Browsing by Author "Singh, Meharvan"
Now showing 1 - 20 of 46
- Results Per Page
- Sort Options
Item A guanidine dietary supplement influences pH sensitivity and NSAID activity in acid-sensing ion channels(2015-12-01) Agharkar, Amruta S.; Gonzales, Eric B.; Singh, Meharvan; Luedtke, Robert R.The acid-sensing ion channels (ASICs) are proton sensitive, sodium channels that belong to the epithelial sodium channel/ Degenerin family of ligand-gated ion channels. Activation of the ASIC1a subtype in the central nervous system increases neurodegeneration after ischemic stroke while ASIC3 subtype in the peripheral nervous system is involved in perception of pain. They are emerging targets for ischemic stroke, pain and inflammation. However, we lack selective ligands to target ASICs. In order to gain a better understanding of the channel and to develop selective ligands we must first determine how ASICs are modulated by synthetic as well as endogenous guanidine compounds. This study investigates whether a guanidine dietary supplement, creatine, modulates ASICs. Creatine has been shown to protect from ischemia and benefits patients suffering from muscular dystrophy, osteoarthritis, and fibromyalgia. Furthermore, pain medications such as nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit ASICs. Since supplements and NSAIDs are available over-the-counter, the significant amount of the population would consume them simultaneously.However, the interactions of combination of creatine and NSAIDs on ASICs still remain elusive. Here we sought to determine if creatine would modulate ASIC1a and ASIC3 proton sensitivity and if the combination of creatine and NSAIDs would inhibit ASIC3. Our results indicate that, creatine reduces human ASIC1a (hASIC1a) steady-state desensitization and increases their recovery from desensitization. Creatine also slows down the open-state desensitization of hASIC1a. The efficacy of hASIC1a is increased by creatine at higher concentrations. This indicates that, creatine increases channel's reactivation from desensitization by stabilizing the closed conformation of hASIC1a. Creatine's effect on rat ASIC3 (rASIC3) was calcium dependent. Creatine reduced proton sensitivity of rASIC3 in the nominal calcium environment. As previously reported, NSAIDs inhibited steady-state current of rASIC3 which is involved in pain perception. However, creatine reduced NSAIDs efficacy on rASIC3. To summarize, the creatine's effect depends on the desensitized state of hASIC1a and creatine increases the availability of channels for opening. While in rASIC3, creatine reduces proton sensitivity in nominal Ca2+ and antagonizes NSAIDs inhibitory effect. Thus, the use of creatine should be monitored in diseased states and when it is consumed along with NSAIDs.Item AMP-Activated Protein Kinase (AMPK) signaling regulates the age-related decline of hippocampal neurogenesis(2018-05) Wang, Brian S.; Jin, Kunlin; Hodge, Lisa M.; Singh, Meharvan; Sumien, Nathalie; Yang, ShaohuaAging is the progressive decline of physiological function and increased vulnerability to disease and death. By the year 2050, 2 billion people will be over the age of 60. Accompanying this, the incidence of age-associated neurological diseases is expected to rise. Thus, there is an urgent need to find therapies to promote healthy brain aging. The finding that neurogenesis continues into adulthood allows us to target endogenous neurogenesis as a potential therapeutic. However, the number of stem cells can decrease by about 80% in the aged brain and is a main cause for the decrease in brain function. The reasons for the age-related decline in neurogenesis can be due to intrinsic factors such as cell metabolism, which have been studied but its role in neurogenesis remains largely unexplored. Interestingly, neural stem cells (NSCs) possess metabolically different characteristics from their differentiated progeny, suggesting the need for a shift in cellular metabolism to accommodate the requirements for neurogenesis. In the process of the metabolic shift, the AMP-activated protein kinase (AMPK) plays a pivotal role for controlling stem cell proliferation and differentiation as a cell's master metabolic regulator. Additionally, AMPK has been reported to control the functions of signaling pathways that regulate the aging process, which suggests its potential involvement in the age-related decline of neurogenesis. Therefore, we hypothesize that inhibition of AMPK signaling activation (phosphorylation) in the old brain will cause a concomitant increase in hippocampal neurogenesis. Our specific aim is to establish whether AMPK signaling plays a critical role in the age-related decline of hippocampal neurogenesis. Our objectives for this aim are to (i) determine the expression pattern of AMPK in the subgranular and subventricular zones of young-adult and old mice using immunohistochemistry and Western blotting; and (ii) examine the impact of loss or gain of AMPK activation on hippocampal neurogenesis in young-adult and old mice using pharmacological agents Compound C (AMPK inhibitor) and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, AMPK activator). Our results show that (i) AMPK subunit isoforms are differentially expressed in the neurogenic regions – most are localized to the cytoplasm in the subgranular zone (SGZ) with the exception of α2 and β1, while most isoforms are found in the nucleus in the subventricular zone (SVZ) except α1; (ii) AMPK signaling activation was significantly increased in the SGZ and SVZ; and (iii) short-term