Browsing by Subject "Neurosciences"
Now showing 1 - 20 of 21
- Results Per Page
- Sort Options
Item Cellular Mechanisms in the Ocular Actions of Endothelin(1996-12-01) White, Karen A.; Thomas YorioWhite, Karen A., Cellular Mechanisms in the Ocular Actions of Endothelin. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 1996, 151 pp., 25 tables, 23 figures, references, 111 titles. Endothelins are a family of regulatory peptides which could have important implications in the regulation of aqueous humor outflow and intraocular pressure (IOP). The objectives of this dissertation were to investigate the cellular mechanism of endothelin (ET) receptor interactions in ocular tissues focusing on their effect on second messengers such as phospholipase C (PLC) and calcium, and their interactions with phospholipase A2 (PLA2) in ciliary muscle cells. The hypothesis was that in human ciliary muscle (HCM) cells, endothelin-1 (ET-1), via the ETA receptor and a pertussis toxin sensitive G-protein, activates PLC, which in turn stimulates calcium mobilization. Independent of this pathway, ET-1 also activates PLA2 and increases the release of prostaglandins. These two pathways provide a cellular second messenger balance that influences ciliary smooth muscle contraction. The current study demonstrated that ET-1 and endothelin-2 (ET-2) stimulate calcium mobilization in HCM cells via an ETA receptor subtype. It appears that the increase in intracellular calcium ([CA2+]) is the result of ET coupled to PLC via a pertussis toxin sensitive G-protein. A biphasic calcium response is elicited with ET stimulation consisting of a transient increase in [Ca2+]I which appears to be primarily due to release of intracellular stores, followed by a lower sustained phase which appears to be dependent on the influx of extracellular calcium. Endothelin-1 also appears to stimulate an increase in prostaglandin E2 (PGE2) formation through activation of PLA2. Furthermore, it appears that the effects of ET-1 on PLC and calcium are independent of the ET-1 effects on PGE2 production, such that the ET-1 induced increase in [CA2+]I are coupled to the PLC signaling pathway, whereas increase in PGE2 production appears to be the result of an ETA receptor coupled to PLA2. Whether there are different subtypes of ETA receptors or the receptor is coupled through different G-proteins is uncertain. Endothelin-1 and Big ET-1 immunoreactivity was also observed in both HCM and human nonpigmented ciliary epithelial (HNPE) cells. This is the first time that ET-1 and Big ET-1 immunoreactivity has been detected in the HCM cells, suggesting that these cells have the capability to synthesize both peptides. Furthermore, the increase in ET-1 and Big ET-1 immunoreactivity upon stimulation with TNF-α suggests that cytokines may be important regulators of ET synthesis and release. The findings of this research aid in the understanding of the mechanism of action whereby ETs regulate aqueous humor dynamics and IOP. Through a better understanding of the cellular actions of ET, insight is gained into the development of new ocular selective agents acting at the ET receptor.Item Identifying Unique Therapeutic Targets To Rescue Retinal Ganglion Cells From Degeneration After Optic Nerve Crush(2014-05-01) Sharma, Tasneem P.; Clark, Abbot F.Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of cellular and molecular events resulting in neuronal apoptosis and regenerative failure. The pathogenic mechanisms and gene expression changes associated with these detrimental events can be effectively studied using a rodent optic nerve crush (ONC) model. The purpose of this study was to use a mouse ONC model to: (a) evaluate changes in retina and ON gene expression, (b) identify neurodegenerative pathogenic pathways, (c) discover potential new therapeutic targets, and (d) evaluate the neuroprotective and axogenic properties of one selected therapeutic target on axotomized RGCs in vitro and the optic nerve crush (ONC) mouse model in vivo. Meta-analysis of altered gene expression (≥1.5 changes and ≤1.5 changes, p [less than] 0.05 demonstrated 29 up- and 20 downregulated retinal gene clusters and 82 up- and 42 down-regulated optic nerve clusters. Regulated gene clusters included regenerative change, synaptic plasticity, axonogenesis, neuron projection, and neuron differentiation related genes. Expression of selected genes (Vsnl1, Syt1, Synpr and Nrn1) from retinal and ON neuronal clusters was qualitatively and quantitatively examined for their relation to axonal neurodegeneration by immunohistochemistry and qRT-PCR. Axotomized RGCs treated with recombinant hNrn1 (selected target) significantly increased survival of RGCs by 29% (n=8, p [less than] 0.01) and neurite outgrowth of cultured neurons by 261% compared to controls in cultured neurons (n=5-7, p [less than] 0.05). RGC transduction with AAV2-CAG-hNRN1 prior to ONC promoted RGC survival (42%, n=5-8, p [less than] 0.05) and significantly preserved ERG RGC function by 41% until 28 dpc (n=6, p [less than] 0.05) compared to the control AAV2-CAG-GFP transduction group. These ONC induced neuronal loss and regenerative failure associated clusters can be extrapolated to changes occurring in other forms of CNS trauma or in clinical neurodegenerative pathological settings. In conclusion, this study identified potential therapeutic targets to address two key mechanisms of CNS trauma and neurodegeneration: neuronal loss and regenerative failure and presented Nrn1 as a potential therapeutic target for CNS neurodegenerative diseases.Item Lal, Harbans, Ph.D.(1994-02-08) Lal, Harbans; Hailey, BlakeDr. Lal, Professor and Chairman of Pharmacology, began his career with TCOM in 1980. He shares his early goals as faculty, his research, and his outlook on the school's future. Interviewed by Blake Hailey, February 8, 1994Item Mechanisms of Glucocorticoid-induced ocular hypertension(2003-08-01) Zhang, Xinyu; Thomas YorioZhang, Xinyu, Mechanisms of glucocorticoid-induced ocular hypertension. Doctor of Philosophy (Pharmacology & Neuroscience). August 2003; 163p; 4 tables; 24 figures; 102 titles. Glucocorticoids, frequently used anti-inflammatory and immunosuppressive agents, are associated with ocular hypertension and glaucoma. Endothelin-1 (ET-1) is also implicated in glaucoma pathology and optic neuropathy as its concentration is elevated in glaucoma patients and in animal models of glaucoma and chronic administration of ET-1 produces damage to the optic nerve head in rats. Glucocorticoids have been reported to regulate the expression of ET-1 gene and ET receptors in the cardiovascular