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.

Age-related neurologic disease is a significant and growing burden on our society. Although the largest share of research effort has typically been devoted to the common neurodegenerative illnesses (such as Alzheimer’s disease, or AD), the reality is that nearly all cases of neurodegenerative disease possess elements of mixed pathology. Vascular contributions to cognitive impairment and dementia (VCID) is a complex form of dementia, combining aspects of vascular disease and other forms of dementia, such as Alzheimer’s disease. This pathology is heterogeneous and can include cerebral amyloid angiopathy (CAA), hemorrhages, white matter infarcts, and changes to the neurovascular unit. Given the heterogeneous nature of VCID, we hypothesized that we could further elucidate mechanisms that drive dementia in VCID by examining pathology in mouse models and use this data to guide the study of human autopsy cases. Using a mouse model of VCID, we identified NHE1, a sodium hydrogen exchanger that was upregulated in these mice, as a possible candidate for a factor involved in cerebrovascular disease in humans. We saw a significant age effect of NHE1 in cases with Down syndrome (DS), leading us to further examine cerebrovascular pathology in individuals with DS. People with DS are at a high risk of developing cognitive impairment and dementia after the age of 50. In fact, virtually all adults with DS develop the neuropathology for an AD (beta-amyloid (Aß) senile plaques and tau neurofibrillary tangles) diagnosis by the age of 40 due to a triplication of chromosome 21. We found that these individuals develop CAA and microhemorrhages as a function of age, and that these rates are as severe as sporadic AD, despite an age difference of ~30 years. We also found that individuals with DS have different microglial morphologies than controls or individuals with AD. This data indicates that people with DS develop significant cerebrovascular and AD pathology, indicative of VCID. Overall, we found that mixed pathologies, specifically VCID, is an important contributor to the development of dementia and should be studied further to better understand how this pathology drives cognitive impairment. Further, it is clear that mouse models map imperfectly onto complex human diseases, and that significant work remains to be done towards achieving an adequate model of VCID.

Glycogen is the sole carbohydrate storage molecule found in mammalian cells and plays an important role in cellular metabolism in nearly all tissues, including the brain. Defects in glycogen metabolism underlie the glycogen storage diseases (GSDs), genetic disorders with variable clinical phenotypes depending on the mutation type and affected gene(s). Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy and a non-classical GSD. LD typically manifests in adolescence with tonic-clonic seizures, myoclonus, and a rapid, insidious progression. Patients experience increasingly severe and frequent epileptic episodes, loss of speech and muscular control, disinhibited dementia, and severe cognitive decline; death usually ensues in the second decade of life. LD, like one- third of all epilepsy disorders, is intractable and resistant to antiseizure drugs. A hallmark of LD is the accumulation of intracellular, insoluble carbohydrate aggregates known as Lafora bodies (LBs) in brain, muscle, and other tissues. LBs are a type of polyglucosan body, an insoluble aggregate of aberrant glycogen found in some GSDs and neurodegenerative disorders. Like most GSDs, LD is an autosomal recessive genetic disorder. Approximately 50% of LD patients carry mutations in the epilepsy, progressive myoclonus 2A (EPM2A) gene encoding laforin, a glycogen phosphatase. Remaining patients carry mutations in EPM2B, the gene that encodes malin, an E3 ubiquitin ligase. Laforin and malin play important roles in glycogen metabolism. In the absence of either enzyme, glycogen transforms into an insoluble, hyperphosphorylated and aberrantly branched polysaccharide reminiscent of plant starch. This abnormal polysaccharide precipitates to form LBs and has pathological consequences in the brain. Since a definitive LD diagnosis requires genetic testing, whole exome sequencing has been increasingly used to diagnose LD. As a result, numerous cases of more slowly progressing or late-onset LD have been discovered that are associated with missense mutations in EPM2A or EPM2B. Over 50 EPM2A missense mutations have been described. These mutations map to many regions of the laforin X-ray crystal structure, suggesting they produce a spectrum of effects on laforin function. In the present work, a biochemical pipeline was developed to characterize laforin patient mutations. The mutations fall into distinct classes with mild, moderate or severe effects on laforin function, providing a biochemical explanation for less severe forms of LD. LBs drive LD pathology. As a result, LBs and glycogen metabolism have become therapeutic targets. Since LBs are starch-like, and starch is degraded by amylases, these enzymes are potential therapeutics for reducing LB loads in vivo. However, amylases are normally secreted enzymes. Degradation of intracellular LBs requires a cell-penetrating delivery platform. Herein, an antibody-enzyme fusion (AEF) technology was developed to degrade LBs in vitro, in situ in cell culture, and in vivo in LD mouse models. AEFs are a now putative precision therapy for LD, potentially the first therapeutic to provide a significant clinical benefit. Prior to this work, LD was considered a homogenous disorder and treatments were only palliative. The data herein support a spectrum of clinical progression, a potential therapy for LD, and mechanistic insights into LD pathophysiology. This work illustrates how personalized medicine, both in diagnosis and treatment, can be achieved through basic biochemical approaches to human disease.

