Karen O'Malley - US grants

The goal of this project is to investigate genetic mechanisms involved in neuronal plasticity. Specifically, I propose to examine, at the molecular level, mechanisms involved in regulating neurotransmitter genes in dissociated sympathetic ganglion cultures. A cloned probe for tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, will be used to study the regulation and expression of its gene in vitro. Tyrosine hydroxylase mRNA levels and rate of transcription will be determined under various conditions known to influence the enzyme levels. In particular, I will determine the effects of nerve growth factor, glucocorticoids, elevanted K+ and various other factors on synthesis rates and mRNA levels for this enzyme. This data will be correlated with tyrosine hydroxylase enzyme levels determined in matched cultures. These studies will be extended to investigate the regulation of tyrosine hydroxylase under conditions inducing a cholinergic phenotype. Experiments will focus on what role conditioned medium, glucocorticoids and epidermal growth factor play in eliciting this phenotypic switch. Also, I will examine whether these are post-transcriptional or post-translational effects using the TH cloned probe and enzyme number determinations. Results from these studies will further our knowledge of how cells become committed to differentiation and what are some of the factors involved.

The PI is proposing to utilize a novel expression system (baculovirus vectors) to determine the functional significance of the various isoforms of tyrosine hydroxylase. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis and, as such, is a crucial and pivotal point in many physiological processes. Recently, the PI's laboratory has isolated full length cDNAs of the multiple human subtypes. To investigate whether these different isotypes have altered functions, the PI will subclone the cDNAs into the baculovirus expression vectors. This system will generate large quantities of the different forms of TH to permit kinetic and structural analyses. This system will also provide the means to use site- directed mutagenesis to assess the role of particular amino acids in phosphorylation, catalysis, cofactor binding, etc. Results from these studies will delineate structural requirements for TH regulation and expression and will help understand mechanisms of activation and possible roles in atypical catecholamine function.***//

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand at the molecular level the factors involved in the tissue-specific expression of neuronal genes. In this application the investigators propose to use this information in conjunction with advances in molecular genetics to create animal models of Parkinson's Disease (PD) in which gene expression is spatially and temporally controlled. A bottleneck in taking full advantage of these powerful tools however, lies in the lack of appropriate promoters to target genetic switches to specific populations of neurons. Thus, a prerequisite for using genetic manipulation to unravel brain function is the development of new tools to target brain regions of interest. Because preliminary experiments indicate that the reporter gene, eGFP, can be targeted to the tyrosine hydroxylase (TH) locus where it is co-expressed with endogenous TH, the investigators have hypothesized that the TH locus can be combined with inducible elements to generate a targeted switch in vivo. If subsequent experiments show that catecholaminergic properties have not been changed in the TH/eGFP heterozygotes, the tetracyline (TET) regulatory system transactivator derivative, rtTA2s-M2, will be swapped for eGFP using homologous recombination. TH/TET transactivator lines will be evaluated by crossing with TET-responsive reporter gene mouse lines. If reporter genes are appropriately regulated, these animals will serve as an invaluable resource for turning gene expression on and off in catecholaminergic cells. A second major conceptual goal is to use this resource to develop an animal model of Parkinson's disease. Defects in the ubiquitin-proteosome pathway have been genetically linked to Parkinson's disease including a loss of function of the E3 ligase, Parkin. The co-chaperone CHIP is a positive regulator of Parkin mediating its ability to ubiquitinate its substrates. Dominant-negative (DN) CHIP derivatives block endogenous CHIP activity contributing to impaired proteosome function, increased levels of aberrant proteins, and unfolded protein response. In order to test the hypothesis that overexpression of DN-CHIP in catecholaminergic cell types can lead to an animal model of Parkinson's Disease, transgenic lines carrying a TET-responsive DN-CHIP minigene will be generated and crossed with the TH-driven TET transactivator. Offspring will be evaluated for induction of unfolded protein response (BiP, CHOP10, caspase 12 induction) and the loss of catecholaminergic cells. Conceivably, these animals will serve as a useful model of Parkinson's disease and as an aid in determining the underlying mechanisms of this response. Knowledge gained from these studies may offer new routes for therapeutic intervention.

