCPIP expression increased glial fibrillary acid protein expression and also caused distinct morphological changes, both indicative of glial differentiation. Similar results were observed with MCP-1 treatment. Interestingly, APP expression decreased in response to MCPIP. Instead, we found APP activity regulates expression of both MCP-1 and MCPIP. Furthermore, inhibition of either p38 MAPK or JAK signaling pathways significantly reduced APP's effect on MCP-1 and MCPIP. These data demonstrate the role APP has in glial differentiation of NT2 cells through MCP-1/MCPIP signaling. It is possible that increased APP expression after CNS injury could play a ii role in MCP-1 production, possibly promoting astrocyte activation at injured site. We next investigated the effect that MCP-1 has on NT2 cell migration. Studies have shown that when neuroprogenitor cells are transplanted into the brain they migrate towards damaged areas, suggesting that these areas express factors that recruit migrating cells. Generally, after neuronal injury there is a neuroinflammatory response that results in increased chemokine production. We demonstrate that MCP-1 significantly induces the migration of NT2 neuroprogenitor cells. Activation of intracellular cyclic adenosine monophosphate (cAMP) or protein kinase C with forskolin and phorbol 12-myristate 13-acetate (PMA), respectively, was able to completely abolish the MCP-1 induced migration. Contrarily, neither extracellular signal-regulated kinase or p38 mitogen activated protein kinase was required for NT2 cells to respond to MCP-1. Interestingly, APP's ability to activate MCP-1 expression was shown to play a role in NT2 cell migration. We showed that NT2 cells expressing APP were capable of inducing migration of other neuroprogenitor cells. Utilizing a MCP-1 neutralizing antibody we discovered that APP induced migration was not caused solely by increased MCP-1 production. Interestingly, APP increased expression of several C-C chemokines: MCP-1, Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES), and macrophage inflammatory protein alpha (MIP-1 alpha). This demonstrates the unique role APP has in regulating chemokine production, which directly affects cell migration. Taken together, this study provides us with a greater understanding of the mechanisms involved in both glial differentiation and migration of NT2 neuroprogenitor cells.

As the most widely consumed drug around the globe, there is a vast array of contradicting research available on caffeine. One of the most debated and researched topics on caffeine is its effect on the brain. Meanwhile, the data on the neurological condition of migraine has information scattered throughout countless research articles and experiments. Although neither migraine or caffeine are completely understood by the medical world, this analysis attempts to give a more coherent understanding of the relationship between the two. This is done by first understanding the known and theorized mechanisms of caffeine as well as the pathologies of migraine. Discussions on channelopathies, current migraine medications, and case studies will be presented. After much background research, we hypothesized that caffeine could excite neurons at physiological concentrations to the point of activation. This was tested by targeting the transcription factor cFos using immunocytochemistry in vitro. The protein cFos was identified due to its rapid translation—just 15 minutes after stimuli—to indicate activation. In addition to a control culture, three different caffeine concentrations were tested on the neurons: 50 micromoles— average plasma level after 1-2 cups of coffee consumption, 100 micromoles—average plasma level after 5-6 cups of coffee also believed to be the therapeutic amount to defend against neurological diseases such as Alzheimers Disease, and 250 micromoles—the average plasma level considered to be toxic in humans. Indeed, we saw a 53.8% increase in cFos expression in the neurons as 100 micromolar of caffeine was added and exposed to the cell cultures for 24 hours. In order to ensure the results obtained in this study were physiologically relevant in vivo, known toxic levels were tested for in vitro neurotoxicity. It was found in vitro that at the non toxic plasma concentrations of 50 micromolar and 100 micromolar of caffeine did not display cellular death as tested by Trypan Blue viability testing, Crystal Violet morphologies, and fleurojade immunochemistry that tests for degeneration. Each of these experiments identified a significant death increase as the toxic level of 250 micromoles of caffeine were utilized. This allowed us to theorize that the activation of neurons found in these experiments due to caffeine exposure would apply the same effect in vivo.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an X-linked late-onset neurodegenerative disorder caused by a noncoding trinucleotide repeat expansion in the FMR1 gene. This gene produces fragile x mental retardation protein (FMRP), an RNA binding protein whose targets are involved in brain development and synaptic plasticity. One of the proposed mechanisms of FXTAS pathogenesis is an RNA gain-of-function in which the repeat expansion causes toxic mRNA that sequesters important proteins in the cell, interfering with their functions. Another suggested method of pathogenesis is through a mutant protein called FMRpolyG. This protein results from repeat-associated non-AUG (RAN) translation, in which the expanded repeats are translated where they otherwise would not be. This protein co-localizes with intranuclear inclusions and nuclear membrane proteins, causing disorganization of the nuclear lamina in FXTAS patient brain samples and neurons differentiated from FXTAS patient-derived induced pluripotent stem cells (iPSCs). iPSC technology involves reprogramming an adult somatic cell back to an embryonic-like state, allowing it to be differentiated into all cell types. A limit with iPSCs, though, is modeling late-onset disorders because the cells lose all age-related features during reprogramming. Progerin, a truncated form of the lamin A protein, has been used to age neurons differentiated from Parkinson Disease (PD) patient-derived iPSCs. Progerin-mediated aging was found to unmask PD-like phenotypes in those neurons, making it a promising technology for modeling late-onset disorders such as FXTAS. In this study, we investigated the link between the aging process and FXTAS pathogenesis in neurons differentiated from FXTAS patient-derived iPSCs with the use of progerin. Progerin transduction was successful in aging the FXTAS neurons. The presence of FMRpolyG was confirmed and an interaction with Lap2b was observed. In some neurons, there was also an observed interaction between FMRpolyG and progerin. Overall, this data suggests that there is an interaction between the mutant FMRpolyG protein and the nuclear membrane during aging, which may contribute to the cell death that causes neurodegeneration in FXTAS patients.

