Dana E. Selley - US grants

DESCRIPTION: (Applicant's Abstract) Opioid analgesic drugs, including heroin and morphine, form a major class of drugs with potential for abuse. Different opioid agonists exhibit different efficacies in various biological systems, and agonist efficacy may not only determine the response produced by the drugs, but also contribute to the development of tolerance to and dependence upon the drug. The proposed project will examine the signal transduction mechanisms underlying agonist efficacy and intrinsic efficacy at mu and delta opioid receptors in two well-established cell culture model systems: mMOR-CHO cells that have been transfected with the mouse mu opioid receptor, SK-N-SH neuroblastoma cells, which contain primarily, mu receptors, and NG108-15 neuroblastoma x glioma cells which contain only delta opioid receptors. Agonist-stimulated [35S]-GTPgS binding, and agonist displacement of [3H]antagonist binding, will be used to measure agonist efficacy and intrinsic efficacy. Efficacy will be defined as maximal stimulation of [35S]GTPgS binding and intrinsic efficacy as both the maximal stimulation and the ratio of Ki in competition binding assays to ED50 in [35S]GTPgS assays. Scatchard analysis of agonist-stimulated [35S]GTPgS binding will be determined whether agonists of different efficacies change the affinity or number of activated G-proteins. Catalytic amplification factors will be calculated by comparing the receptor Bmax to the agonist-stimulated GTPgS binding Bmax, and the role of GDP and sodium in regulating agonist efficacy will be examined. The second phase of the project will examine the relationship between drug efficacy and receptor desensitization and downregulation after chronic drug treatment of cells. In these experiments, cells will be chronically treated with agonists of different efficacies to determine how much the response is decreased, to what extent cross-desensitization to the response produced by drugs of different efficacies develops, and the signal transduction mechanisms underlying the loss in agonist efficacy after chronic drug treatment. In the last phase of the proposed project, the contribution of receptor:transducer ratio and specific transducer reserve to agonist efficacy will be addressed using irreversible antagonists, pertussis toxin, and molecular transfection. A cDNA encoding a cloned Gia2 subunit will be transfected into cells to determine the contribution of specific transducer reserve to opioid agonist efficacy. Thus, the studies in this project are aimed at a mechanistic understanding of how opioid agonist efficacy is produced, and how desensitization effects agonist efficacy, at the level of the receptor-transducer interaction.

The discriminative stimulus properties of alcohol may contribute to its abuse and addiction potential in humans. Animal models of alcohol discrimination have indicated the involvement of serotonin (5-HT) receptors, including the G/i/o-coupled 5-HT/lB/D receptors, in mediating the discriminative stimulus properties of alcohol. The main hypothesis of the proposed pilot study is that the acquisition of ethanol discrimination in rats is associated with changes in 5-HT/l receptor activity. A technique has been developed in our laboratory, whereby receptor-coupled G-protein activity is measured by agonist-stimulated [35S]GTPgammaS autoradiography in tissue sections. This technique allows quantification of the biochemical activity of G-protein-coupled receptors with a high degree of anatomical resolution. This methodology is ideally suited to the study of the effects of the acquisition of ethanol discrimination on 5-HT1 receptor-coupled G-protein activity. Regions where changes in 5-HT1 receptor-coupled G-protein activity are observed autoradiographically will be dissected, and more quantitative biochemical and pharmacological analysis will be performed using both agonist-stimulated [35S]GTPgammaS binding and receptor binding in membranes. This approach, which combines behavioral pharmacology with neuroanatomy and signal transduction biochemistry, may elucidate the neuroadaptive role of 5-HTl receptors in the development of ethanol discrimination. This pilot project directly addresses the Center aims regarding the possible cellular mechanisms underlying ethanol's discriminative stimulus effects. It will provide import pilot data that can supplement the studies performed in Project 3 of the Center, as well as provide information for the R01 work conducted by Dr. Grant.