DESCRIPTION (Applicant's Abstract): The long term goal of this project is to define the consequences of the interaction of cocaine with presynaptic dopaminergic systems. Many of the behavioral effects of cocaine are attributed to its ability to block the reuptake of dopamine in mesolimbic neurons. Consequently, increased extracellular dopamine may enhance postsynaptic transmission and/or modulate presynaptic dopamine receptors known to inhibit neurotransmitter synthesis as well as further release. The specific aim of this application is to dissect this system by focussing on presynaptic events associated with cocaine's purported modulation of dopamine autoreceptors. Towards this end, model neuronal systems have been engineered by transfecting immortalized mesencephalic dopamine producing cell lines with cloned D2 and D3 receptors. To test the hypothesis that the D2-like receptors subserve different autoreceptor roles, the transfected cell lines are being systematically tested for receptor mediated effects on synthesis and release. Previously we have shown that agonist stimulation of D2 and D3 receptors leads to subtype specific reductions in dopamine release and synthesis. These data imply specific roles for each receptor and suggest the effects of cocaine may vary depending upon receptor subtype. Studies outlined in this proposal will 1 ) test the hypothesis that the differential autoreceptor effects of D2 and D3 receptors are due to distinct coupling mechanisms; 2) determine the mechanistic basis for D3 desensitization; 3) test the hypothesis that in primary cultures of neurons the dopamine D2 receptor subtype can serve all these functions: modulation of firing rate, dopamine synthesis and dopamine release; and 4) test the acute and chronic effects of cocaine on autoreceptor function in transfected cell lines and primary dopaminergic cultures. Taken together these studies will significantly extend our ongoing efforts to understand the complex receptor/presynaptic effector interactions involved in the reinforcing and behavioral sensitization effects of cocaine.

DESCRIPTION (provided by applicant): Oxidative stress is a major factor in Parkinson's Disease (PD). Dopamine (DA) itself is easily oxidized to quinone derivatives and reactive oxygen species (ROS) that impair energy metabolism and form adducts with proteins such as upsilon-synuclein. Because pharmacological depletion of DA in animal models is confounded by non-specific peripheral and central nervous system effects, the role of DA oxidation in nigral cell death has been previously impossible to address. Thus a key unanswered hypothesis in this field is that DA oxidation is a major contributor to the death of dopaminergic neurons in PD. The proposed studies address several aspects of this hypothesis including the interaction of known environmental factors in triggering DA oxidation. Specifically, the hypothesis that the DA-releasing potential of the parkinsonism-inducing drug, MPP+, is due to its ability to exchange with DA and/or to reduce intracellular pH gradients will be addressed using newly derived mice expressing enhanced green fluorescent protein from a dopaminergic locus (TH+/eGFP). Primary cultures derived from these animals as well purified synaptosomal and vesicular preparations from dopaminergic terminal fields will be used in combination with fluorescent and radioactive probes to determine the temporal aspects of DA release, intracellular membrane changes, ROS formation, ATP loss, etc in response to toxin treatment. In addition, the hypothesis that DA oxidation contributes to the death of dopaminergic cells will be directly tested in vivo using animals genetically engineered to have different levels of DA production. Behavioral, oxidative and immunocytochemical criteria will be used to establish the role of DA in both the acute and chronic MPTP model of PD. To test whether DA depletion prevents ROS, new methodologies to detect in situ ROS will be used with a battery of antibodies directed against nitrotyrosine, nitrated alpha-synuclein, etc. to temporally evaluate ROS formation following acute or chronic MPTP administration in DA deficient and wild type animals. Taken together, the proposed studies will determine whether DA oxidation plays a central role in the death of DA synthesizing cells and provide insights impossible to obtain from standard animal models. Knowledge of the source and cascade of events surrounding DA-induced free radical formation will help answer risk-benefit controversies surrounding the use of dopamine replacement therapies as well as facilitate the development of new drugs and/or treatment strategies in the pathogenesis of PD.