The development of the vertebrate eye is tightly controlled by precise genetic regulations. From a single ocular primordium to bilateral eyes with complex structures and cell types, it requires intensive proliferation and migration for cells in both the ectoderm and mesoderm to accomplish ocular morphogenesis, and during this process cell differentiation and interaction takes place to establish the complex composition of ocular cell types and cellular connections. Genetic defects can lead to severe abnormalities in eye morphogenesis and cell differentiation during ocular development. A tremendous amount of work has been done to identify both intrinsic and extrinsic factors that regulate ocular development. However, much more work is needed to fully understand this complex process. Sox4 is known as a transcription activator that regulates cell survival and differentiation in multiple embryonic tissues during development. Evidence of its requirement during ocular development has recently emerged, but the mechanism by which Sox4 regulates ocular development is far from elucidated. Chapter 1 of this dissertation provides an overview of different stages in embryonic eye development and known genetic interactions during each stage. It also reviews recent knowledge about SoxC proteins and their roles in ocular development. Chapter 2 presents data characterizing the expression profile of the zebrafish sox4 co-orthologs, sox4a and sox4b, in the developing eye. Additionally, it presents data from morpholino-mediated sox4 knockdown in zebrafish, which indicate that Sox4 deficiency leads to defects in choroid fissure closure through elevation in the Hedgehog (Hh) signaling pathway. Sox4 knockdown causes upregulation of the Hh ligand indian hedgehog b (ihhb), which alters the proximal-distal boundary of the optic vesicle and inhibits choroid fissure closure. Chapter 3 presents data reporting the generation of sox4 mutant zebrafish lines using the CRISPR/Cas9 genome editing system. Characterization of one sox4a maternal zygotic (MZ) mutant line confirms Sox4’s role in negative regulation of Hh signaling and reveals new evidence that maternal and zygotic sox4 are both critical for ocular development. Chapter 4 presents data demonstrating that sox4 is required for rod photoreceptor neurogenesis. Rod photoreceptor terminal differentiation is delayed in both sox4 morphants and sox4 CRISPR mutants, while rod progenitor and precursor cells are properly specified. In Chapter 5, the roles of Sox4 in regulating ocular development are summarized based on the results, and implications of the results are discussed to expand our understanding of the genetic regulation of ocular morphogenesis and retinal neurogenesis.