but not long-term pharmacological inhibition of AMPK signaling could partially rescue hippocampal neurogenesis in the old brain. Taken together, these results indicate that AMPK is a critical mediator in the regulation of downstream processes for the age-related decline in hippocampal neurogenesis.Item Astrocytes & Ischemic stroke(2014-08-01) Roy Choudhury, Gourav; Yang, Shaohua; Singh, Meharvan; Schreihofer, DerekAlthough less appreciated, recent findings introduced critical contributions of astrocytes to numerous CNS functions, like neurogenesis, synaptogenesis, ion homeostasis, neurotransmission, and blood brain barrier formation. Their active participation in the progression of specific CNS pathologies has garnered major attention and culminated in thorough investigation of astrocyte function in brain. Reactive astrogliosis, characterized by increases in glial fibrillary acidic protein (GFAP) and cellular hypertrophy, describes the extensive structural and functional changes that astrocytes undergo in response to tissue injury. Despite of extensive investigation, the molecular mechanism of reactive astrogliosis in ischemic stroke still remains elusive. p38 MAPK is a well studied signal transducing pathway known to be involved in modulating cell type specific responses to ischemic injury. The first study presented in the dissertation delineates the involvement p38 MAPK signaling pathway in reactive astrogliosis after ischemic stroke. Results showed that astrocyte specific deletion of p38 MAPK attenuated oxygen-glucose deprivation (OGD)-induced increase in GFAP expression in primary astrocytes in vitro. Additionally, inhibition of p38 MAPK (SB239063/genetic deletion) slowed astrocyte migration without affecting astrocyte proliferation. In vivo deletion of p38 MAPK from astrocytes attenuated reactive astrogliosis after permanent middle cerebral artery occlusion in mice. These findings strongly indicated that p38 MAPK plays a critical role in reactive astrogliosis after ischemic stroke. During ischemic stroke, astrocyte dysfunction causes extensive cell death through excitotoxicity, disruption of ion and water homeostasis. Restoration of astrocyte function thus may be beneficial to ischemic tissue in the long term. Methylene blue (MB), a metabolic enhancer, has been well studied and known to improve cellular respiration, glucose metabolism and attenuate superoxide production by efficient electron transport in mitochondria. In the second part of this dissertation we determined the effect of MB in astrocytes under oxygen glucose deprivation (OGD) and reoxygenation stress and the underlying protective mechanisms. Our studies demonstrated that MB improved astrocyte bioenergetics and promoted astrocyte survival following OGD and reoxygenation. In conclusion both the studies presented, provide a unique perspective of the importance of astroglial response in ischemic injury and how its modulation can benefit the healing and recovery of the brain following ischemic injury.Item Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, NeerajKaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.Item Characterization of the interactions of guanidine compounds with the human GABA-A ρ1 receptor(2015-12-01) Snell, Heather D.; Gonzales, Eric B.; Dillon, Glenn H.; Singh, MeharvanThis dissertation investigates the activity of guanidine compounds GMQ, and amiloride and its derivatives on the human GABA-A ρ1 receptor, compounds classified as antagonists for the heteromeric GABA-A αβγ receptor. The GABA-A ρ receptor possesses many differences in kinetics, expression, and pharmacology from the heteromeric GABA-A αβγ receptors. Many GABA-A αβγ receptors ligands interact differently, or fail to interact with, the GABA-A ρ receptor. Thus the activity of these guanidine compounds on the GABA-A ρ1 receptor remains unknown. Based on the differential pharmacology displayed by the GABA-A ρ receptors, we propose that GMQ and amiloride would interact with the GABA-A ρ1 receptor as agonists, different from their activity on the heteromeric GABA-A αβγ receptors. Importantly, our data demonstrates GMQ and amiloride interacts with the GABA-A ρ receptors as negative and positive allosteric modulators, respectively. The 15’ residue of the second transmembrane domain of the GABA-A ρ1 receptor is important in the positive allosteric modulatory mechanism, and the accessibility of the guanidine group on the guanidine compound is integral in the positive allosteric modulation mechanisms of amiloride and its derivative 5- (N,N-Hexamethylene) amiloride (HMA). The investigation of novel compounds that interact with the GABA-A ρ receptor differently from GABA-A αβγ receptor would contribute to a better understanding of the GABA-A ρ receptor structure and the production of novel therapeutics specific for the GABA-A ρ receptor. Particularly, the GABA-A ρ receptor is implicated in retinal hypoxic disorders such as diabetic retinopathy. These guanidine compounds could be utilized as a back-bone for the production of compounds that could alleviate the pathologies caused by advanced stages of diabetic retinopathy.Item Chronic testosterone deprivation sensitizes the middle-aged rat brain to damaging effects of testosterone(2020-05) Smith, Charity; Schreihofer, Derek A.; Cunningham, Rebecca L.; Singh, Meharvan; Yang, Shaohua; Jones, Harlan P.Levels of the testosterone (T) fall in aging men. Recently, the number of men obtaining testosterone replacement therapy (TRT) has increased dramatically. However, other consequences of aging, such