neuroprotectants; oxidative stress; Parkinson's disease; dopamine; neurons; cell death; free radical scavengers; nonhuman therapy evaluation; methylphenyltetrahydropyridine; disease /disorder model; biological signal transduction; free radical oxygen; 6 hydroxydopamine; apoptosis; tissue /cell culture; laboratory rat; calcium indicator; dyes; behavior test; immunocytochemistry; stereotaxic techniques;

DESCRIPTION (provided by applicant): This is an application for renewal of the Systems and Molecular Neurobiology Training Program (T32 GM08151) in the Division of Biology and Biomedical Sciences at Washington University. This grant provides critical support for graduate education in the Neuroscience Program, which is one of the finest in the world. At its heart are long-standing commitments to excellence in research, to interdisciplinary education, and to providing its students with superb training in their courses and in the laboratory. It is also an especially broad program, combining expertise in molecular, cellular and systems-level approaches to the study of neural function and dysfunction. Continued diversification has allowed it to remain at the forefront of developments on many different research areas. Continued innovation makes the educational program an exciting one.

[unreadable] DESCRIPTION (provided by applicant): Nuclear Ca2+ plays a critical role in many cellular functions although its mode (s) of regulation is unclear. Recently, we have shown that the metabotropic glutamate receptor, mGlu5, mobilizes nuclear Ca2+ independent of cytosolic Ca2+ regulation. Immunocytochemical, ultrastructural, and subcellular fractionation techniques revealed that the metabotropic glutamate receptor, mGlu5, was localized to nuclear membranes in heterologous cells as well as midbrain and cortical neurons. Nuclear mGlu5 receptors expressed in heterologous cell types could bind agonist and isolated nuclei responded to agonist with rapid, oscillatory [Ca2+] elevations that were blocked by antagonist. Because these results challenge existing paradigms as to the role of intracellular receptors and have important ramifications in intracellular signaling, they are applicable to the purpose of the R21 mechanism. The goal of the current application is to establish a native, physiological system with which to test these novel concepts further. Because preliminary results indicate that mGluR5 receptors are expressed on both nuclear and plasma membranes in striatal cultures, these preparations will be used to examine the hypothesis that nuclear mGlu5 receptors can regulate nuclear Ca2+ in neurons. Specifically, cultures will be loaded with a calcium indicator whose spatio-temporal distribution will be analyzed in the confocal microscope. Following treatment with specific agonists and antagonists, cultures will be fixed, stained, and field re-located to determine whether nuclear oscillatory responses, if any, are associated with nuclear mGlu5 receptors. Striatal nuclei, isolated in situ, will be analyzed in a similar fashion. Subsequent experiments will address what ligand is activating nuclear receptors and how it is doing so. Given that mGlu5 receptors play pivotal roles in synaptic plasticity, neuronal development and modulation of synaptic transmission, the mechanisms by which they do so are of critical importance. Direct nuclear calcium regulation represents a novel signaling strategy by which intracellular receptors such as mGlu5 may play a pivotal role in generating and shaping intracellular Ca2+ signals [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Metabotropic glutamate receptors (mGluRs) are a diverse and abundant class of G protein-coupled receptors (GPCRs) that mediate the transmitter and modulatory actions of glutamate, the most abundant and ubiquitous transmitter in the vertebrate CNS. Based on the important role these receptors have in synaptic plasticity, neuronal development, and the modulation of synaptic transmission, it is likely that appropriate functioning of these receptors is a critical determinant of the cell biology underlying mental health and disease. In this application, we propose a series of experiments to continue investigating the structural basis and functional consequences of mGluR dimerization. We discovered this phenomena, shown it is due to a homodisulfide between cys129 of two mGlu5 polypeptides, and have demonstrated that, surprisingly, covalent dimerization is not critically essential for agonist binding or signal transduction through mGlu5, but is important for receptor stability. To explore in more detail the functional role of covalent mGluR dimerization, a series of in vitro and in vivo experiments are proposed here. We will undertake a detailed comparison between the biochemical, kinetic, and cell biological properties of wt and dimerization-deficient receptors in heterologous expression systems. We will produce transgenic mouse lines in which the mutant receptor incapable of covalent dimerization replaces the wt. These animals will be used for in vivo studies on the function of the receptor, including the responses of the animals to hyperthermia and ischemia, and as a source of tissue and cells expressing the mutant receptor in situ, which will be used for in vitro experiments. We have also shown that there are non-covalent associations between mGlu5 polypeptides. Here we propose a set of experiments to ascertain the specific parts of the molecule mediating this dimerization. Related studies will explore how the cell achieves subtype-specific assembly of mGluR dimers. We anticipate that these investigations of mGluR dimerization will deepen our understanding of neural communication and signal transduction, processes that underlie brain function and dysfunction.