Obesity and drug abuse share common neural circuitries including the mesocoticolimbic and striatal dopamine reward system. In the current study, a rat model of diet-induced obesity (DIO) was used to determine striatal dopamine function, impulsivity and motivation as neurobehavioral outcomes and predictors of obesity. For the outcome study, rats were randomly assigned a high-fat (HF) or a low-fat (LF) diet for 8 wk. Following the 8-wk HF-diet exposure, rats were segregated into obesity-prone and obesity-resistant groups based on maximum and minimum body weight gain, respectively, and neurobehavioral outcomes were evaluated. For the predictor study, neurobehavioral antecedents were evaluated prior to an 8-wk high-fat diet exposure and were correlated with subsequent body weight gain. Striatal D2 receptor density was determined by in vitro kinetic analysis of [3H]raclopride binding. DAT function was determined using in vitro kinetic analysis of [3H]dopamine uptake, methamphetamine-evoked [3H]dopamine overflow and no net flux in vivo microdialysis. DAT cell-surface expression was determined using biotinylation and Western blotting. Impulsivity and food-motivated behavior were determined using a delay discounting task and progressive ratio schedule for food-reinforcers, respectively. Relative to obesity-resistant, obesity-prone rats exhibited 18% greater body weight, 42% lower striatal D2 receptor density, 30% lower total DAT expression, 40% lower in vitro and in vivo DAT function, 45% greater extracellular dopamine concentration, and 2-fold greater methamphetamine-evoked [3H]dopamine overflow. Obesity-prone rats exhibited higher motivation for food, but were less impulsive relative to obesity-resistant rats. Neurobehavioral antecedents of DIO included greater motivation for high-fat reinforcers in rats subsequently shown to be obesity-prone relative to obesity-resistant. Impulsivity, DAT function and extracellular dopamine concentration did not predict the DIO-phenotype. Thus, motivation for food is linked to both initiation and maintenance of obesity. Importantly, obesity results in decreased striatal DAT function, which may underlie the maintenance of compulsive food intake in obesity.

The ZNF9 gene on chromosome 3 encodes the cellular nucleic acid binding protein (CNBP), a ubiquitously expressed, 177 amino acid (≈19.5kDa) protein that is highly conserved among vertebrates. The function of the protein is largely unknown, however an expansion in the first intron of the protein results in myotonic dystrophy type 2 (DM2), a multisystemic disease featuring cardiac arrhythmia, muscle wasting, cataracts, and a range of neuropathologies. Remarkably, we recently discovered that CNBP is involved in regulating the activity of β-secretase, the enzyme that produces the first cleavage event in the generation of the amyloid-β peptide (Aβ). The progressive fibrillization and deposition of Aβ is widely believed to be the primary causal factor in the development of Alzheimer’s disease (AD), and AD-like pathology in individuals with Down syndrome (DS). DS provides a unique model for evaluating how these factors change in the aged brain as compared to young brain, and how such changes affect the proportion of DS patients with AD. In the AD brain, both BACE1 and BACE2 increased from an early stage of disease; in DS brains, BACE1 significantly decreased (p<0.04) with age, whereas BACE2 was unchanged, even though the gene for BACE2 is located within the DS obligate region of chromosome 21. BACE1 and BACE2 activity levels were highly correlated in this series (r2 = 0.95), indicating that there may be a higher degree of shared regulation than previously believed. This implicates regulators of BACE as potentially critical for the development of AD, and our data suggests that CNBP may be one such regulator. In AD, CNBP increases early in the disease process, a change that does not occur in the normal aging process or in DS. CNBP and BACE protein levels were correlated in these cases (p<0.001), while there was no relationship between CNBP and age, or CNBP and Aβ, in either the human or mouse brain, indicating that CNBP does not increase as a consequence of normal aging. Thirty day overexpression of CNBP following adeno-associated viral delivery in murine gastrocnemius muscle resulted in an increase in BACE1 protein (p<0.01) and a consequential increase in Aβ production (p<0.01). Other experiments indicated that CNBP overexpression did not affect the half-life of BACE1 mRNA or protein, but resulted in an increase in BACE1 translation. These data indicate that CNBP is an important regulator of β-secretase, and may play an important role in the onset and progression of AD.

Endogenous bioelectric fields guide morphogenesis during embryonic development and regeneration by directly regulating the cellular functions responsible for these phenomena. Although this role has been extensively explored in many peripheral tissues, the ability of electric fields to regulate wound repair and stimulate regeneration in the mammalian central nervous system (CNS) has not been convincingly established. This dissertation explores the role of electric fields in regulating the injury response and controlling the regenerative potential of the mammalian CNS. We place particular emphasis on their influence on astrocytes, as specific differences in their injury-induced behaviors have been associated with differences in the regenerative potential demonstrated between mammalian and non-mammalian vertebrates. For example, astrocytes in both mammalian and non- mammalian vertebrates begin migrating towards the lesion within hours and begin to proliferate after an initial delay of two days; subsequently, astrocytes in non-mammalian vertebrates support neurogenesis and assume a bipolar radial glia-like morphology that guides regenerating axons, whereas astrocytes in mammals do not demonstrate robust neurogenesis and undergo a hypertrophic response that inhibits axon sprouting. To test whether injury-induced electric fields drive the astrocytic response to injury, we exposed separate populations of purified astrocytes from the rat cortex and cerebellum to electric field intensities associated with intact and injured mammalian tissues, as well as to those electric field intensities measured in regenerating non-mammalian vertebrate tissues. Upon exposure to electric field intensities associated with uninjured tissue, astrocytes showed little change in their cellular behavior. However, cortical astrocytes responded to electric field intensities associated with injured mammalian tissues by demonstrating dramatic increases in migration and proliferation, behaviors that are associated with their formation of a glial scar in vivo; in contrast, cerebellar astrocytes, which do not organize into a demarcated glial scar, did not respond to these electric fields. At electric field intensities associated with regenerating tissues, both cerebellar and cortical astrocytes demonstrated robust and sustained responses that included morphological changes consistent with a regenerative phenotype. These results support the hypothesis that physiologic electric fields drive the astrocytic response to injury, and that elevated electric fields may induce a more regenerative response among mammalian astrocytes.