as increased oxidative stress, may result in detrimental effects when combined with TRT, including an increased stroke risk. Whether such a delay would alter the effects of TRT on stroke is not known. We hypothesized that a delay TRT following castration in middle-aged male rats would result in increased oxidative stress and a reduction in the neuroprotective effects of testosterone following stroke. We evaluated the effects of testosterone treatment after short (2 week) and long-term testosterone deprivation (10 weeks) in middle-aged male rats on cerebral ischemia, oxidative stress and cognitive function. Our data suggest testosterone treatment after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia, however in the absence of injury improves cognition. Both effects are regulated by oxidative stress.Item Clinical Significance of Annexin A2 in Predicting Poor Prognosis in African American Women with Triple-Negative Breast Cancer(2017-05) Gibbs, Lee D.; Vishwanatha, Jamboor K.; Basha, Riyaz; Lovely, Rehana S.; Mathew, Porunelloor A.; Singh, MeharvanTriple-negative breast cancers (TNBC) are identified by the absence of these three major receptors that drive most breast cancer subtypes. TNBC is the most aggressive breast cancer subtype and studies have shown that the incidence of TNBC is much higher in premenopausal African American (AA) women and woman of African descent in comparison to woman of European descent. TNBC in AA women has been associated with worst overall survival after controlling for socioeconomic factors, treatment latency, and tumor receptor expression. This suggests that the clinical outcome of TNBC in AA women may result more from biological differences than access to adequate healthcare. Utilizing a large archived breast cancer cohort of genome sequencing information and the evaluation of these targets in breast tissue and serum can lead to recognition of reliable biological markers that have tremendous potential to enhance detection, treatment, and prognosis. Our previous studies have shown that Annexin A2 (AnxA2), a 36 kda calcium-dependent phospholipid binding protein, is abundantly expressed in TNBC. We have shown AnxA2 to play multiple roles in TNBC by regulating cellular functions; including plasminogen activation, angiogenesis, proliferation, migration, invasion, and metastasis. AnxA2 is one of the most identified proteins expressed in exosomes (small vesicles that are secreted from tumors as metastatic regulators). We have previously demonstrated exosomal AnxA2 contribution to metastasis of TNBC cells in vivo. The proposed study will determine the correlation of AnxA2 with poor prognoses in AA TNBC patients, and establish the clinical significance of exosomal AnxA2 in contributing to the poor clinical outcomes seen in AA TNBC patients. Three specific aims were addressed in this work. Aim 1- Determine the association of secreted exosomal AnxA2 with TNBC amongst AA patients. Aim 2 - Evaluate AnxA2 expression in TNBC tissue samples amongst a breast cancer patient cohort of various breast subtypes. Aim 3 - Determine the correlation of AnxA2 gene expression with poor pathological, prognostic variables and race/ethnicity in TNBC patients through in silico analysis.Item Connexin 43 as a Mediator of Androgen and Estrogen-induced Protection against Oxidative Stress in Astrocytes(2018-03-14) Rybalchenko, Nataliya; Singh, Meharvan; Kubelka, NicholasPurpose: Connexin 43 (Cx43) is a transmembrane protein highly expressed in astrocytes, whose expression and/or channel permeability can impact cell viability in the presence of oxidative stress. Androgens and estrogens are also known to protect against oxidative stress. However, it remains unknown whether these hormones alter Cx43 channel expression or permeability as a mediator of their protective effects. Here, we propose that the androgen, dihydrotestosterone (DHT) and the estrogens, E2 and 3betadiol (the latter being a metabolite of DHT), protect against oxidative stress in astrocytes through the regulation of Cx43 expression and/or permeability. Hypothesis: Reducing the permeability of Cx43 gap junctions or Cx43 hemichannels enhances the protective efficacy of E2, DHT, or 3betadiol against oxidative stress in cerebral cortical astrocytes. Methods: Using cortical astrocytes from neonatal female C57/Bl6 mice and the astrocytic C6 glioma cell line, we assessed changes in Cx43 mRNA expression, Cx43 permeability, and cell viability following treatment with 100 nM E2, DHT, or the DHT metabolite, 5-alpha-androstane-3-beta,17-beta-diol (3betadiol), and compared it to the effect of the vehicle (DMSO) control, in the presence or absence of the metabolic and oxidative stressor, iodoacetic acid. Changes in Cx43 mRNA expression relative to GAPDH were assessed by RTPCR; The MTT assay was used to assess cell viability under conditions of metabolic/oxidative stress, with or without the pharmacological manipulation of Cx43 channels; changes in Cx43 hemichannel permeability were assessed using the Ethidium Bromide dye uptake assay. Results & Conclusions: While only E2 transiently increased astrocyte Cx43 mRNA expression, E2, DHT, 3betadiol each blocked IAA induced increase in Cx43 hemichannel permeability to an extent similar to the hemichannel antagonist Boldine. Given that Boldine-induced hemichannel blockade significantly protected cells from oxidative stress, hemichannel blockade may also be a component of these steroids’ protective mechanisms. These data indicate that enhanced Cx43 hemichannel permeability not only inhibits