[unreadable] DESCRIPTION (provided by applicant): G-protein coupled receptors are well known for converting an extracellular signal into an intracellular response. Emerging data, however, suggest that some receptors are primarily localized on intracellular membranes where they may play a unique role in the cell's physiology. Recently we have shown that activation of mGlu5 metabotropic glutamate receptors expressed on striatal nuclear membranes leads to rapid, sustained nuclear calcium responses that can be blocked by receptor specific antagonists. Current results demonstrate that both sodium-dependent and independent transporters are involved in moving agonist across both plasma and nuclear membranes as inhibition of either transport system blocks agonist-induced nuclear calcium changes. Remarkably, non-transported agonists induce rapid, transient calcium responses in striatal neurons whereas transported ligands induce long, sustained calcium plateaus. Finally, ligand stimulation of nuclear receptors initiates at least one signaling cascade that is known to alter gene transcription and regulate many paradigms of synaptic plasticity. Because these findings represent a radical departure from traditional models emphasizing cell surface receptors and their ligands, they have important cellular ramifications both in terms of the concept, i.e. that nuclear receptors can regulate nuclear calcium, as well as for mGlu5-specific functions throughout development and in association with synaptic plasticity. The goal of the current application is to utilize our established striatal system together with our newly characterized pharmacological tools to determine the long term consequences of intracellular receptor activation. Using both a candidate gene and an unbiased genomic approach, we will test the primary hypothesis that activation of cell surface versus intracellular mGlu 5 receptors leads to differential changes in gene expression. Inasmuch as mGlu5 receptors are also involved in the pathophysiology of various neurodegenerative and neuropsychiatric disorders such as Parkinson's disease, drug addiction, anxiety and schizophrenia they represent attractive targets for drug discovery. Future studies targeting drugs to cell surface versus intracellular receptors might lead to new therapeutic tools for these disorders. Thus these studies are highly applicable to the purpose of the R21 mechanism. The metabotropic glutamate receptors play fundamental roles in modulating neuronal excitability, synaptic transmission, and various metabolic functions in both health and disease. Given that many of these receptors are largely intracellular, this application seeks to determine the functional consequences of intracellular metabotropic glutamate receptors. Future studies targeting drugs to cell surface versus intracellular receptors might lead to new therapeutic tools for various neurodegenerative and neuropsychiatric disorders such as Parkinson's disease, drug addiction, anxiety and schizophrenia. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): It is estimated that almost 30% of people in the US endure chronic pain. While certain drugs can control some types of pain, many have limitations as well as dose-limiting side effects. Thus safer, more specific drugs would have high clinical utility. Recent studies have shown that the metabotropic glutamate receptor, mGluR5, plays a role in the development and maintenance of chronic pain such that pharmacological antagonism of mGluR5 is thought to be analgesic. Intriguingly, mGluR5, not only plays a critical role on the plasma membrane but also an unknown but profound role on nuclear membranes of the spinal cord neurons that are associated with chronic inflammatory pain. For example, increased levels of mGluR5 are found on nuclear membranes of spinal cord dorsal horn neurons in rats with neuropathic pain. To date the specific role of intracellular mGluR5 has been completely overlooked in any neuronal type including dorsal horn neurons, despite studies showing 60-90% of the receptor is on intracellular membranes. Thus a clearer understanding of the specific role played by spinal intracellular mGluR5 in chronic inflammatory pain could provide a strong rationale for the development of restricted antagonists as novel pain therapeutics. It is therefore the goal of this proposal to examine the role of spinal intracellular mGluR5 in chronic inflammatory pain. Specifically we hypothesize that intracellular mGluR5 plays an important role in modulating chronic pain models. Using pharmacological, biochemical, and molecular techniques, we address the following questions: Does blockade of cell surface mGluR5 with an impermeable antagonist (LY393053) prevent downstream sequelae of glutamate induced inflammation or neuropathic injury; and 2) what are the underlying signaling mechanisms associated with intracellular mGluR5-dependent chronic inflammatory pain. These studies will reveal the cellular and molecular mechanisms by which mGluR5 modulates chronic pain, and will clearly define the role of intracellular receptors in an in vivo setting. Subsequent studies wll assess downstream targets altered by mGluR5 signaling. Inasmuch as mGluR5 is involved in both peripheral and central sensitization it represents an attractive target for drug discovery. Future studies targeting drugs to cell surface versus intracellular receptors might lead to new therapeutic tools for chronic inflammatory pain.