system. However in the eye, the interactions between glucocorticoids and ET-1 have been implicated in the regulation of intraocular pressure and contribute to glaucoma pathology. Therefore, the purpose of the investigations described herein was to determine the novel mechanisms that may be involved in the regulation of intraocular pressure by glucocorticoids with interactions with ET-1 and ET receptors in NPE cells, a source of ET-1, and in TM cells where both glucocorticoids and ET-1 effect aqueous humor outflow. The hypothesis was that ET-1 exacerbates the actions of glucocorticoids on TM cells and contributes to increased outflow resistance. Furthermore, individual sensitivities to glucocorticoids differ considerably. About one in every three people in the general population is considered potential steroid responders while almost all primary open angle glaucoma (POAG) patients are steroid responders and develop ocular hypertension after ocular administration of glucocorticoids. The molecular mechanisms underlying the higher glucocorticoid responsiveness among POAG patients remain unknown. The glucocorticoid receptor beta isoform (hGRβ) has become a candidate for glucocorticoid resistance in some diseases, especially in asthma, based on the reports of its negative activity. The purpose of this segment of the investigations was to test the hypothesis that glucocorticoid responsiveness was regulated by the expression of hGRβ in TM cells. We demonstrated that dexamethaosone (Dex), a synthetic glucocorticoid, increased ET-1 synthesis and release from human non-pigmented ciliary epithelial (HNPE) cells. Dex also suppressed ETB receptor protein expression and attenuated ET-1 mediated increase in nitric oxide (NO) while Dex had no effect on ETA receptor expression and ETA receptor mediated intracellular Ca2+ mobilization in TM cells. The increase in the release of ET-1 from HNPE cells with a concomitant decrease of ETB receptor protein expression and ETB receptor mediated NO release by Dex in TM could result in an increase in the contraction and decrease in relaxation of trabecular meshwork thus reducing the intratrabecular space. Such actions by ET-1 may exacerbate Dex effects on the outflow pathway leading to increased outflow resistance and consequently elevated intraocular pressure that typically is associated with glucocorticoids. We have also found a significant difference in hGRβ levels among normal versus glaucomatous TM cell lines, with the POAG TM cell lines having lower hGRβ receptor expression. This is coincidence with the fact that in the normal population, there is a low rate of glucocorticoid responders as compared to almost all POAG patients considered as glucocorticoid responders. Overexpression of hGRβ in TM cells, produced by transfecting a hGRβ expression construct, inhibited Dex-induced expression of myocilin, a glaucomatous gene, supporting the contention that hGRβ acts as a negative regulator of glucocorticoid activity. In addition, we studied the machinery of cytoplasm to nuclear transport of hGRβ. We identified that a chaperon protein, hsp90, is a requirement for the nuclear translocation of hGRβ. In conclusion, we have described a novel-signaling pathway for glucocorticoids through the regulation of ET-1 and ET receptors in the anterior segment which have consequences on aqueous humor outflow. We have also demonstrated a possible molecular mechanism by which glucocorticoid responsiveness in POAG patients is achieved as a result of the low level of nuclear hGRβ receptor isoform expression. Furthermore, we have, for the first time, identified hap90 as a chaperon protein for the translocation of hGRβ from the cytoplasm to the nucleus.Item NEUROPROTECTIVE PROPERTIES OF SIGMA-1 RECEPTOR IN GLAUCOMA(2014-05-01) Mueller, Brett H.; Thomas YorioGlaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) that affects over 70 million individuals worldwide. Glaucoma pathology is manifested as cupping of the optic disk, damage to the nerve fiber layer, and visual field deficits. The final pathological step of this disease contributing to visual field loss is the apoptosis of retinal ganglion cells (RGCs). Currently, the only therapeutic agents that are used to treat glaucoma are IOP lowering drugs. However, even when IOP is brought within normal range, a significant number of patients still have progression of visual deficits. Currently, there are no treatment options that have the ability to sustain the viability of RGCs during the disease process of glaucoma. Therefore, neuroprotective drugs that protect RGCs need to be developed as adjunct therapeutic agents to IOP lowering drugs. The sigma-1 receptor (σ-1r) is a non-opioid receptor that has been shown to have the ability to bind to benzomorphans, steroids, and psychotropic drugs. This receptor is ubiquitously expressed throughout the entire body; however, the endogenous ligand and function of σ-1r is not yet known. Several in vitro and in vivo studies have demonstrated the neuroprotective effects of σ-1r stimulation in several models of retinal neurodegenerative diseases including glaucoma and diabetic retinopathy. Numerous studies have linked the neuroprotective effects of σ-1r to its ability to block cytotoxic calcium ion influx through ligand gated and voltage gated ion channels, modulation of ER stress, maintenance of mitochondrial homeostasis, and stimulation of pro-survival intracellular signaling pathways. However in primary RGCs, there have been no studies demonstrating σ-1 receptor mechanism of action. The only proposed neuroprotective mechanism of action of σ-1r that has been performed in retinal flat mounts is blockage of calcium ion influx through activated NMDA receptors. This present research project investigated the mechanism of neuroprotective effects of σ-1rs in primary RGCs, particularly involving L-type voltage gated calcium channels (VGCCs) and activation of extracellular-signal-regulated kinases (ERK 1/2). We demonstrated that VGCCs were activated using KCl (20mM). Pre-treatment with a known L-type VGCC blocker produced a 57% decrease in calcium ion influx through activated VGCCs (following depolarization by KCl). In addition, calcium imaging showed that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Treatment with a σ-1r antagonist, BD1047, produced a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs. This confirms that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms of action of both these agents. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in primary RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in primary RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in primary RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in primary RGCs implies that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs Another signaling pathway that was studied as a potential target of σ-1r mediated neuroprotection was the MAP kinase pathway, in particular, ERK phosphorylation as an index of cell survival. RGCs subjected to oxygen and glucose deprivation (OGD) for 6 hours induced 50% cell death in primary RGCs and inhibited pERK1/2 expression by 65%. Cell death was attenuated when RGCs were treated with pentazocine under OGD and pERK1/2 expression was increased by 1.6 fold compared to OGD treated RGCs without pentazocine treatment. The co-treatment of with an ERK1/2 inhibitor PD098059 with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. These results established a link between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. In conclusion, we have established two novel mechanisms underlying σ-1 receptor mediated neuroprotection in primary RGCs. These findings suggest that activation of the σ-1 receptor in RGCs has a role in calcium regulation and the activation of the ERK1/2 pathway. In addition, this study also demonstrates the robust neuroprotective effects of σ-1 receptor in RGCs when subjected to OGD. These data also provide evidence suggesting that σ-1 receptor may be a therapeutic target to protect RGCs during ocular neurodegenerative diseases like glaucoma.Item Oxidative Stress Alters IP3 Receptor Function in the Neuronal Cell Line HT22(2008-05-01) Longoria, Sandra; Peter Koulen; Kati Prokai; Tina MachuSandra Longoria., Oxidative Stress Alters IP3 Receptor Function in the Neuronal Cell Line HT22, Master of Science (Biomedical Sciences), May 2008, 72 pp., 25 Figures. Oxidative stress contributes to the genesis of several neurodegenerative disorders such as Alzheimer’s Disease (AD). Oxidants such as, tert-butyl hydrogen peroxide (tBHP), have been used in in vitro models of neurodegeneration to induce oxidative stress. Small changes in the regulation of the intracellular calcium (Ca2+) concentration can contribute to brain aging and increase vulnerability of neurons to cellular and functional damage in neurodegenerative diseases. In neurons, inositol 1, 4, 5-trisphosphate (IP3) is a second messenger that is generated through receptor activity at the plasma membrane. IP3 receptors (IP3R) are located on endoplasmic reticulum (ER) membranes and are intracellular calcium channels (ICC) that release Ca2+ into the cytoplasm in response to activation by their ligand IP3. The goal of the present study was to measure the contribution of ICCs to Ca2+ dysregulation in neurons experiencing oxidative stress. I tested the hypothesis that oxidative stress induced with tBHP causes increased intracellular Ca2+ release via activation of IP3 receptors. I used the murine hippocampal cell line HT22, as a model for neuronal oxidative stress. Immunocytochemistry and Ca2+ imaging experiments were performed to identify areas of altered IP3R expression and activity under normal conditions and induced oxidative stress. tBHP treatment increased expression and Ca2+ release activity of neuronal IP3 receptors. My findings support that oxidative stress as seen in a number of neurodegenerative diseases negatively affects regulation of Ca2+ release through increased expression and activity of IP3 receptors.Item Pharmacological Assessment of Novel Phenylacetamide as a Sigma 1 Receptor Ligand(2014-05-01) Malik, Maninder; Robert LuedtkeThe symptoms of psychosis have been categorized as positive, negative and cognitive. Traditionally drug discovery in psychiatric disorders has focused on positive symptoms of the disease. However, cognitive impairment is equally prevalent and represents a major impediment to the recovery of patients. Hence, research on the drug discovery and development that can improve overall quality of life of patients with neuropsychiatric conditions is important. The main aim of this project was to evaluate a selective and potent sigma 1 receptor phenylacetamide (LS-1-137) as a potential pharmacotherapeutic agent for treating neuropsychiatric disorders and associated cognitive impairment. The sigma 1 receptor is an endoplasmic reticulum (ER) resident protein located on the interface of ER and mitochondria. The sigma 1 receptor is a 25 KDa protein that shares no amino acid sequence homology with any other known mammalian proteins. The research being done on these novel receptors suggests that rather than being a classical receptor-signaling unit sigma 1 receptors act as a molecular chaperone. Several studies suggest sigma 1 receptor ligands modulate abnormal neurotransmission that contributes to the pathogenesis of several CNS disorders. In recent studies by our laboratory, it was found that our novel sigma 1 receptor ligand, LS-1-137 (developed by our collaborators Mach and colleagues) exhibit neuroprotection both in vitro and in vivo. In this project we further characterized LS-1-137 as a potential treatment option for cognitive and neuropsychiatric disorders. Our fundamental hypothesis is that sigma 1 receptor selective compounds represent a potential neuroprotective pharmacotherapy for treating psychosis and cognitive deficits associated with neuropsychiatric disorders. LS-1-137 was evaluated in a 2, 5-dimethoxy-4-iodoamphetamine (DOI) induced head twitch response (HTR) model and a scopolamine-induced cognition impairment model. Our findings suggest that LS-1-137 attenuates the DOI-induced HTR and alleviates scopolamine-induced impairment in learning. Our in vitro data suggest that LS-1-137 is an agonist at sigma 1 receptors and triggers the release of BDNF from rat astrocytes. Furthermore, rotarod and swim test studies indicated that unlike currently prescribed neuroleptics, LS-1-137 does not compromise the agility or muscular coordination of animals. Therefore, in this dissertation we have assessed a novel pharmacotherapeutic agent that may treat the psychosis and cognitive dysfunction associated with neuropsychiatric disorders.Item Presenilins Modulate Cellular Activity of Ryanodine Receptors(2012-12-01) Payne, Andrew J.; Peter KoulenPayne, Andrew J., Presenilins Modulate Cellular Activity of Ryanodine Receptors. Doctor of Philosophy (Biomedical Sciences), December, 2012, 160 pp., 7 tables, 39 figures, bibliography 241 titles. Ryanodine Receptors (RyRs) are large, endoplasmic reticulum (ER) intracellular calcium channels in excitable cells. RyRs are major cellular mediators of calcium-induced calcium release and crucial regulators of intracellular calcium