Now showing items 1-7 of 7

• #### Central Nervous System Regulation Of Metabolic Suppression In Arctic Ground Squirrels

The main focus of this dissertation is central nervous system regulation of metabolic suppression in hibernating mammals in general, and the Arctic ground squirrel (Urocitellus parryii) as a model for seasonal hibernation. Hibernation is a unique physiological, morphological, and behavioral adaptation to overcome the periods of resource limitation. Metabolic suppression seen in torpor during hibernation has several biomedical applications. A multitude of studies have revealed the role of the central nervous system in regulating hibernation, including a role for neurotransmitters and neuromodulators. Previous studies have shown that the neuromodulator adenosine mediates altered thermoregulation during induction of torpor in facultative hibernators, but it is not clear how adenosine influences torpor in seasonal hibernators. The main focus of the current project was to test the hypothesis that a seasonal change in purinergic signaling is necessary for the onset of spontaneous torpor in the Arctic ground squirrel. My dissertation reports that adenosine meets all of the necessary requirements for an endogenous mediator of torpor in the hibernating Arctic ground squirrel. A progressive increase in sensitivity to adenosine A 1 receptors mediated signaling defines the seasonal transition into the hibernation phenotype. I show that adenosine A1 receptor activation is necessary and sufficient to induce torpor in the Arctic ground squirrel. Glutamate is an excitatory neurotransmitter which is widely studied in hibernation research. My dissertation demonstrates that N-methyl-D-aspartate type glutamate receptors, located in the periphery or circumventricular organs, are involved in inducing arousal from torpor in the hibernating Arctic ground squirrel. This dissertation also presents evidence that dietary restriction sensitizes adenosine A1receptors in rats through an increase in surface expression in thermoregulatory regions of the brain (hypothalamus). This contributes to the decline in body temperature and respiratory rate in animals subjected to a restricted diet, which mimics a torpor-like effect.
• #### Ceramide Metabolism Regulates A Neuronal Nadph Oxidase Influencing Neuron Survival During Inflammation

Inflammation is a major component of acute and chronic pathologies of the central nervous system, including psychiatric disorders. Microglia respond to pathogens, injury, and toxins by secreting inflammatory mediators including pro-inflammatory cytokines in an event known as neuroinflammation. This thesis research investigated a link between neuroinflammation and oxidative stress, and ultimately neurodegeneration. The cytokine tumor necrosis factor alpha was shown to stimulate a neuronal NADPH oxidase (NOX), specifically by stimulating the production of ceramide and ceramide-1-phosphate via Mg 2+-neutral sphingomyelinase (Mg2+-nSMase) and ceramide kinase. Intriguingly, glucosylceramide blocked NOX activation, linking ceramide neutralization directly to a decline in oxidative stress. Most importantly, NOX activity interfered with actin and sphingosine kinase-1 via oxidation, demonstrating a positive and detrimental feedback mechanism that impedes neuronal survival pathways. Interestingly, crude extracts from wild Alaskan bog blueberries showed the ability to interfere with Mg2+-nSMase, demonstrating a specific neuroprotective property of the berry. Altogether, this thesis research defined a key neuronal pathway linking inflammation to oxidative stress via ceramide metabolism, potentially allowing for future therapeutic development to improve neuronal function and survival.
• #### Investigation Of The Allosteric Modulators Desformylflustrabromine And 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid (Hepes) Interactions On Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop super family of ligand gated ion channels. Dysregulation of nAChRs can lead to pathologies such as Alzheimer's disease, Parkinson's disease, Autism and nicotine addiction. Possible new therapeutic avenues are positive allosteric modulators (PAMs). The natural product desformylflustrabromine (dFBr), a tryptophan metabolite of the marine bryozoan Flustra foliacea, was found to be PAM of alpha4beta2 nAChR. Evaluation of our synthetic water soluble dFBr salt by two-electrode voltage clamp of Xenopus laevis oocytes expressing human nAChR confirmed that synthetic dFBr displayed similar properties as the natural product. Low concentrations of the synthetic dFBr enhanced ACh's efficacy on alpha4beta2 receptors. At higher dFBr concentrations, dFBr inhibited ACh potentiated responses. On alpha7 receptors, dFBr inhibited ACh induced currents. Further pharmacological characterization of dFBr revealed that dFBr was able to enhance partial agonist potencies and efficacies. Evaluation of dFBr on antagonists showed no effect on antagonist inhibition. The mechanisms of biphasic modulation (potentiation and inhibition) of dFBr on alpha4beta2 nAChR were also investigated. Enhanced efficacy of ACh induced currents by dFBr appeared to be accomplished by dFBr stabilization of the open receptor conformation by destabilization of the desensitized state. The inhibition of ACh potentiated currents by dFBr appeared to involve open-channel block. To better understand dFBr mechanisms, its putative binding site was examined. Alanine mutations were made in non-orthosteric clefts on the beta2+ and alpha4- faces. Results revealed residues located on these faces are involved in ACh induced conformational change of the receptor. In addition our data supports our hypothesis that allosteric modulation by dFBr interacts with residues located on the beta2+ and alpha4- faces. The new novel actions of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) as a alpha4beta2 stoichiometric PAM was discovered and characterized. We showed that HEPES, a common buffering agent, potentiated the high ACh sensitivity alpha4beta2 receptor while only inhibiting the low ACh sensitivity alpha4beta2 receptor. Mutagenesis results suggested that residue beta2D217 is a critical residue in the HEPES binding site. Results from these studies will aid in the development of therapeutic ligands that will assist in the treatment of diseases where nAChRs are dysregulated.
• #### Investigations into model systems of neurodegeneration: Organotypic brain slice culture and in vivo microdialysis