ABSTRACT The cost and consequences of CNS disorders such as addiction, autism, anxiety, and depression place an enormous burden on American society. Despite significant progress, treatments for these disorders are limited and have many side effects. Thus safer, more specific drugs would have high clinical utility. One high level drug-able target is the metabotropic glutamate receptor, mGlu5, which plays a critical role in all of these disorders. Indeed, several mGlu5 negative allosteric modulators (NAMs) are currently in clinical trials with varying degrees of efficacy. Besides the chemical scaffold, drug efficacy is also determined by cellular properties and receptor location. Because our previous work has shown that 60-90% of mGlu5 is located on intracellular membranes where it couples to distinct signaling systems, mGlu5 location may play a key role in its ability to be modulated. In fact our new data show that the intracellular receptor is necessary for establishing hippocampal and striatal LTD and sufficient in blocking chronic pain behaviors. Caveats exist, though, since many of these initial antagonists have off- target effects, variable efficacy, and rapid clearance. To overcome these issues we have obtained five new highly selective mGlu5 NAMs from Eli Lilly, Inc. Our initial data included in this application indicate that at least one of these NAMs only blocks cell surface mGlu5 whereas others are freely permeable. Using pharmacological, molecular and genetic tools as well our unique combination of permeable and impermeable agonists and antagonists, here we propose to test whether these novel NAMs block cell surface or intracellular mGlu5 in the striatum and the hippocampus, regions relevant to many CNS disorders. NAMs that differentially regulate mGlu5 will be further tested in ex vivo models of synaptic plasticity. As postsynaptic mGlu5 activation is required for hippocampal and striatal endocannabinoid-mediated LTD, the proposed studies will also determine which receptor pool contributes to presynaptic endocannabinoid-LTD. New regulators like the NAMs tested here will 1) further the concept that the cellular context of receptor modulation may alter its efficacy; 2) determine whether intracellular mGlu5 underlies fundamental aspects of synaptic activity; and 3) provide a critical tool (s) for future in vivo CNS applications. At the therapeutic level, the occurrence of a functional intracellular receptor opens the door to selectively tailoring agonists and/or antagonists to either receptor pool. Future studies targeting drugs to cell surface versus intracellular receptors might lead to new therapeutic tools for addiction, autism, anxiety, and other mGlu5-modulated disorders.