the protective effects of E2 or DHT, but that the protective effects of E2 and DHT may be mediated, at least in part, by the regulation of Cx43 hemichannel permeability.Item Connexin 43 Contributes to Estrogen Protection against Oxidative Stress in Cortical Astrocytes(2019-05) Kubelka, Nicholas K.; Singh, Meharvan; Uht, Rosalie M.; Schreihofer, Derek A.; Yang, Shaohua; Planz, John V.Age-related brain disorders are associated with the decline in the ability of brain cells to cope with homeostatic challenge. Although all major brain cell types have the capacity to respond to homeostatic challenges, astrocytes are particularly well-equipped to counteract these challenges. Here, we focused on Connexin 43 (Cx43) as a protein that is not only highly expressed in astrocytes, but whose expression is critical to inter-cellular communication that in turn, can influence cell viability. Most studies to date have focused on the expression (i.e., abundance) of Cx43. However, a critical limitation of these studies is that they did not thoroughly examine functionality of the Cx43 channels. In particular, there is a paucity of data describing the differential contributions of Cx43-containing hemichannels versus Cx43-containing gap junctions to cellular functions. We hypothesized the astrocyte Cx43 hemichannel as a yet unreported target of androgens and estrogens based on three notions. First, our laboratory has determined that astrocytes are a relevant and important target of such gonadal steroid hormones as estrogens (e.g., 17[beta]-estradiol (abbreviated herein as estradiol or E2)) and androgens (such as DHT), through which these hormones promote healthy brain cell function. Second, oxidative stress is associated with an increase in Cx43 opening. Finally, the Cx43 gene promoter contains functional estrogen response element (ERE) half sites, and estradiol, as well as other estrogenic compounds, decrease Cx43 channel opening in peripheral (non-CNS) tissue. Based on these notions, we hypothesized that gonadal androgens and estrogens will inhibit Cx43 hemichannel opening in cortical astrocytes as well. My data revealed that while E2, dihydrotestosterone (DHT), and the estrogenic metabolite of DHT (3[beta]diol) all protect primary cortical astrocytes from the mixed metabolic/oxidative insult, iodoacetic acid (IAA), only DHT decreased astrocyte Cx43 mRNA expression. Consistent with their cytoprotective effects, however, all three steroids decrease astrocyte Cx43 hemichannel opening, and antagonized the increased opening of Cx43 hemichannels induced by IAA. In an effort to pursue the mechanism by which these steroids reduced Cx43 hemichannel opening, we evaluated the phosphorylation of Cx43 at two key residues, Ser 368 and Tyr 265. Phosphorylation at these residues is associated with channel closing, and as such, we predicted that the three hormones would increase the phosphorylation of Cx43 at one or both of these residues. Whereas Tyr265 phosphorylation was unaffected any of the three hormones, DHT significantly reduced the phosphorylation of Cx43 at Ser368. These observations may indicate that while all three steroids contribute to astrocyte protection through a mechanism that involves blocking astrocyte Cx43 hemichannel opening, DHT may induce molecular changes in the astrocytes that are distinct from those induced by estradiol or 3[beta]diol. The knowledge gained through the experiments conducted not only enhance our understanding of how Cx43 hemichannels and Cx43 gap junctions influence astrocyte function and viability but also define Cx43 hemichannels as relevant targets of gonadal steroid hormone induced regulation of cell viability. Such knowledge may facilitate the development of more precise therapeutics (i.e., selectively targeting Cx43 hemichannels without activity at Cx43 gap junctions in the same cells or tissue), the benefit of which would be to better treat age-associated neurodegenerative disorders as well as disorders of peripheral tissueItem Detection of Androgen Receptors by Flow Cytometry(2008-05-01) Dutta, Mayurika; McClain, Robert; Singh, Meharvan; Hall, StanDutta, Mayurika, ‘Detection of androgen receptors by Flow Cytometry’. Internship Practicum report, Biotechnology, May 2008, 80 pp., 1 table, 18 figures. The use of androgen therapy is expanding given the documented potential benefits like increasing bone mineral density, muscle mass and strength. Androgen therapy also has potential risks including increasing the likelihood of prostate cancer and cardiovascular disease. So, we need a way to differentiate those who are likely to be benefitted by the therapy and those that are not. Data from Dr. Meharvan Singh’s lab has shown that activation of intracellular androgen receptors triggers cell survival pathways, while activation of the membrane androgen receptor suppresses cytoprotective pathways, and thus promotes cell death. We propose to develop a diagnostic kit that measures the relative ratio of intracellular androgen receptors and membrane androgen receptors, which is predicted to gauge relative risks or benefits associated with androgen therapy.Item Development of a mouse model to study the long-term effects of chemotherapy on brain function(2023) Trinh, Oanh; Vann, Philip; Davis, Delaney; Luedtke, Robert R.; Basha, Riyaz; Singh, Meharvan; Sumien, NathaliePurpose: While remaining an effective life-saving intervention for cancer patients, chemotherapy has been associated with many neurotoxic side effects, including chemotherapy-related cognitive impairments (CRCI). Chemotherapy