homeostasis. Disruption of RyR function has been described in pathologies of dysregulated calcium such as Alzheimer’s disease (AD). Presenilins (PS1 and PS2) are ER transmembrane proteins expressed in the central nervous system mediating calcium homeostasis and Notch signaling, and act as the proteolytic core of γ-secretase in amyloid cleavage. Previous single channel electrophysiology studies described a direct interaction between RyR and the N-termini of presenilin 1 (PS1NTF) and presenilin 2 (PS2NTF) that resulted in differential modulation of the RyR open probability and mean Ca2+ current at the RyR single channel level. We herein tested the hypothesis that PS1NTF and PS2NTF functionally modulate RyRs in a physiologically relevant in vitro model resulting in changes to RyR-mediated intracellular calcium release. Confocal microscopy, microfluorimetry, and coimmunoprecipitation studies confirmed a physical interaction between RyRs and PS-NTFs in human neuroblastoma SH-SY5Y cells, an in vitro AD model. Live cell fluorescent calcium imaging was used to quantify the effects of overexpression of PS1NTF or PS2NTF on Ca2+ release from RyR mediated stores. PS1NTF was found to increase RyR gating to the full open state at physiologically normal calcium concentrations. Verifying the previous electrophysiology data, PS2NTF had no effect on RyR at physiological calcium concentrations. Mutagenesis of critical cysteine residues on the PSNTFs was applied to determine the effect of specific structure-function differences between PS1 and PS2 molecules that underlie the isoform specific modulation of RyR calcium release. Mutations to PS2NTF removing disulfide bridging cysteines recapitulated PS1NTF-like regulation of RyR Ca2+ release. Our findings indicate that PS1NTF and PS2NTF bind RyRs differentially. Our results indicate a novel mechanism of intracellular calcium regulation by the PS-RyR interaction and a novel target for the treatment of AD, neurodegenerative disorders, and diseases controlled by RyR and PS functions.Item Rat Naphthalene Cataract Studies: Mechanisms and Prevention(1994-06-01) Xu, Guo-Tong; Thomas YorioXu, Guo-Tong, Rat Naphthalene Cataract Studies: Mechanisms and Prevention. Doctor of Philosophy (Biomedical Sciences/Pharmacology), June, 1994, 134 pp., 16 tables, 34 figures, references, 153 titles. The mechanism of naphthalene-induced cataract in rats and the preventive action of AL01576 (an aldose reductase inhibitor, ARI) were studied in both in vivo and in vitro systems. In the in vivo studies, cataracts were induced in five strains of rats (2 pigmented, 3 albino) by naphthalene feeding (1g/kg/day). The cataractous changes occurred in 1 week as watercleft and spoke-like opacities which merged to form a shell-like opacity in the deep cortex by 3 weeks. Semi-quantitation of the opacities with an arbitrary six-score grading system showed little difference in the cataract development between the pigmented and albino strains. Major biochemical changes observed were a decrease of 20%-30% in GSH by one week of feeding, the appearance of disulfide cross-linking of lens proteins by 3 weeks, and a more than ten fold increase in the content of protein-GSH mixed disulfide. Neither damage to lens membrane functions as measured by 3H-choline or 86Rb uptake or loss of Na+/K+-ATPase activity was detected AL01576 (10 mg/kg/day) completely prevented the naphthalene-induced lens changes in both pigmented and albino rats. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats and suggest that tyrosinase action on naphthalene metabolites (such as 1- or 2- naphthol) is not involved in this cataract formation. The in vitro “naphthalene cataract” was established by exposing rat lens to each of 5 potential naphthalene metabolites in organ culture system (in modified TC-199 medium) for 48 hrs. When naphthalene dihydrodiol was used, both the morphological and biochemical changes in the lens were very similar to those observed in lenses of naphthalene-fed rats, and AL01576 completely blocked these in vitro changes as it did in vivo. Other naphthalene metabolites (1,2-dihydroxynaphthalne, 1-naphthol, 2-naphthol and 1,2-naphthoquinone) caused changes which were different from those induced by naphthalene in vivo and one of them was prevented by AL01576. Therefore, naphthalene in vivo and none of them was prevented by AL01576. Therefore, naphthalene dihydrodiol is the key naphthalene metabolite which reaches the lens via blood and aqueous humor and causes cataract when it is metabolized to 1,2-naphthoquinone. This mechanism is further supported by the detection of naphthalene dihydrodiol in the lens and aqueous humor of naphthalene-fed rats. Examples of various classes of ARI (AL01576, AL04114, Sorbinil and Tolrestat) were compared for their effects on the formation of naphthalene cataract and a dual cataract induced with simultaneous feeding of galactose and naph-thalene. Both AL01576 and AL04114 (spirohydantoin derivatives) completely prevented the changes in the lenses of naphthalene-fed rats. However, Sorbinil (another spirohydantoin ARI) demonstrated a much weaker efficacy in this model and the carboxylic acid ARI, Tolrestat, showed no efficacy at all. In the dual cataract, Tolrestat prevented galactose cataract formation and reduced the lens dulcitol accumulation but showed no protection against the shell-like opacity caused by naphthalene. On the other hand, AL01576 protected the lens from the cataractogenic action of both compounds. These results rule out the involvement of aldose reductase in naphthalene cataract formation. Furthermore, AL04114 (not a cytochrome P-450 inhibitor) showed a similar efficacy as AL01576 (a inhibitor of cytochrome) in naphthalene cataract prevention. Therefore, the inhibition of cytochrome P-450 may not be involved in the prevention of this cataract. Based on these findings and the fact that AL01576 prevents the changes induced by naphthalene dihydrodiol (ND) but not 1,2-naphthoquinone (NQ), a new mechanism for rat naphthalene cataract formation is proposed: naphthalene is converted by cytochrome P-450 to ND, which reaches the eye via the blood and penetrates into the lens. By the action of dihydroxynaphthalene which autoxidizes to form NQ and H2O2 production and thus causes cataract. AL01576 and AL04114 inhibit DDD activity, block NQ and H2O2 production and thus prevent the cataract formation.Item Regulation of intracellular calcium channels by their associated proteins homer 1 and presenilin 1(2006-05-01) Hwang, Sung-Yong; Koulen, Peter; Dillon, Glenn; Singh, MeharvanSung-Yong, Hwang, Regulation of intracellular calcium