The ketogenic diet is commonly used to control epilepsy, especially in cases when medications cannot. The diet typically consists of high fat, low carb, and adequate protein and produces a metabolite called acetoacetate. Seizure activity is characterized by glutamate excitotoxicity and therefore glutamate regulation is a point of research for control of these disorders. Acetoacetate is heavily implicated as the primary molecule responsible for decreasing glutamate in the synapse; it is believed that acetoacetate interferes with the transport of glutamate into the synaptic vesicles. The effects on synaptic transmission at glutamatergic synapses was studied in relation to the ketogenic diet in Drosophila larvae for this thesis. Various measures of synaptic transmission were conducted. Acetoacetate decreased neurotransmission at the synapse. It was also found that acetoacetate has direct effects on the postsynaptic membrane, which indicates a novel role for the metabolite.

HIV is capable of causing a set of neurological diseases collectively termed the HIV Associated Neurocognitive Disorders (HAND). Worsening pathology is observed in HIV+ individuals who use opioid drugs. Memory problems are often observed in HAND, implicating HIV pathology in the hippocampus, and are also known to be exacerbated by morphine use. HIV-1 Tat was demonstrated to reduce spatial memory performance in multiple tasks, and individual subsets of CA1 interneurons were found to be selectively vulnerable to the effects of Tat, notably nNOS+/NPY- interneurons of the pyramidal layer and stratum radiatum, PV+ neurons of the pyramidal layer, and SST+ neurons of stratum oriens. Each of these interneuron subsets are hypothesized to form part of a microcircuit involved in memory formation. Electrophysiological assessment of hippocampal pyramidal neurons with Tat and morphine together revealed that Tat caused a reduction in firing frequency, however, chronic morphine exposure did not have any effect. When morphine was removed after chronic exposure, non-interacting effects of Tat and morphine withholding on firing frequency were observed, suggesting that a homeostatic rebalancing of CA1 excitation/inhibition balance takes place in response to chronic morphine exposure independently of any Tat effects. Additionally, differential morphological effects of Tat and morphine were observed in each of the three major dendritic compartments, with SR being less affected, suggesting complex circuit responses to these insults reflecting local change and potentially changes in inputs from other brain regions. Behaviorally, Tat and morphine interactions occur in spatial memory, with morphine potentially obviating Tat effects.

The development of the nervous system is a multi-level, time-sensitive process that relies heavily on cell differentiation. However, the molecular mechanisms that control brain development remain poorly understood. We generated a knockout (KO) mouse for the cilia associated gene Spag17. These animals develop hydrocephalus and enlarged ventricles consistent with the role of Spag17 in the motility of ependymal cilia. However, other phenotypes that cannot be explained by this role were also present. Recently, a mutation in Spag17 has been associated with brain malformations and severe intellectual disability in humans. Therefore, we hypothesized that Spag17 plays a crucial role in nervous system development. To investigate this possibility, we first characterized the spatiotemporal expression of Spag17 in the developing brain by using Beta-galactosidase staining and immunohistochemistry. Results showed Spag17 expression in the spinal cord in embryonic E11. By E11.5-12.5 the expression extends to the rhombic lip from the developing hindbrain, as well as to the forebrain and midbrain regions. E14.5-15.5 embryos exhibit an intense expression in the developing ventricles as well as the cerebellum. From E17.5 to birth (P0), the gene is more broadly expressed. We then used a global Spag17 KO mouse model to characterize the function of Spag17 during brain development. Immunohistochemical studies performed in brain sections from E15.5 and P0 time points showed increased expression of the neural progenitor marker Nestin, and reduced expression of mature neuron marker NeuN, increasing positive trend with the young neuron marker Tuj1. Altogether, these findings reveal that Spag17 has a unique spatiotemporal distribution and may be critical for the maturation of neural progenitor cells.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease leading to decreased lung volume and eventual respiratory failure. At present, the median post-diagnosis lifespan is between three and six years. Myofibroblasts are collagen-secreting cells essential for wound healing, but also implicated in the fibroproliferation and extra cellular matrix deposition commonly seen in IPF. The nitric oxide (NO) signaling pathway is implicated in protomyofibroblast to myofibroblast transition and regulation. Previous work has shown that in pulmonary myofibroblasts, endothelial nitric oxide synthase (eNOS) is the primary NOS isoform expressed. The current study used cultured rat pulmonary myofibroblasts between passages two and five as a cell model. The cells were grown in normal growth media (DMEM + 10% FBS) or serum starved (DMEM + 0% FBS) to induce cellular differentiation. In this study, immunocytochemistry was used to show localization of eNOS is dependent on cellular differentiation, with protomyofibroblasts expressing eNOS primarily in the nucleus and protomyofibroblasts expressing eNOS in the perinuclear region. We also show catalytic activity and localization of eNOS are correlated by visualizing nitric oxide production in the cells using a permeable fluorescein chromophore. By using western blot analysis on fractionated cell lysates we found eNOS expressed in the nucleus under normal growth conditions. eNOS is at least partially regulated by intracellular calcium (Ca2+) and calmodulin (CaM). Western blot analysis using native eNOS and phospho-specific eNOS antibodies on fractionated cells treated with the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate (PMA) with and without addition of its antagonist ethylene glycol tetraacetic acid (EGTA) was conducted to investigate PKC’s role in eNOS regulation by phosphorylation. Indeed, PKC activation was found to mitigate expression in the nucleus, while inhibition of the activator restored the activity expression above basal levels. This finding correlates with previous data from our lab showing a decrease in activity in myofibroblasts treated with PMA and assayed amperometrically with an NO electrode.