exposure leads to a decline in learning, memory, processing speed, attention, and executive functions, which may persist for more than 20 years post-treatment, impairing the quality of daily lives of survivors. Childhood cancer survivors are particularly vulnerable to chemotherapy and have been impacted in their educational achievements, employment, social relationships, and even life expectancy. Most common childhood cancers are often treated with the folate-inhibitor methotrexate (MTX). Our study aimed at establishing a tumor-free mouse model of MTX-induced brain impairments. We hypothesized that early exposure to MTX would induce impairment in cognition, as well as motor and affective functions. Methods: Male and female C57BL6/J postnatal day 15 pups received intraperitoneal injections of saline or MTX (2 mg/kg) once a day for 3 days. Pups were weaned on PND21, and subsets were behaviorally characterized at 1 or 7 months after MTX exposure (n=6-8 for 1.5 months old, and n=11-13 for 8 months old) for motor, affective and cognitive functions using a comprehensive behavioral test battery. Results: At 1.5 months, coordination and motor learning was significantly impaired in males and improved in females. All other measures did not reveal any other significant effects, however trends of impaired motor and cognitive functions could be discerned. At 8 months, there were no effects of MTX on motor, affective and some cognitive functions. However, MTX exposure led to an impairment on spatial learning and memory and increased swimming speed. Conclusions: Studies at 1.5 months will need to be repeated to increase power and ascertain conclusions on brain functions. Early exposure to MTX treatment led to long-term impairments in both male and female mice and could be used as a model to test interventions to limit CRCI.Item Effect of Progesterone on Calcium Signaling of Hippocampal Neurons(2006-05-01) Hwang, Ji-Yeon; Koulen; Singh, Meharvan; Yang, ShaohuaJi-yeon Hwang, Effects of Progesterone on Calcium Signaling of Hippocampal Neurons. Master of Science (Pharmacology and Neuroscience), May 2006, 74 pp., 18 Figures. Progesterone (P4) is one of the steroid hormones responsible for female sexual behavior. It has been recently show that P4 plays also multiple roles in the central nervous system (CNS) including neuroprotection. Calcium (Ca2+) is involved in numerous cellular processes in nerve cells such as neurotransmitter release and cell death. In the present studies, we present evidence that P4 increases the activity of IP3R-mediated Ca2+ release within nerve cells leading to cell survival and neuroprotection. The purpose of the present study is to identify the subcellular distribution of all IP3Rs and other signaling proteins including Akt and phosphor-Akt, in the primary hippocampal neuron and to test the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca2+ signaling in neurons. We observed that primary hippocampal neurons express predominantly IP3R type 1, 2, and 3. The cellular distribution of all IP3R isoforms as well as Akt and phospho-Akt was increased in primary hippocampal neurons by P4 treatment. In addition, phospho-Akt was translocated to nucleus in response to P4. P4-pretreated neurons showed potentiated IP3R-mediated intracellular Ca2+ responses. Acute application of P4 resulted in transient elevations of intracellular Ca2+ concentrations. Our results will contribute to establishing potential pharmacological approaches for the treatment of pathological conditions characterized by a dysregulation of cellular Ca2+ concentrations such as Alzheimer’s disease.Item ERK/MAPK Pathway Regulation of GABA-A Receptor Function.(2009-12-01) Dohi, Akiko; Singh, MeharvanGABA-A receptor is a ligand-gated ion channel that conducts negatively charged chloride ions. Influx of this ion leads to hyperpolarization of neurons; thus, suppression of the neuronal excitability. Alterations in GABAergic neurotransmission may contribute to depression and anxiety. While neurosteroids can regulate the responsiveness of the GABAA receptor in allosteric manner, certain intracellular signaling pathways can also regulate the function of the GABA-A receptors through phosphorylation of its subunits. One pathway that is regulated by both neurotrophic factors and steroid hormones is the ERK/MAPK pathway. This pathway is involved in cell proliferation, maturation, and even, cell death. The role of this pathway in the regulation of the GABAA receptor, however, is not well studied and is the subject of my dissertation. Initial studies conducted by the Singh and Dillon laboratories showed that pharmacological inhibition of the ERK/MAPK pathway potentiated the α1β2γ2 configuration of the GABAA receptor, expressed in HEK-t cells. This suggested that the ERK/MAPK pathway was involved in the negative regulation of the GABAA receptor function. In silico analysis revealed that the Thr 375 residue within the α1 subunit was a plausible target of the ERK/MAPK pathway. As a result, I hypothesized that the activation of the ERK/MAPK pathway inhibited GABAA receptor function through the direct phosphorylation of the Thr 375 residue, resulting in receptor internalization. Supporting this hypothesis was data showing that mutation of the Thr 375 residue to Alanine prevented the enhancement of GABA-gated currents elicited by inhibiting the ERK/MAPK pathway. However, using the HEK-t cell line transfected with the α1β2γ2 configuration of the GABAA receptor, I determined that the activation of the ERK/MAPK pathway by HGF did not