channels by their associated proteins homer 1 and presenilin 1. Doctor of Philosophy (Pharmacology and Neuroscience), May, 2006, 184 pp., 4 tables, 20 illustrations, 74 titles. In neurons, Calcium (CA2+) serves as a critical intracellular messenger that regulates a variety of cellular processes such as gene expression, neurotransmitter release, cell death, and synaptic plasticity. Therefore, it is essential for neurons to control their Ca2+ levels tightly. Ca2+ is released within the cell from intracellular stores such as the endoplasmic reticulum by activation of intracellular Ca2+ channels (ICCs) such as the inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs). Each of these two groups of ICC has three isoforms. A number of associated proteins of these two ICCs that were shown to modulate activity of the respective channel have been identified. Homer 1, a synaptic scaffolding protein not only physically associated with IP3R type1 (IP3R1), but also changes the activity of IP3R1, suggesting that Homer 1 is involved in intracellular Ca2+ signaling. Based on the similarity in amino acid sequence and molecular and physiological properties among IP3R isoforms and the fact that IP3R type 3 (IP3R3) contains the proline-rich motif (PPxxFr) that is required for the interaction with Homer, it was hypothesized that Homer 1 associates with IP3R3, leading to changes in the channel activity. Presenilin 1 (PS1) is a transmembrane protein, being expressed in cell body, dendrites, and axon in the neuron. Mutations in PS1 account for most cases of early-onset familial Alzheimer’s disease (AD). PS1 was shown to associate with RyRs and to modulate their channel activity. Therefore, it was hypothesized that specific regions of PS-1 bind to RyR type 2 (RyR2), a major isoform in the brain, resulting in changes in the channel activity. Homer 1c was shown to associate with IP3R3, leading to a decrease in channel activity. A specific region of PS1 that interacts with RyR2 was identified to increase the channel activity of RyR2. Results of the present study contributed to the understanding of the nature of intracellular Ca2+ signaling as well as the mechanisms of action by which ICCs are regulated by their associated proteins. These findings provide the rationale for novel strategies to study neurological disorders including AD and epilepsy that are mediated by Ca2+ dysregulation.Item Renal Hypertension Impairs Coronary Hyperemia During Exercise(2003-08-01) Williams, Maurice A.; Patricia GwirtzWilliams, Maurice A., Renal Hypertension Impairs Coronary Hyperemia During Exercise. Doctor of Philosophy (Biomedical Sciences), August, 2003, 103 pp., 2 tables, 10 illustrations, bibliography, 180 titles. High blood pressure (hypotension) is a common disease that greatly impacts cardiovascular disease and quality of life making it a high priority for early detection and treatment. Hypertension is a major risk factor for coronary artery disease, heart failure, stroke and sudden death. The incidence of hypertension is increasing as the population ages. Exercise intolerance or exertional fatigue is a common complaint of patients with hypertension. We tested the hypothesis that the acute onset of renovascular hypertension results in a sustained, elevated sympathetic adrenergic stimulation of the heart which blunts the coronary hyperemic response and reduces the cardiac contractile response to exercise. Studies were conducted in chronically instrumented dogs before and after the acute onset of renosvascular hypertension of only 2 weeks. This degree of hypertension would normally go undetected or untreated by physicians. Short-term hypertension blunted coronary blood flow at rest and during each level of submaximal exercise. Hypertensive dogs showed a significant reduction in cardiac pump function during submaximal exercise compared to the responses in the normotensive dogs. These changes were very dramatic for such a short term of a mild hypertension. These studies were designed to examine mechanisms mediating the blunted coronary hyperemic response during exercise which imposes a limit on cardiac function. The results of these experiments addressed fundamental questions regarding alterations in neurohumoral control of cardiac contractile function and the mechanisms by which neurohumoral control of the heart is altered by hypertension. These studies should also clarify the mechanistic rationale for medical therapies to treat patients with hypertension.Item Risk for Stroke Among Migraine Sufferers(2001-05-01) Hall, Rebecca G.; Antonio Rene; Manuel BayonaHall, Rebecca G., Risk for Stroke Among Migraine Sufferers. Master of Public Health (Epidemiology), May, 2000, 27 pp., 9 tables, references, 33 titles. The objective of this study was to investigate, using the National Health Interview Survey (NHIS), whether those who suffer from migraine or severe headache do. Odds ratios were calculated for stroke among migraine sufferers compared to those who do not suffer from migraine. Results were adjusted for age, gender, and race. Risk factors for stroke were also analyzed. The crude odds ratio for stroke among migraine sufferers compared to non-migraine sufferers is 2.17, increasing 3.77 with age-adjustment. These results suggest that vascular events that are associated with migraine may also be associated with an increased risk for stroke. Discovering the mechanism that generates this relationship has widespread implication to the population and may save taxpayers billions of dollars annually be leading to better treatments for and possible prevention of migraine.Item Role of catecholaminergic A2 neurons of nucleus of the solitary tract(NTS) in cardiovascular and respiratory adaptations to chronic intermittent hypoxia (CIH) in rats(2014-05-01) Bathina, Chandra Sekhar; Steve MifflinThis study examined the role played by the catecholaminergic A2 neurons of the nucleus of the solitary tract (NTS) of adult male Sprague- Dawley rats in the increased mean arterial pressure (MAP) noticed following exposure to chronic intermittent hypoxia (CIH), a rodent model to simulate arterial hypoxemic conditions occurring in humans suffering from sleep apnea. In one study, we tested the hypothesis that tyrosine hydroxylase (TH) knockdown in NTS reduces the sustained elevation in MAP noticed in the rats exposed to CIH. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs with a GFP reporter having either short hairpin RNA for TH (shRNA) or scrambled virus (scrambled) were injected into caudal NTS. shRNA through formation of RNA-induced silencing complex reduced the amount of TH levels in the NTS. Virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). CIH increased MAP and HR during the day in both the scrambled (n= 14, p Experiments were also conducted to understand the molecular level changes occurring in the A2 neurons, following CIH exposure. mRNA expression changes occurring in the A2 neurons were analyzed by novel technique of laser capture microdissection (LCM) by labeling the A2 neurons using adeno-associated virus with TH promoter attached to green fluorescent protein (GFP). A2 neurons are found to express mRNA of angiotensin receptor subtypes AT1a and AT1b. Moreover, excitatory amino acids (EAAs) like glutamate released from chemoreceptor afferents during chronic intermittent hypoxia (CIH) are found to modulate the activity of the neurons in the region of NTS. The aim of this study was to assess the effect of CIH on the mRNA expression levels of AT1a, AT1b and EAAs receptor subunits in the A2 neurons. We utilized commercially available adeno associated virus (AAV) vector mediated delivery of green fluorescent protein (GFP) labeled tyrosine hydroxylase promoter (AAV-GFP-TH), which will incorporate into the TH genome and express GFP with the TH expression to label the A2 neurons. 7 virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). Laser capture microdissection was performed to capture the A2 neurons from caudal NTS. Total RNA from these neurons was extracted and the gene expression for different genes were assessed by quantitative real time reverse transcription polymerase chain reaction and compared between the control and CIH rats using 2-ΔΔct method. CIH is found to decrease AT1a (p=0.002; control - 1.08 ± 0.13, n=7; CIH – 0.48 ± 0.07, n= 6) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPA) receptor subunit GluR2 (p=0.03; control - 1.11 ± 0.24, n=7; CIH- 0.52 ± 0.12, n= 6) and increase transcription factor FosB (p=0.03; control - 1.14 ± 0.25, n=7; CIH- 1.97 ± 0.25, n= 5) mRNA expression levels in the A2 neurons. These results suggests that there is increase in activity of these neurons following CIH and a possibility of these neurons becoming more calcium permeable as GluR2 is found to resist calcium permeability. Western blot studies were also conducted from the whole NTS punches, to study the changes in protein levels of the genes studied using LCM. The changes in TH protein levels were not significant in both caudal and sub-postremal NTS (P [greater than] 0.05). GluR1 and GluR2 protein level changes were not significant in the caudal NTS, however, there was a significant decrease (P As the mRNA analysis of A2 neurons suggested, there might be changes occurring in the calcium permeability of A2 neurons following CIH, attempts were made to do calcium imaging studies on the A2 neurons. There was difficulty in the colocalization of GFP with the fura-2AM, the calcium imaging dye. So, calcium imaging was conducted on the NTS neurons of sham Sprague-Dawley rats and CIH exposed rats. 30 μM AMPA application caused a 340/380 ratio change of 0.17 ± 0.01 (n=5) in control rats and this change was significantly higher 0.55 ± 0.13 in CIH rats. The probability of neurons responding to AMPA application was considerably higher in CIH rats. CNQX treatment of the slices abolished the changes in intracellular calcium in neurons from both control and CIH rats, demonstrating that the responses noticed after AMPA application were AMPA receptor mediated. Increases in intracellular calcium levels following 500 μM potassium chloride applications validate the fact that the neurons were viable. Further studies on quantifying the phosphorylated GluR1 and GluR2, subunits of AMPA receptors are required to explain the driving force behind this uniform increase in intracellular calcium levels of NTS neurons after CIH. We conclude that the sustained hypertension observed during CIH can be prevented by TH knockdown and this mechanism might involve paraventricular nucleus (PVN) of forebrain, hypothalamo-pituitary adrenal axis (HPA axis) or intermediolateral cell column (IML) of spinal cord. A2 neurons also undergo molecular alterations that might increase their calcium influx in to the neuron and vise-versa.Item Role of Serine/Threon protein Phosphateases in Estrogen Mediated Neuroprotection(2007-08-01) Yi, Kun DonThe Signaling pathways that mediate neurodegeneration are complex and involve a balance between phosphorylation and dephosphorylation of Signaling and Structural proteins. Estrogens have a variety of mode of action including transducing signaling events including the activation and/or supression of survival pathways. The purpose of this study was to delineate the role of protein phosphatases (PP) in estrogen neuroprotection. We assessed the role of PP in neuroprotection mediated by estrogen and its analogues. We also determined the role of estrogen receptors (ER) and MAPK signaling. Okadaic Acid (OA) and calyculin A (CA), non-specific serine/threonine PP inhibitors, were exposed to cells at various concentrations in the presence or absence of 17beta-estradiol, 17alpha-estradiol, the enantiomer of 17beta-estradiol (ENT E2), 2-(l-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (ZYC3, non-ER binding estrogen analog) and/or glutamate. OA and CA caused a dose-dependent decrease in cell viability. None of the estrogen and its analogues showed protection against neurotoxic concentrations of either OA or CA, while all estrogens attenuated glutamate toxicity. However in the presence of these PP inhibitors, estrogen mediated protection against glutamate toxicity was lost. Glutamate treatment caused a 50% decrease in levels of PP1, PP2A and PP2B protein; while, co-administration of estrogen or its analogues with glutamate prevented the decrease in PP1, PP2A, and PP2B levels. In addition, PP2A and calcineurin activities were significantly suppressed with treatment of glutamate and/or OA; while the presence of these estrogens attenuated the decreases in PP activity. Moreover, an increase in reactive oxygen species, protein cabonylation, lipid peroxidation, caspase-3 activity, and mitochondrial dysfunction were evidence in both glutamate and OA mediated cell death. Estrogens attenuate these increases in glutamate-mediated cell death, but were ineffective in OA-induced neuronal death. Furthermore, gluatmate treatment caused a persistent increase in phosphorylation of ERK1/2 that corresponds with the decrease protein levels of PPs. Treatment of estrogens protect cells against glutamate-induced oxidative stress and excitotoxicity through an ER-independent mediated mechanism that serves to preserve phosphatase activity in the