The mechanisms behind neurodegeneration in disease and injury have yet to be fully defined. Many in vitro and in vivo model systems, have been developed to investigate the mechanisms of neurotoxicity and its relation to human disease and injury. There are a few resounding connections between most types of neurological disorder; namely oxidative stress and inflammation. The glutamate receptor agonist, N-methyl-D-aspartate, can be used to imitate excitotoxicity during stroke as it overstimulates the glutamate receptor, leading to rises in intracellular calcium levels, which in turn lead to oxidative stress within the cell. Amyloid-beta protein (Abeta) a useful in many of its isoforms in creating in vitro model systems of Alzheimer's disease (AD). Abeta can directly cause the production of potentially harmful free radicals. This study investigates the formation of model systems of neurodegeneration: in vivo microdialysis and organotypic brain slices culture in order to assess the role of oxidative stress and inflammation morphologically and biochemically. The effect of melatonin, an endogenous antioxident, on oxidative stress associated with NMDA and Abeta neurotoxicity was determined through morphological analysis and biochemical markers of oxidative stress. This study reports that both NMDA and Abeta(25--35) cause oxidative stress in an organotypic brain slice culture model system of stroke and Alzheimer's disease as established by: (1) morphological analysis of tissue and ultrastructure, (2) redox-active assay, (3) heme-oxygenase assay, (4) 8-hydroxyguanosine assay and (5) interleukin IL-1beta and IL-6 assay (Abeta only) These investigations also demonstrate that melatonin can attenuate the oxidative stress associated with NMDA and Abeta exposure. These findings expand upon previous evidence from cell culture analysis of oxidative stress induced by NMDA and Abeta. Therefore, this evidence supports the theory that oxidative stress is involved in neurodegeneration in both excitotoxicity in stroke and in Abeta-mediated damage in Alzheimer's disease, and that endogenous antioxidant treatment may be a useful therapeutic approach in such injury and disease.
• #### Nmda Receptors In Hibernating Arctic Ground Squirrels