influence the peak amplitude of the GABA-gated currents. Further, the potentiation of the GABA-gated currents was apparently not due to internalization of the receptor. Collectively, while we believe that the Thr 375 within the α1 subunit is relevant to the effect of ERK/MAPK pathway inhibition, it was not a direct target of the ERK/MAPK pathway.Item Exploring Trabecular Meshwork Molecular Pathogenic Mechanisms In Primary Open Angle Glaucoma And Glucocorticoid Induced Glaucoma(2016-08-01) Bermudez, Jaclyn Y.; Clark, Abbot F.; Mao, Weiming; Singh, MeharvanIn a normal functioning eye, the aqueous humor, a fluid secreted by the ciliary body, drains through the trabecular meshwork (TM), a multilayered tissue in the anterior segment of the eye. The TM is the initial site of damage in glaucoma. Damaged TM results in higher aqueous humor outflow resistance and causes elevated IOP, the latter of which leads to optic nerve damage. Numerous clinical studies have shown that lowering IOP can prevent neuronal damage and slow/stop the progression of the disease. In the glaucomatous TM (GTM), there is excessive extracellular matrix protein deposition, cytoskeletal changes and altered cell function. The transforming growth factor β (TGFβ) pathway is activated by TGFβ2 which has been found to be more abundant in the GTM. Additionally, formation of cross-linked actin networks (CLANs) in the GTM is increased compared to non-glaucoma TM. Primary open angle glaucoma (POAG), glucocorticoid-induced glaucoma (GIG) and glucocorticoid-induced ocular hypertension (GCOHT), share similar pathophysiologies. GC-OHT differs from POAG in that about 40% of the population develops GC-OHT after topical treatment with glucocorticoids however, the mechanism that differentiates steroid responders from non-responders is unknown. In our studies we have explored trabecular meshwork molecular pathogenic mechanisms that are responsible for the disease pathology. We have studied epigenetics as a regulatory mechanism for increasing TGFβ2 expression. We have also used proteomics to determine proteins that are associated with CLANs. Lastly, we studied genes that are differentially expressed in glucocorticoid responders versus non-responders in our bovine model of GC-OHT. Overall, our research has enhanced our understanding of the TM and the molecular mechanisms that play a role in glaucoma. We hope to use this information to find new disease modifying therapies.Item Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents(2007-05-01) Martin, Shelley E.; Basu, Alakananda; Forster, Michael; Singh, MeharvanMartin, Shelley E., Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents. Master of Science (Pharmacology and Neuroscience), May, 2007, 53 pp., 7 figures, 2 tables, bibliography, 95 titles. Β-amyloid1-42 (Aβ42) has been implicated in the pathogenesis of Alzheimer’s disease (AD); however, the amount of this peptide in the brain does not correlate well with the presence or severity of AD. This project tested the hypothesis that individual differences exist in susceptibility to Aβ42 neurotoxicity arising from the differences in the expression of α7 nicotinic acetylcholine receptors α7 nACHRs). This hypothesis was tested in primary neuronal cultures derived from inbred mouse strains which differ in expression of α7 nAChRs. Also, the ability of nicotinic agents to modulate Aβ42 toxicity was examined. Significant strain differences in susceptibility to Aβ42 toxicity were found; however, these were not related to levels of α7 nAChRs. Additionally, strain differences were found in the ability of α7-selective partial agonist, an α7-selective antagonist and a α4β2 nAChR-selective antagonist to protect against this toxicity. Inbred strains of mice may be useful in uncovering the pathophysiology of AD.Item Geographic Analysis of Trauma Readmissions in North Texas(2016-12-01) Sanchez, Derick J.; Gwirtz, Patricia A.; Berg, Rance E.; Singh, MeharvanDue to the high cost and increased risk of mortality associated with unplanned patient readmissions, research has been aimed to identify risk-factors in patients with high hospital utilization and recidivism. The primary aim of this study was to characterize readmissions across multiple institutions in patients initially admitted to a single urban Level I trauma center. Analysis was carried out to test the hypothesis that a patient’s geographic location of residence can be used to predict readmission rates. Data was queried from a regional database that is comprised of more than 150 hospitals in the North Texas region. Patient ZIP code and county of residence were analyzed using binary logistic regression to determine significance of predictability of readmission by patient geography. Additional variables such as demographics, diagnosis, Elixhauser comorbidities, and insurance were also analyzed to create a full clinical and geographic regression model describing patterns in readmissions.Item Glucocorticoid Receptor Mediated CRH Gene Repression(2015-12-01) Bhave, Shreyas A.; Uht, Rosalie M.; Singh, Meharvan; Gonzales, Eric B.The hypothalamic pituitary adrenal (HPA) axis is an important neuroendocrine system which mediates the mammalian stress response. Dysregulation of the HPA axis, often due to failed glucocorticoid receptor (GR) mediated negative feedback, is tightly associated with many psychiatric disorders including depression. Glucocorticoids attenuate the activated HPA axis partly by suppressing Corticotropin releasing hormone (CRH) gene (crh) expression. Though regulation of crh is a critical