Cardiovascular disease (CVD) is the leading cause of death in the United States. Nearly half of the people diagnosed with heart failure (HF) die within 5 years of diagnosis. Brain abnormalities secondary to CVD have been observed in many discrete regions, including the hippocampus. Nearly 25% of patients with CVD also have major depressive disorder (MDD), and hippocampal dysfunction is a characteristic of both diseases. In this study, the hippocampus and an area of the hippocampal formation, the dentate gyrus (DG), were studied in a canine model of HF. Using this canine HF model previously, we have determined that myocardial infarction with mitral valve regurgitation (MI/MR) + spinal cord stimulation (SCS) can preserve cardiac function. The goal of this study was to determine if the SCS can also protect the brain in a similar fashion. Both the entire hippocampus and the DG tissues were dissected from canine brains and analyzed. These findings provide strong evidence that, in addition to the cardioprotective effects observed previously, SCS following MI/MR induces neuroprotective effects in the brain.

Infertility is one of the major problems of obesity. Studies have shown that administration of leptin reversed obesity-induced infertility in rats and mice. Coenzyme Q10 (CoQ10) is an antioxidant and also supplies the energy needed for ovulation and embryo development. We hypothesized that leptin when combined with CoQ10 could greatly improve obesity-induced infertility. The results showed a significant decrease in food intake, body weight, and the regular estrous cycle was restored after treatment with leptin+CoQ10. There was a significant increase (p10 significantly (p10 can improve fertility in obese infertile female rats. This study could provide a novel therapeutic strategy for the treatment of infertility and formulation of new drugs for the treatment of obesity-induced infertility in females.

Neuropathic pain affects up to 50% of the 29 million diabetic patients in the United States. Neuropathic pain in diabetes manifests as a disease of the peripheral and central nervous systems. The prevalence of type 2 diabetes is far greater than type 1 (90%), yet the overwhelming focus on type 1 models this has left the mechanisms of pain in type 2 diabetes largely unknown. Therefore I aimed to improve the current mechanistic understanding of pain associated with type 2 diabetes using two preclinical rodent models: Zucker Diabetic Fatty rats and db/db mice. In addition, I highlight the translational importance of simultaneous measurement of evoked/sensory and non-evoked/affective pain-related behaviors in preclinical models. This work is the first to show a measure of motivational-affective pain in a model of type 2 diabetes. I used methodological approaches including: (1) immunohistochemical and calcium imaging to assess stimulus-evoked sensitization; (2) measurement nociceptive behaviors and evoked sensory thresholds as well as pain affect using novel mechanical conflict avoidance and conditioned place preference/aversion assays; (3) pharmacological and genetic manipulation of methylglyoxal, TRPA1, AC1, and PPARγ. I hypothesized that the thiazolidinedione class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists would reduce neuropathic pain-like behavior and spinal neuron sensitization in traumatic nerve injury and type 2 diabetes. As PPARγ is a nuclear receptor, and already targeted clinically to promote cellular insulin sensitization to reduce hyperglycemia, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. In two separate research aims, I challenged this view and tested whether the PPARγ agonist pioglitazone would (1) rapidly alleviate neuropathic pain through a non-genomic mechanism and (2) reduce painful sensitization in nociceptive and neuropathic pain models independent from lowering blood glucose. I aimed to investigate the contribution of the glucose metabolite methylglyoxal to painful type 2 diabetes. I tested the hypothesis that methylglyoxal produces nociceptive, evoked, and affective pain that is dependent on activation of the sensory neuron cation channel TRPA1 and the secondary messenger enzyme AC1. I also tested whether pioglitazone or the novel methylglyoxal scavenging peptide GERP10 could alleviate painful type 2 diabetes.