face of oxidative/excitotoxic insults resulting in attenuation of the persistent phosphorylation of ERK1/2 associated with neuronal death.Item Role of ΔFosB in nucleus of the solitary tract (NTS) in cardiovascular adaptations to chronic intermittent hypoxia (CIH) in rats(2015-08-01) Wu, Qiong; Mifflin, Steve W.; Cunningham, J. Thomas; Schreihofer, Ann M.Chronic intermittent hypoxia (CIH) rodent model is widely utilized to study obstructive sleep apnea (OSA) associated disease such as hypertension. Arterial chemoreceptor is activated by CIH, and leads to increased sympathetic nerve discharge, resulting in elevated arterial pressure. The central neuronal mechanisms of CIH induced hypertension are barely understood. The nucleus of the solitary tract (NTS) receives the first synaptic inputs from arterial chemoreceptor afferents. Transcription factor ΔFosB is increased in the NTS after a 7 day-CIH exposure. We hypothesize that NTS ΔFosB could mediate neuronal plasticity, contribute to CIH induced hypertension. Three specific aims were addressed. Aim 1: To determine the relationship between NTS ΔFosB and CIH hypertension. Viral constructs were delivered into NTS to functionally block ΔFosB (ΔJunD group). Mean arterial pressure (MAP) was measured in day time when rats were exposed to intermittent hypoxia and night time when they were in normoxia. The increase in MAP observed in ΔJunD and sham groups during day time was dampened in ΔJunD group during night time, indicating the contribution of ΔFosB to the sustained component of CIH associated hypertension. Aim 2: To determine the time-course of induction of ΔFosB immunoreactive NTS neurons during CIH exposure. Rats were separated into normoxia, 1 day, 3, 5, 7 days CIH, and 1 day, 3, 7 days recovery after 7 days CIH groups. ΔFosB immunoreactivity increased within 1 day CIH, and maintained this elevation throughout 7 days of CIH. 1 day recovery was sufficient to reduce ΔFosB immunoreactivity to normoxia level. Therefore, ΔFosB under CIH develops rapidly. Aim 3: To determine the function of ΔFosB in glutamatergic transmission after CIH. Miniature excitatory post-synaptic current (mEPSC) properties of NTS neurons of rats exposed to either different days of CIH or room air were compared. CIH increased mEPSC amplitude but not frequency, suggesting a post-synaptic site of effect. Additionally, functional blockade of NTS ΔFosB with ΔJunD decreased mEPSC amplitude back to normoxia level. Finally, overexpression of NTS ΔFosB increased mEPSC amplitude to similar levels as CIH. These results suggest that ΔFosB in NTS neurons mediates molecular adaptations which might play an important role in CIH associated hypertension.Item Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli(1997-12-01) Jung, Marianna E.; Walls, Cleatus; Downey, H. Fred; Forster, MichaelJung, Marianna E., Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli. Doctor of Philosophy (Biomedical Sciences), December 1997, 150 pp, introduction, 2 chapters, discussion, bibliography, 109 titles. This study compared gender differences in the anxiogenic stimuli induced by either a GABA-A antagonist, pentylenetetrazol (PTZ) or by a 5-HT1b/2 agonist, m-chlorophenylpiperazine (m-CPP) before and during ethanol withdrawal (EW). Rats were trained to discriminate either PTZ (16mg/kg, IP) or m-CPP (1.2 mg/kg, IP) from saline in a two lever choice task for food reward. Male and female rats were gonadectomized or sham-operated, and ovariectomized (OVX) female rats were tested during replacement treatment with 17β estradiol (2.5 mg, 21 day release, sc). The dose-response for the discrimination of the interoceptive stimulus (IDS) produced by PTZ (0-16 mg/kg) or m-CPP (0 to 1.2 mg/kg) was measured under all hormonal conditions. For m-CPP trained rats, latency to first lever-press response was also tested. Results: sham and estradiol-replaced female rats had higher ED50s for discrimination of the PTZ or m-CPP IDS than intact males or OVX rats. There is a dose-related impairment of operant responding after mCPP injection. Sham and estradiol replaced OVX rats showed an increased delay to the initiation of response after m-CPP injection as compared to sham or castrated male rats or OVX rats that showed no effect at the doses tested. Rats then received a chronic ethanol diet (6.5%) for 10 days. At twelve hours of ethanol withdrawl, they were tested for lever selection after saline injection. Fewer sham female and estradiol-replaced female rats responded on the drug lever during acute EW as compared to sham male, castrated or OVX rats. In general, the anxiogenic drug lever selection of OVX rats resembled that of male rats but was restored toward that of sham female rats by estradiol replacement. Castration did not alter the response of male rats to either PTZ or mCPP. Serum β –estradiol concentrations were determined by radioimmunoassay for sham, OVX, and estradiol-replaced female rats. The concentration was significantly higher in hormone-replaced female rats than in OVX. The estradiol concentration in sham female rats showed a cyclic pattern over 4 consecutive days, but this pattern did not correlate with any difference in IDS. Blood ethanol concentration (BEC) was determined using head space gas chromatography. BEC was higher in intact female rats than in intact male rats after ethanol injection (2 g/kg, ip), but did not differ during EW. Conclusions: females produce less anxiogenic IDS in response to either GABA inhibition or 5-HT1b/2 activation, but are more impaired by m-CPP in their ability to initiate operant responses than male rats. In addition, fewer intact females developed a spontaneous IDS during EW than males which is not the result of lower BEC. Estrogen appears to play a trophic role in altering responsiveness to anxiogenic stimuli.Item Synergy 2007: Annual Research Report(2007-01-01)Item Synergy 2011: Annual Research Report(2011-01-01)Item The Role of a Membrane Androgen Receptron in the Brain(2007-02-01) Gatson, John Wayne; Simpkins, James; Koulen, Peter; Basu, AlakanandaGatson, Joshua Wayne, The Role of a Membrane Androgen Receptor in the Brain. Doctor of Philosophy (Biomedical Sciences), February 2007, pp187, 34 illustrations. In the brain, depending on the insult type, androgens have been shown to protect from or exacerbate the levels of cell death. This discrepancy is partly due to the array of receptors that androgens may activate during injury. For example, activation of intracellular androgen receptors (AR) leads to the activation of pro-survival pathways and protects from various toxins such beta-amyloid. In contrast, previous studies