component of the HPA axis, the molecular mechanisms are poorly understood. In this study, we sought to investigate the molecular mechanism by which GR regulates crh expression. The results indicate that Histone deacetylase (HDAC) 1 and methyl CpG binding protein 2 (MeCP2) interact with GR forming a putative complex and that this interaction is GR-ligand, Dexamethasone (Dex), dependent. Furthermore, results indicate that DNA methyltransferase (DnMT) 3b also interacts with GR and this interaction is Dex dependent. Next we tested the role of MeCP2 and DNA methylation in GR mediated crh repression. The results suggest that MeCP2 is necessary for maintenance of basal crh levels. Decreased levels of MeCP2 are associated with the increased crh expression and Dex fails to repress crh in the absence of MeCP2. Then we examined the role of DNA methylation in crh regulation. The data suggest that Dex increases promoter methylation at specific sites, and that inhibition of methylation at these sites by 5-Aza-2-deoxycytidine (5-AzaDC) is associated with increased expression of crh. The results also suggest that inhibition of DNA methylation abrogates Dex mediated repression of crh. In fact, Dex activates crh in the face of reduced promoter methylation. While 5-AzaDC and Dex do not alter the protein levels of GR and MeCP2, inhibition of DNA methylation decreases the ability of GR and MeCP2 to occupy the crh promoter. Taken together, the data indicate that Dex mediated repression of crh is mediated through the formation of a putative complex and requires MeCP2 and site specific promoter methylation. The findings from this study add novel aspects to the molecular mechanism of GR mediated crh repression. This will lead to better treatment and management of depression which affects nearly 10% of the US population.Item HIV-1 Impairment via UBE3A and HIV-1 Nef Interactions Utilizing the Ubiquitin Proteasome System(MDPI, 2019-11-27) Pyeon, Dohun; Rojas, Vivian K.; Price, Lenore; Kim, Seongcheol; Singh, Meharvan; Park, In-WooMolecular basis of HIV-1 life cycle regulation has thus far focused on viral gene stage-specificity, despite the quintessence of post-function protein elimination processes in the virus life cycle and consequent pathogenesis. Our studies demonstrated that a key pathogenic HIV-1 viral protein, Nef, interacted with ubiquitin (Ub)-protein ligase E3A (UBE3A/E6AP), suggesting that interaction between Nef and UBE3A is integral to regulation of viral and cellular protein decay and thereby the competing HIV-1 and host cell survivals. In fact, Nef and UBE3A degraded reciprocally, and UBE3A-mediated degradation of Nef was significantly more potent than Nef-triggered degradation of UBE3A. Further, UBE3A degraded not only Nef but also HIV-1 structural proteins, Gag, thus significantly inhibiting HIV-1 replication in Jurkat T cells only in the presence of Nef, indicating that interaction between Nef and UBE3Awas pivotal for UBE3A-mediated degradation of the viral proteins. Mechanistic study showed that Nef and UBE3A were specific and antagonistic to each other in regulating proteasome activity and ubiquitination of cellular proteins in general, wherein specific domains of Nef overlapping with the long terminal repeat (LTR) were essential for the observed actions. Further, Nef itself reduced the level of intracellular Gag by degrading a cardinal transcription regulator, Tat, demonstrating a broad role for Nef in the regulation of the HIV-1 life cycle. Taken together, these data demonstrated that the Nef and UBE3A complex plays a crucial role in coordinating viral protein degradation and hence HIV-1 replication, providing insights as to the nature of pathobiologic and defense strategies of HIV-1 and HIV-infected host cells.Item Impact Of Culture Conditions on Primary Astrocyte Phenotype(2019-05) Prah, Jude K.; Yang, Shaohua; Singh, Meharvan; Forster, Michael J.; Yan, Liang-Jun; Fudala, RafalAlthough previously thought to be passive support cells in the central nervous system (CNS), recent findings introduced critical contributions of astrocytes to numerous CNS functions like energy metabolism, ion and water homeostasis, blood brain barrier formation and neurotransmission. Their dysfunction has been implicated in the initiation and progression of specific CNS pathologies with astrocyte now given serious attention as cellular target for neuroprotection and treatment of neurological disorders. In spite of the aforementioned advances, our understanding of the mechanisms and pathways regulating astrocytic function, dysfunction and astrogliosis is still rudimentary. This is as a result of the complex interwoven nature of different cells in the CNS. Because of the complexities of the brain structure and function in vivo, methods of in vitro primary culture that overcome the influence of complex brain environment provide critical tools for understanding brain cell function at the cellular and molecular levels. The current primary astrocytes cultures are mostly maintained in serum-containing hyperglycemic medium which is non-physiological and produces astrocyte with a reactive, morphological and functional phenotype different from in vivo quiescent astrocytes. The first study presented in the dissertation delineates a serum free astrocyte culture condition that maintains primary astrocytes in a quiescent state. Results showed that primary astrocytes isolated from the cerebral cortex of postnatal day 1 C57BL6 mice and cultured in an astrocyte base medium supplemented with fibroblast growth factor (FGF2) and