Hibernation is a unique physiological state characterized by suppressed metabolism and body temperature that is interrupted by multiple, brief periods of arousal throughout the hibernation season. Blood flow fluctuates during hibernation and arousal in a reperfusion-like manner without causing neurological damage. Previous studies show that hippocampal slices from hibernating animals tolerate experimental oxygen nutrient deprivation and N-methyl-D-aspartate (NMDA) toxicity better than slices from euthermic animals. However, the cellular mechanisms underlying these examples of tolerance remain unclear. Tolerance to NMDA toxicity suggests that modulation of NMDA receptors (NMDAR) contributes to intrinsic tissue tolerance in slices from hibernating Arctic ground squirrels (hAGS, Spermophilus parryii). NMDAR are one subtype of glutamate receptors. NMDAR play critical roles in excitatory synaptic transmission, synaptic plasticity, learning and memory, and excitotoxicity. NMDAR1 (NR1) is a fundamental subunit of NMDAR and required for receptor function. The main focus of the current project was to test the hypothesis that NMDAR are down-regulated in hAGS compared with interbout euthermic AGS (ibeAGS) and to explore the potential mechanisms of this down-regulation. NMDAR function can be modulated by protein phosphorylation, subunit composition, and internalization. Hence, the aim of chapter 2 was to determine the distribution of NRl in hAGS and ibeAGS using immunohistochemistry. The aim of chapter 3 was to examine NMDAR function in cultured hippocampal slices from hAGS, ibeAGS, and rats using calcium imaging, and to investigate potential modulation of NMDAR such as phosphorylation and internalization for altered function using western blot analysis. Given that synaptic remodeling and functional changes after arousal from hibernation, and NMDAR play an important role in learning and memory, the aim of chapter 4 was to address the effects of hibernation on learning and memory in AGS using an active avoidance task. Here, we report that NMDAR in hAGS are down-regulated via decreased phosphorylation of NR1. This down-regulation is not due to changes in NR1 distribution and internalization. In addition, the fraction of NR1 in the functional membrane pool in AGS is less than in rats. These findings provide evidence that modulation of NMDAR contributes to neuroprotection observed in hAGS.
• #### Pharmacology Of A Novel Class Of Allosteric Modulators For The Alpha4 Beta2 Sub-Type Of Neuronal Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of a large family of ligand gated ion channels that mediate inhibitory and excitatory neural transmission in the central nervous system (CNS). The nicotinic subfamily has been implicated in a range of neurological disorders including autism, Alzheimer's disease and nicotine addiction; diseases that are currently both challenging and costly to treat. Despite the apparent importance of nAChRs in these disorders, only a limited number of drugs are currently available for altering nicotinic signalling in the CNS. No drug therapies are currently available that specifically target autism and only a limited number of drugs are available for the treatment of Alzheimer's disease. This thesis presents a novel class of nAChR ligands based on the natural product desformylflustrabromine (dFBr). Desformylflustrabromine (dFBr), a metabolite of the marine bryozoan Flustra Foliacea, was previously identified as an allosteric modulator of the alpha4beta2 subtype of nAChRs. In collaboration with Dr. Richard Glennon at the Virginia Commonwealth University, College of Pharmacy, we developed a synthetic dFBr and evaluated its interaction with two of the most common subtypes of nAChRS, alpha7 and alpha4beta2 (Chapter 2). We confirmed that dFBr is the active component of Flustra Foliacea and identified an additional inhibitory action that becomes evident as dFBr concentrations are increased beyond 10muM. This inhibition was not previously reported. Synthetic dFBr appears significantly more potent at potentiation of alpha4beta2 receptors then reported for the natural extract and shows only inhibitory action on alpha7 receptors. Multiple analogues of dFBr were designed and synthesized to determine the structure activity relation (SAR) for dFBr's action on alpha4beta2 receptors (Chapter 3). We identified three analogues capable of potentiating responses of acetylcholine. The majority of compounds inhibited responses on both alpha4beta2 and alpha7 receptors. The data presented here provide important information for determining a preliminary pharmacophore for dFBr and provide direction for the design of additional analogues on the path to development of more potent and potentially therapeutically useful analogues. To better understand the relationship of dFBr to other nAChR modulators, we also compared the action of dFBr to that of physostigmine, zinc and 17 beta-estradiol (Chapter 4). These compounds are thought to act at three different binding sites on nAChRs. All three compounds increase responses of alpha4beta2 receptors to acetylcholine. Our data show that dFBr is distinct from the clinically used modulator physostigmine but suggests similarities in mechanism with zinc and 17-beta-estradiol. These data provide important information regarding the mechanism of dFBr modulation and provide direction for future site directed mutagenesis studies that will identify the dFBr binding site. Identification of the binding site is critical for the development of receptor models that will facilitate computer assisted drug design.
• #### Stabilization of secondary structure of synthetic Alzheimer beta-amyloid protein analogs in the presence of aluminum (III) ions

The gradual accretion of fibrillar protein deposits in a tissue or organ is a hallmark of all amyloidogenic diseases. These deposits accumulate as senile plaques and cerebrovascular deposits in the brain and are characteristics of Alzheimer's disease. A majority of the brain amyloid deposits consist of a 40 amino acid protein, the Alzheimer $\beta$-protein, A$\beta$P, which in a soluble form is ubiquitous in biological tissues. In order to provide a more detailed understanding of the structural transformations of soluble A$\beta$P, sequence analogs derived from $\beta$1-40, and having His $\to$ Arg, and scL-Asp- $\to$ scD-Asp substitutions were synthesized. The kinetic variations of $\beta$1-40 and $\beta$6-25 were studied using amide circular dichroism spectroscopy by monitoring ellipticity changes of the peptide backbone. In both peptides, the gradual loss of secondary structure was a multiphasic process which was also dependent on concentration. The circular dichroism titrations with metal ions revealed the involvement of at least two ions in the conformational transitions of $\beta$1-40 and $\beta$6-25. The association of Al(III) with scL-Asp $\to$ scD-Asp derived analogs caused surprising conformational changes in $\beta$6-25, which were distinct from $\beta$1-40. Microheterogeneous products corresponding to Al(III)-bound peptide species were resolvable on the reversed-phase surface. The association of aluminum was investigated by low field $\sp{27}$Al nuclear magnetic resonance spectroscopy. The signal corresponding to Al(III)-bound peptide species revealed that at least four Al(III) ions were bound to $\beta$1-40 and $\beta$6-25 between pH 5 and 6. Moreover, $\beta$1-40 effectively competed with EDTA to bind with Al(III). This study also describes a strategy which resolved the band broadening in reversed-phase high-performance liquid chromatography of $\beta$1-40 and derived analogs. Chromatographic parameters related to interactive contact area of $\beta$1-40 and derived analogs were determined on reversed-phase matrix. The peptides were bound to the reversed-phase surface in their monomeric form. Slow partition kinetics appear to contribute to significant band broadening, which suggests a secondary retention effect--indicating a conformational change due to unfolding on the stationary phase surface.