Glioblastoma multiforme (GBM) is a debilitating malignant brain tumor with expected patient survival of less than a year and limited responsiveness to most treatments, often requiring biopsy for diagnosis and invasive surgery for treatment. We propose a DNA computer system, consisting of input, computation, and output components, for diagnosis and treatment. The input component will detect the presence of three GBM biomarkers: vascular endothelial growth factor (VEGF), caveolin-1α (CAV), and B2 receptors. The computation component will include indicator segments for each of these genes, and ensure that output is only released if all the biomarkers are present. The output component will consist of the therapeutic agent interleukin-12 (IL-12). This study will designate four groups of animals: untreated tumor-free (control), tumor-inoculated (RG2), treated and tumor-free (DNA), and treated and tumor-inoculated (RG2/DNA). In the RG2 and RG2/DNA groups, we will inoculate adult male Fischer rats with RG2 cells into the striatum to induce tumor growth. Rats in the DNA and RG2/DNA groups will be implanted with the DNA system at the same location via recombinant adeno- associated viral vectors. The effectiveness of the DNA system will be evaluated through tumor size measurements, collected from brain slices stained with hematoxylin and eosin, and survival curve. Additionally, IL-12 localization will confirm the release of the output component. We anticipate that the DNA treatment will result in a decrease in tumor size, leading to smaller tumor size in the RG2/DNA group versus the RG2 group. The control group is expected to survive the longest, followed by the DNA group, then the RG2/DNA group, and finally the RG2 group. In the DNA group, IL-12 is expected to stay localized to the implantation site, remaining in its unreleased stem-loop form. On the other hand, it is expected to be released and active in the RG2/DNA group. This study provides a proof of concept to demonstrate the viability and effectiveness of a DNA system using VEGF, CAV, and B2 receptors as biomarkers and IL-12 as a therapeutic output component in the RG2 model. Further research may include varying several of the parameters used in this study, including amount of RG2 administered, choice of biomarkers, quantity and choice of output component, and choice of animal model. This system provides a promising and innovative new approach that is less invasive than surgery yet is still effective in diagnosing, targeting, and treating GBM.

The works presented in this dissertation provide insights into the mechanisms of chemotherapy-induced cognitive impairment (CICI or “ChemoBrain”) and take steps toward outlining a preventive strategy. CICI is now widely recognized as a complication of cancer chemotherapy experienced by a large percentage of cancer survivors. Approximately fifty percent of existing FDA-approved anti-cancer drugs generate reactive oxygen species (ROS). Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, produces the reactive superoxide radical anion (O2-•) in vivo. Dox treatment results in oxidation of plasma proteins, including ApoA-I, leading to TNF-α-mediated oxidative stress in plasma and brain. TNF-α elevation in brain leads to further central nervous system toxicity including mitochondrial dysfunction, neuronal death, and cognitive impairment. Co-administration of the antioxidant drug, 2-mercaptoethane sulfonate sodium (MESNA), prevents Dox-induced protein oxidation and subsequent TNF-α elevation in plasma without interfering with the cancer-killing ability of Dox. In studies presented in this dissertation, we measured oxidative stress in both brain and plasma of Dox-treated mice both with and without MESNA. MESNA ameliorated Dox-induced oxidative protein damage in plasma, confirming our prior studies, and in a new finding led to decreased oxidative stress in brain. Using novel object recognition (NOR), we demonstrated the Dox administration resulted in memory deficits. Using hydrogen magnetic resonance imaging spectroscopy (H1-MRS) techniques, we demonstrated that Dox administration led to a dramatic decrease in choline(phosphocholine)/creatine (Cho/Cr) ratios in mouse hippocampus. The activities of both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D(PLD) were severely diminished following Dox administration. The activity of PC-PLC was preserved when MESNA was co-administered with Dox. In the absence of TNF-α, MRS-indexed Cho/Cr ratio, PLD activity, and mitochondrial oxygen consumption are preserved in brain, and markers of oxidative stress are reduced. Together with results from our previous studies, these results provide strong evidence that TNF-α is strongly associated, if not responsible for CICI. We also tested the notion that O2-• is responsible for Dox-induced plasma protein oxidation and TNF-α release. O2-• resulted in increased oxidative damage to proteins when added to plasma and increased levels of TNF-α in macrophage culture, providing strong evidence that O2-• is responsible for these Dox-induced toxicities.