Nancy A. Muma - US grants

Alzheimer's disease (AD), the most common cause of dementia in middle and late life, is characterized by progressive impairments in memory, language, visuospatial perceptions, and behavior. Clinical criteria have been defined for possible and probable AD, but a diagnosis of AD requires the histological demonstration of neurofibrillary tangles and senile plaques. The major constituent of neurofibrillary tangles, neuropil threads, and neurites, is paired helical filaments (PHF), which contain modified tau proteins. Normally, alternative splicing of messenger ribonucleic acid (mRNA) transcribed from a single gene gives rise to variations in tau isoform expression. In the present proposal, variations in tau isoform expression will be examined in two models of neuronal injury (i.e., axonal transection with and without subsequent regeneration, and aluminum-induced neurofibrillary abnormalities), using a combination of in situ hybridization, RNA blotting, and quantitative polymerase chain reaction procedures. The roles that tau isoform expression play in these animal models will lend insight into the functions of various isoforms. Furthermore, the examination of alterations of tau isoform expression that involve-simple models of neuronal injury will lay the foundation for the study of tau in more complex models and, eventually, for studies of this class of protein in aging and neurodegenerative diseases, including AD. The overexpression of mRNA-encoding tau or any of the tau isoforms could contribute to the formation of PHF seen in AD. Structural and functional modulation of proteins is frequently regulated via alternative splicing, therefore, differential involvement of tau isoforms in PHF is quite likely. Studies such as these, which elucidate factors that lead to the formation of abnormal structures in AD, should be useful in the development of new treatment strategies for the cytoskeletal abnormalities that occur in individuals with AD.

DESCRIPTION (Applicant's abstract): This proposal will examine the role that regulatory G protein signaling (RGS) proteins play in modulating G-protein linked signal transduction in vivo. RGS proteins are a novel family of proteins that increase the hydrolysis of GTP bound to the G-alpha subunit and reduce the activation of the second messenger system. Thus, changes in the levels of RGS proteins may mediate increases or decreases in the responsiveness of G protein-coupled receptors. We have demonstrated that overexpression of one particular RGS protein, RGS4, can induce desensitization of one member of the 5-HT2 receptor family in cells in culture. However, it is not known whether RGS4 protein has the same effect on 5-HT2 receptors in mammalian brain in vivo. Antisense oligodeoxynucleotide strategies will be used to test the hypothesis that RGS4 protein regulates 5-HT2A receptor signaling in the hypothalamus. We will test the regulation of 5-HT2A receptor adaptation in the hypothalamic paraventricular nucleus, because it contains abundant levels of RGS4 protein and 5-HT2A receptors, 5-HT2A receptors in this nucleus can be desensitized or become supersensitive with several treatments, and because hormone responses to agonist treatment can be used as a sensitive measure of receptor signaling in the hypothalamus. We will use antisense oligonucleotide strategies to reduce the levels of RGS4 protein to 1) cause supersensitivity of signaling and 2) attenuate the desensitization of 5-HT2A signal transduction, induced by chronic treatment with the 5-HT2A/2C agonist DOI. Lastly, our preliminary data suggest that RGS4 protein expression is decreased during fluoxetine-induced 5-HT2A receptor supersensitivity. Our studies have ruled out several possible mechanisms and have led to the hypothesis that a decrease in the levels of RGS4 proteins could mediate this supersensitivity. Thus, we will examine the correspondence between the time course of the reduction in RGS4 protein expression and of fluoxetine-induced 5-HT2A receptor supersensitivity. The results of the proposed studies will provide new insight into the function of RGS proteins in vivo, and the mechanisms regulating 5-HT2A receptor systems. Given the importance of G protein signaling in mammalian brain, it is essential to understand the regulation of RGS proteins. Because of the prominent role that 5-HT2A receptors play in the etiology of neuropsychiatric disorders, understanding the regulation of adaptive changes in 5-HT2A signaling by RGS proteins will lay the foundation for new targets for therapeutic intervention for these disorders.

DESCRIPTION (provided by applicant): The long-term goal of our studies is to identify the molecular mechanisms of desensitization of serotonin 2A (5-HT2A) receptor signaling. Adaptive changes in post-synaptic 5-HT2A receptor signaling underlie the mechanism of action of several drug treatments for psychiatric disorders including anxiety, schizophrenia, depression, and bipolar disorder. Paradoxically, chronic treatment with both 5-HT2A receptor agonists such as DOI and antagonists including olanzapine desensitize 5-HT2A receptor signaling. Atypical antipsychotics are 5-HT2A receptor antagonists, and although these drugs are widely used, significant numbers of individuals are refractory to drug therapy. To help to resolve this paradox and identify novel targets that regulate 5-HT2A receptor signaling, we plan to investigate the mechanisms by which 5-HT2A receptor signaling is desensitized. We will examine the mechanisms of desensitization of 5-HT2A receptor signaling induced with 5-HT2A receptor agonists (Aim 1), and 5-HT2A receptor antagonists (Aim 2) in cells in culture and in vivo. Our central hypothesis is that agonists induce post-translation modifications while antagonists induce transcriptional changes to cause adaptational changes in 5-HT2A receptor signaling. We will build on our preliminary findings that 5-HT2A receptor agonists induce post- translational modifications to G proteins while chronic treatment with a 5-HT2A receptor antagonist increases RGS7 protein expression via increases in JAK/STAT signaling (STAT being a transcription factor). In addition to modern molecular biology approaches, we use neuroendocrine responses to 5-HT2A receptor-stimulation as an index of desensitization of 5-HT2A receptor signaling in vivo. A major advantage of the neuroendocrine challenge tests is that the results obtained in experimental animals can be rapidly applied to humans since these tests can be performed in humans. Ultimately the purpose of these studies is to identify new targets for therapeutic intervention for psychiatric disorders currently treated with drugs that alter 5-HT2A receptor signaling. By understanding the mechanisms involved in signaling and neuroadaptation, new approaches can be developed to reduce the delay in therapeutic response with antipsychotic and antidepressant therapies and treat individuals refractory to current therapies. PUBLIC HEALTH RELEVANCE: Adaptive changes in post-synaptic 5HT2A/2C receptor signaling may underlie the mechanism of action of several drug treatments for neuropsychiatric. For example, several antipsychotic drugs, such as olanzapine, desensitize both 5-HT2A and 5-HT2C receptors while 5- HT uptake blockers alter the efficacy of 5-HT2A receptor signaling. However, the molecular mechanisms that underlie these adaptive changes in 5-HT2A receptor signaling are not well understood. The purpose of this proposal is to determine the mechanisms involved in the adaptational responses to 5-HT2A receptor agonists and antagonists. By discovering the molecular mechanisms underlying signaling and desensitization of 5-HT2A receptor signaling, new targets for therapeutic intervention will be identified.