epidermal growth factor (EGF) (ABM- FGF2-EGF) have higher process bearing morphologies similar to in vivo astrocytes and different from the flat polygonal fibroblast like morphologies exhibited by astrocytes cultured under the traditional FBS condition developed by McCarthy and de Vellis (1980) (MD-10% FBS). Additionally astrocytes cultured in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP and vimentin, increased glutamine synthase and glutamate transporter mRNA levels compared to astrocytes in the MD-10% FBS condition. These findings strongly indicates that astrocytes cultured in ABM-FGF2-EGF medium compared to the usual FBS medium promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. This media provides a novel method for studying astrocytes function in vitro under physiological and pathological condition. Hyperglycemia could increase neuronal glucose level which leads to neuronal damage in a phenomenal referred to as glucose neurotoxicity. On the other hand the impact of hyperglycemia on astrocytes has been less explored although astrocytes are critical for glucose uptake and metabolism and many primary astrocyte cultures are maintained in high glucose conditions. In the second part of this dissertation we investigated the impact of hyperglycemia on astrocyte phenotype and function. Our studies demonstrated that hyperglycemic levels (25 mM) induce cell cycle arrest, ROS production, cytokine expression and inhibited astrocyte proliferation. High glucose enhanced glycolysis and increased metabolic potentials of astrocyte. In addition high glucose activated AMP-activated kinase (AMPK) signaling pathways and induces reactive astrocyte phenotype. In conclusion both studies presented a unique perspective of how culture conditions influence astrocyte phenotype and experimental outcome. Our study also provided a mechanism which may underline the role of astrocytes in hyperglycemia induced neurological complications.Item INHIBITION OF LET-7I AS A MEANS TO ENHANCE THE NEUROPROTECTIVE EFFICACY OF PROGESTERONE IN THE ISCHEMIC BRAIN(2018-05) Nguyen, Trinh V.; Singh, Meharvan; Basu, Alakananda; Ghorpade, Anuja; Cunningham, Rebecca L.; Yang, ShaohuaThe occurrence of ischemic stroke is relatively rare among pre-menopausal women. Strikingly, this risk doubles every 10 years after the menopausal transition; and women are likely to experience worse outcomes and higher mortality post stroke than men. Since both estrogen (E2) and progesterone (P4) levels decline precipitously following the menopause, this hormonal reduction may, at least partially, contribute to the higher risk and worse outcomes. By inference, these hormones could play a critical role in protecting women against ischemic stroke. In this dissertation project, we focus on P4, the relatively understudied of the two hormones. And while P4 has been shown to be a potent neuroprotectant in various experimental models of stroke, the underlying mechanisms remain unclear. One known mediator of P4's protective function is brain-derived-neurotrophic-factor (BDNF), which has an established role in promoting neuronal differentiation, survival, and synaptogenesis. In addition, emerging literature and data from our laboratory strongly support the indispensable role of glia in P4's neuroprotective program and thus, may also play a significant role in post-stroke recovery. We recently reported that P4 induces a significant release of BDNF from primary astrocytes, through a putative membrane-associated progesterone receptor consisting of progesterone-receptor-membrane-component-1 (Pgrmc1). This receptor is abundantly expressed in various regions of brain and mediates such effects of P4 in the central nervous system (CNS) as anti-apoptotic effects, spinogenesis, and BDNF release. What is not known, however, is how the expression of this receptor is regulated. This dissertation was aimed to elucidate what regulates the expression of Pgrmc1 and BDNF in glia and how such regulation influences the neuroprotective function of P4 in the ischemic brain. Based on the observation that Let-7i regulates the expression of Pgrmc1 in a peripheral cell type, and our in silico analysis that revealed that both Pgrmc1 and BDNF are potential targets of let-7i, we hypothesized that let-7i represses P4's neuroprotective effects by down-regulating the expression of both Pgrmc1 and BDNF in glia, leading to: 1) suppression of P4-induced BDNF release from glia, and 2) attenuation of the beneficial effects of P4 on neuronal survival and markers of synaptogenesis in the ischemic brain. Using primary cortical astrocytes as an experimental model, we found that let-7i negatively regulated the expression of Pgrmc1 and BDNF. This was correlated with a reduction in P4-induced BDNF release from these cells. Under such conditions of reduced expression of both Pgrmc1 and BDNF, P4 was unable to protect primary neurons against oxygen-glucose-deprivation (OGD) or regulate markers of synaptogenesis. In our in vivo model of transient ischemic stroke, we found that protective effects of P4 were greatly enhanced in animals that were concomitantly treated with an inhibitor (antagomir) of let-7i. The combined treatment also enhanced synaptogenesis in the peri-infarct region. Collectively, the data presented here suggested that in the ischemic brain, let-7i negatively influences P4-induced neuroprotection via regulation of the Pgrmc1/BDNF axis. As such, inhibition of let-7i maybe an effective means to enhance the efficacy of P4 in treating ischemic stroke.
- «
- 1 (current)
- 2
- 3
- »