The metabotropic glutamate receptor, mGlu5, plays a fundamental role in many neuronal processes including synapse formation, synaptic plasticity, and changes in synaptic efficacy. Not surprisingly, impaired mGlu5 signaling is implicated in disorders of synaptogenesis such as Fragile X Syndrome (FXS), autism, and obsessive compulsive disorder (OCD). Indeed, genetically or pharmacologically blocking mGlu5 function robustly improves animal models of these disorders; however exploratory clinical trials have exhibited varying degrees of success. Rather than invalidating mGlu5 as a therapeutic target, such results highlight the need for a better understanding of receptor function including its cell and location specificity. For example, we have shown that 60-90% of mGlu5 is located on intracellular membranes where it couples to distinct signaling systems versus its cell surface counterpart. Importantly, intracellular mGu5 is sufficient for establishing hippocampal and striatal long term depression, a form of synaptic learning and memory that is dysfunctional in FXS, autism and OCD. The objective of the proposed research is to develop animal models that will enable testing of both location-specific mGlu5 signaling and the ability of candidate therapeutics to affect receptors on the intracellular membranes versus the cell surface. Our central hypothesis is that the effects of signaling by mGlu5 in vivo are ?location dependent?. In Aim 1, we will take advantage of CRISPR technology to generate two mouse strains: one will incorporate a short C-terminal tag on mGlu5 that will target the receptor solely to the ER and nuclear membranes; and the other will incorporate a short N-terminal epitope on mGlu5 that will target the receptor solely to the cell surface. In Aim 2, we will use these unique animals to capitalize on our recent observation suggesting that intracellular mGlu5, but not cell-surface-localized mGlu5, is critical in synaptic models of learning and memory. We hypothesize that blocking intracellular-restricted mGlu5 will inhibit changes in synaptic plasticity underlying learning and memory as well as motor, social and anxiety-like behaviors whereas blocking cell-surface-restricted mGlu5 will not do so. This would be the first report of location-specific functions of intracellular versus cell surface-localized mGlu5 in vivo. Because mGlu5 is one of a growing number of receptors that signal from inside the cell, the proposed experiments will enhance knowledge of other intracellular receptors as well. Future in vivo studies targeting drugs to intracellular versus cell-surface-localized receptors are expected to lead to the development of new and effective therapeutic tools for FXS, autism, OCD, and other mGlu5-modulated disorders.

ABSTRACT Neuropsychiatric disorders such as autism spectrum disorder (ASD), anxiety and depression cost hundreds of billions of dollars each year in medical, economic and social costs. Despite progress, treatments for these conditions remain limited. Hence safer, more specific drugs would have high clinical utility. One critical, drug-able target is the metabotropic glutamate receptor, mGlu5, antagonists of which robustly improve animal models of these disorders; however the results of initial clinical trials have been mixed. Rather than invalidating mGlu5 as a therapeutic target, such results highlight the need for a better understanding of mGlu5 function, including its subcellular localization. For example, we have shown that 70-90% of mGlu5 is located on intracellular membranes, where it couples to signaling systems distinct from those of its cell surface counterpart. Importantly, intracellular mGu5 is sufficient for establishing long term depression (LTD), a form of synaptic plasticity that is dysfunctional in ASD, anxiety and depression. The objective of the proposed research is to establish the role that intracellular mGlu5 plays by identifying differences in its function on the cell surface vs. inside the cell, in vitro, ex vivo and in vivo. Our central hypothesis is that mGlu5 interacts with different signaling pathways depending on where it is located and that this results in distinct regulation of synaptic processes and related behaviors. To test this hypothesis we have developed a genetically restricted mouse line in which mGlu5 is present only on intracellular membranes (mGlu5IM). We will use this newly derived animal line in the following aims. In Aim 1, we will determine whether mGlu5IM vs. mGlu5WT or mGlu5KO has different effects on signaling pathways in vitro (cultured neurons) by testing proposed candidate genes and by using unbiased transcriptome profiling (RNA-Seq) to identify new pathways. In Aim 2, we will use ex vivo slice preparations to determine whether mGlu5IM vs. mGlu5WT or mGlu5KO variants play unique roles and signal through distinctive pathways in synaptic processes such as LTD. In Aim 3, we will determine whether the variant mice exhibit unique effects on sensorimotor, anxiety-like and depressive-like behaviors as well as on operant models of reward. This would be the first report of location- specific functions of intracellular mGlu5 in vivo. As mGlu5 is one of a growing number of receptors that signal from inside the cell, the proposed experiments will also enhance knowledge of other intracellular receptors. Future studies targeting drugs to intracellular vs. cell- surface-localized receptors are expected to lead to the development of better drugs for mGlu5- modulated disorders such as ASD and depression,