The long term objectives of the proposed studies are to provide novel approaches towards treating women who suffer from hot flashes and mood disorders during menopause. Desensitization of serotonin 1A (5-HT1A) receptor signaling both in midbrain and forebrain regions (hypothalamus, amygdala) may be an underlying mechanism for the therapeutic effects of estrogen and of Selective Serotonin Reuptake Inhibitors (SSRIs), such as fluoxetine (Prozac (r) for depression, anxiety and hot flashes in post-menopausal women. However, unlike SSRIs, which require 7-14 days to induce a therapeutic response and a desensitization of 5-HT1A receptors, estrogen can produce a desensitization of 5-HT1A receptors within 2 days of administration. This competitive renewal will investigate the mechanisms responsible for the desensitization of post-synaptic hypothalamic 5-HT1A receptor systems during treatment with estrogen. The focus of the studies is on regulatory G protein signaling-Z1 (RGSZ-1), a protein that acts as a Galphaz protein GTPase Activating Protein (Gz-GAP) that regulates the interaction between 5-HT1A receptor-coupled Galphaz proteins and effector systems (such as adenylyl cyclase or potassium channels). The overall hypothesis is that estrogen-induced desensitization of 5-HT1A receptor signaling is due to increased expression of RGSZ-1. The following aims will test this hypothesis. Specific Aim 1 will test the hypothesis that estrogen receptor-beta (ER beta) mediates the increased expression of RGSZ-1 protein and mRNA and desensitization of the 5-HT1A receptors. Specific Aim 2 will test the hypothesis that up-regulation of RGSZ-1 mediates the estrogen-induced desensitization of hypothalamic 5-HT1A receptors. Specific Aim 3 will test the hypothesis that a treatment combining SSRIs (such as fluoxetine) with estrogen leads to activation of two complementary mechanisms (reduced Galphaz proteins and increased RGSZ-1 proteins, respectively), leading to a synergistic desensitization of 5-HT1A receptor signaling. The proposed studies may lead to novel targets for medications which affect the regulation of RGSZ-1 to treat women who suffer from hot flashes and mood disorders during menopause

? DESCRIPTION (provided by applicant): Huntington's disease (HD) affects one in every 10,000 people in the US, with a current estimate of ~30,000 patients in the US. HD is a progressive neurodegenerative disease caused by an autosomal dominant mutation coding for a polyglutamine expansion in huntingtin protein. Although the cause of the disease has been identified and genetic tests are available to identify those individuals who carry the mutation and will succumb to the disease, there is currently no therapy to slow or prevent the disease progression, only symptomatic treatments with limited impact. The purpose of the proposed studies is to identify biological probes which can be used for future drug development and eventual testing for their ability to prevent the progressive neurodegeneration in HD. We previously reported in vitro and in vivo proof of concept that disrupting the interaction of mutant huntingtin (mhtt) with calmodulin (CaM) is an outstanding target for treatment in models of HD. We demonstrated that disrupting the binding of mhtt to CaM, with a 46 amino acid peptide, consisting of amino acids 76-121 of CaM, was protective in HEK293 cells transiently expressing a mhtt protein construct, in neuronally differentiated SH- SY5Y cells stably expressing a mhtt protein construct and a transgenic mouse model of HD, R6/2 mice. The goal of the current proposal is to identify selective small molecule inhibitors to disrupt the binding of mhtt to CaM. We have developed and validated a HTS ready, AlphaScreen(r) assay to identify compounds which disrupt the binding of mhtt to CaM. Orthogonal screens will be used to test for selectivity using in vitro assays to test for disruption of the interaction of CaM with other proteins and a cel culture-based assay to evaluate cytotoxicity of the compounds. {Cheminformatic analysis will identify a set of prioritized chemotypes that will be subjected to iterative medicinal chemistry optimization.} Lastly, tertiary screens in a cell culture model of HD will be used to determine whether the chemical probes are neuroprotective against mhtt and disrupt mhtt-CaM interactions in cells. The goal of this project is to identify compounds that disrupt the binding o mhtt to CaM and do so selectively without inhibiting the function of CaM. The top compounds will further protect against both the deleterious effects of mhtt in neuronal cells and the transamidation of mhtt in neurons. The long-term objective of the project, outside of the scope of this proposal is to develop compounds that are protective against the neurodegeneration and other deleterious effects of mhtt in transgenic mouse models of HD and eventually lead to translational clinical testing for HD.