have demonstrated that testosterone causes an increase in lesion size following stroke. The damaging effects of androgens in the brain may be mediated by a membrane-associated AR (mAR), since activation of mAR in peripheral tissue results in a decrease in cell growth and an increase in apoptotic cell death during serum deprivation. Here, I hypothesize that activation of a mAR in cortical astrocytes, suppresses the ERK and Akt signaling pathways and increases cell death in the presence of a metabolic and oxidative stressor. In this study, we found that glia express both isoforms of the AR (AR-B and AR-A) and that dihydrotestosterone (DHT) elicits ERK and Akt phosphorylation in rat glioma (C6) cells. The effect of DHT on the activation of these signaling pathways is AR dependent, since flutamide blocked this effect. In contrast to the intracellular receptor, we concluded that DHT-BSA (membrane impermeant form of DHT) binds to DHT displaceable sites on the plasma membrane. Also, treatment with DHT-BSA in the C6 cells resulted in a significant decrease in phosphor-ERK and Akt levels, suggesting the existence of two different pathways through which DHT can influence the activity of these signaling pathways. With respect to cell survival, the C6 cells and primary cortical astrocytes were treated with the metabolic and oxidative insult, iodoacetic acid (IAA), in the presence or absence of DHT, DHT-BSA, or estradiol. Following treatment, DHT and estradiol protected the glia from IAA-induced toxicity, whereas DHT-BSA caused a significant increase in cell death in the presence of a sublethal concentration of IAA. These results indicate that activation of the intracellular pathway is protective and activation of a membrane pathway is damage-inducing during injury, further supporting our results from the ERK and Akt signaling studies. To further characterize this mAR in the brain, we decided to look for indices of apoptosis such as caspase activation and TUNEL staining. It was found that DHT-BSA treatment in the presence of IAA, resulted in an increase in caspase-3/7 activation and increased TUNEL staining. In addition, PKC-delta mediated DHT-BSA-induced cell death, since antagonism of PKC-delta with rottlerin afforded protection. In conclusion, we have partially characterized a novel mAR in astrocytes during injury. Here, the damaging effects of androgens, at least in astrocytes, may in fact be mediated by a mAR, which may be a therapeutic target stroke or reperfusion injury.Item The Role of Advanced Glycation End Products in Brain Aging(2007-10-01) Thangthaeng, Nopporn; Michael J. Forster; Tina MachuThangthaeng, Nopporn, The Role of Advanced Glycation End Products in Brain Aging. Doctor of Philosophy (Biomedical Sciences), October, 2007, 178 pp., 9 tables, 6 figures, bibliography, 213 titles. Glycoxidation is a process of post-translational modification of proteins, involving both glycation and oxidation that ultimately generated advanced glycation end products (AGEs). Glycoxidation, which pay promote oxidative stress and disrupt protein structure and function, is hypothesized to be responsible for pathological conditions related to aging, diabetes, neurodegenerative diseases, and degenerative ophthalmic diseases. Previous studies have demonstrated that AGEs accumulate in the brains of aged animals and humans, yet few studies have directly addressed the possibility that AGEs are a cause of age-related brain dysfunction. Therefore, the overall purpose of the present studies was to examine the role AGEs in normal brain again and the associated decline in cognitive and psychomotor function. In order to achieve the goals, two different approaches were taken. The first approach involved (i) determining whether or not AGEs accumulated in different regions of the brain as a function of age and (ii) determining whether these changes were correlated with individual differences in the ability of old mice to perform in tests of cognitive and psychomotor function. Age-associated accumulation of CML, a predominant form of AGEs in vivo, and expression of receptor for AGEs (RAGE) protein, inferred from densitometry quantification of immunoblots in different regions of the brain, were assessed by comparing groups of 8-or 25-month old mice. The 25-month-old mice were administered a series of behavioral tests to assess cognitive and psychomotor function prior to assessment of glycation status. In the second approach, groups of mature (6 mos) and older mice (18 mos) were fed with a control diet or a diet enriched with galactose (49% of caloric content), an intervention that was expected to promote formation of AGEs. The mice were subsequently tested for impairment of their cognitive and psychomotor functions after 8 weeks on the assigned diet. Upon completion of the behavioral tests (after 14 weeks on diet), amounts of CML and RAGE protein were assessed through densitometric analyses of the immunoblots. The main findings from the first approach were that (i) there was a robust increase in CML content and expression of RAGE protein in the aged mouse brain that occurred in a region-specific manner; (ii) the relative amounts of CML and RAGE were not closely associated with the degree of age-related impairment of mice tested for brain function. The main findings from the second approach were that high dietary galactose: (i) failed to induce aged-like behavioral impairments in young/mature mice; (ii) exacerbated age-related impairment of some psychomotor functions and (iii) had no significant effects on glycation status or oxidative damage. Comparison of the experimental outcomes from the first and second approaches was complicated by a difference in the fat content of the diets fed to the mice in the two studies, which had an apparent effect on the amounts of AGEs and protein oxidation present in young mice. However, considering the results of the two studies independently warrants the following conclusions: (i) Amounts of AGEs do not predict individualized brain aging as assessed by neurobehavioral impairment and may instead by largely reflective of chronological age. (ii) Diets enriched with galactose may produce deleterious effects in older mice that do not involve a change in oxidative damage or glycation status. Overall, these studies provide little support for a specific role of glycoxidation in normal brain aging. It is impossible that the extent of accrual of AGEs in the normally aging brain is insufficient to affect cellular function, whereas larger accumulations of AGEs may be associated with various pathological conditions discussed in the literature.