DESCRIPTION (provided by applicant): Cannabinoid (CB) type-1 receptors (CB1) in the central nervous system (CNS) mediate the psychoactive effects of delta-9-tetrahydrocannabinol, the major active constituent in marijuana. CB1 receptors also mediate many effects of the lipid-derived endogenous cannabinoids (endocannabinoids). This endocannabinoid system plays important roles in regulating motor activity and coordination, short-term memory, pain perception, metabolic homeostasis and drug reward and craving. CB1 receptors can be regulated by post-translational modification and protein-protein interactions, which can alter functional activity, cellular localization and expression levels of these receptors. These processes play a role in limiting the duration of action of CB agonists and in the development of tolerance or dependence upon repeated administration of CB agonists. The proposed project will investigate the function of a newly discovered CB receptor-interacting protein, CRIP1a, which binds to the distal C-terminus of CB1 receptors and attenuates constitutive (basal) activity of these receptors. Preliminary findings also suggest that CRIP1a can alter agonist-induced CB1 signaling in a ligand- and signaling pathway-dependent manner. Preliminary data indicate that CRIP1a can inhibit agonist- induced downregulation or desensitization of CB1 receptors, and that CRIP1a is co-localized with CB1 receptors, particularly in CNS glutamatergic neurons. The following specific aims are proposed to investigate the function of CRIP1a: 1) develop novel cell lines and siRNA constructs as tools to determine the effects of CRIP1a on acute and chronic activation of CB1 receptors and 2) develop a CRIP1a knockout mouse line as a novel tool to investigate effects of CRIP1a on physiological function, behavior and CB pharmacology in vivo. Biochemical and cell imaging approaches will be used to determine effects of co-expression or siRNA-mediated knockdown of CRIP1a in cell models on CB1 receptor-mediated G-protein association (co-immunoprecipitation) and activation (GTP3S binding), and interaction with the regulatory protein 2-arrestin. Effects of CRIP1a on CB1 receptor desensitization, downregulation and internalization will then be examined in these cell models. A CRIP1a gene knockout mouse line will be created using a "flox" approach. Knockout mice will be subjected to basic health assessment and in vivo phenotyping, followed by determination of effects of the knockout on the pharmacological potency of CB agonists in tests of hypothermia, hypolocomotion, catalepsy and antinociception. Anatomical and biochemical studies will then be conducted to determine effects of CRIP1a knockout on CB1 receptor levels, G-protein activation and cellular localization in the CNS. These studies will provide valuable data concerning the role of CRIP1a in the regulation of CB1 receptor-mediated signal transduction associated with functional responses in animals. This work will provide novel target leads for development of drugs that selectively regulate the activity of CB1 receptors for the treatment of drug addiction and other diseases in which the endocannabinoid system is a critical modulatory component. PUBLIC HEALTH RELEVANCE: CB1 cannabinoid receptors mediate many of the effects of marijuana and interact with naturally occurring marijuana-like substances in the brain. This system is important in the regulation of appetite, pain perception, memory, movement and coordination, and seems to play a role in the rewarding effects of several addictive drugs. The proposed project would study a newly discovered protein, called CRIP1a, which interacts with CB1 receptors and appears to modulate their function. These studies will investigate the role of CRIP1a in the regulation of CB1 receptors using genetically modified cultured cell lines and mice in which the CRIP1a gene has been inactivated, to increase our understanding of the effects of marijuana in the brain and perhaps provide a novel target for development of drugs that selectively regulate the activity of CB1 receptors.

DESCRIPTION (provided by applicant): Cannabinoid CB1 receptors in the CNS mediate psychoactive effects of marijuana and are a critical component of the endogenous cannabinoid system, which is involved in motivation and reward, motor control, appetite, learning and memory, and pain and thermoregulation. Control of motor and motivational behaviors by the endocannabinoid system is thought to be largely mediated by CB1 receptors in the striatum/basal ganglia (striatum/BG). Within this system, CB1 receptors have been found to interact with dopaminergic neurotransmission, and are colocalized with D2 receptors in subpopulations of medium spiny GABAergic neurons. Both CB1 and D2 receptors are G-protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase (AC) activity via Gi/o activation, and under certain circumstances can stimulate adenylyl cyclase through Gs/olf activation. The G-protein gamma subunit type 7 (Gng7) is highly expressed in striatum/BG relative to other brain regions, and has been shown to play a role in D1 receptor stimulation of adenylyl cyclase and to regulate G-alpha-olf expression as determined in Gng7 knockout (KO) mice. However, potential involvement of Gng7 in inhibitory regulation of AC by GPCRs has not been examined. Our preliminary findings indicate that AC inhibition by striatal CB1 and D2 receptors is diminished in Gng7 knockout mice, suggesting a role for this G-gamma subunit in inhibitory Gi/o signaling. Alternatively, Gng7 KO could, by concomitant reduction in G-olf, alter the proportion of striatal AC activity that can be inhibited by Gi/o. The proposed R03 project will test the hypothesis that CB1 and D2-mediated inhibition of AC is attenuated in Gng7 knockout mice because G-gamma-7 plays a role in inhibitory Gi/o signaling. Aim 1 will determine the extent of loss of adenylyl cyclase inhibition by CB1 and D2 receptors in subregions of the striatum/BG in Gng7 knockout mice, including caudate-putamen, nucleus accumbens and globus pallidus, as well as other CB1 and D2 containing regions such as prefrontal cortex and hippocampus. Cerebellum, which expressed CB1 receptors but does not express Gng7, will serve as negative control region. Aim 1 will also determine whether the effect of loss of Gng7 on AC inhibition is limited to CB1 and D2 receptors or is seen with other striatal GPCRs (e.g. opioid). Aim 2 will determine whether CB1- or D2-stimulated Gi/o-protein activity is diminished in regions in which AC inhibition is diminished in Gng7 KO mice, as determined by agonist-stimulated [35S]GTPgammaS binding. Whether any reduction in G-protein activation is due to a loss in receptor expression will be tested by immunoblotting of CB1 and D2 receptors. Aim 2 will also test the alternative hypothesis that attenuated AC inhibition is due to concomitant reduction in G-olf in Gng7 KO mice, by determining G-protein activation and AC inhibition by CB1 and D2 receptors in heterozygous G-alpha-olf KO mice. These studies will serve as a basis for a future a R01 application that will more extensively investigate the relationship between CB1 and D2 receptors in the regulation of striatal function, and the contribution of this regulation to control of motor and motivational behavior. These studies will help to determine molecular mechanisms contributing to neuropsychiatric disorders such as substance abuse and schizophrenia, as well as motor disorders. PUBLIC HEALTH RELEVANCE: Marijuana (cannabis) and other abused drugs produce their rewarding effects through the regulation biochemical signaling pathways in a brain region known as the striatum. This region is also involved in control of voluntary movement and habitual behavior, and is also under the control of a chemical messenger known as dopamine. A key protein in these signaling pathways in striatum is the G-protein gamma type 7 subunit. This project will determine the role of this protein in the biochemical effects of cannabinoid and dopamine receptor activation by studying mice that have been genetically altered to not express this protein. These studies will provide information that contributes to our understanding of the neural mechanisms of drug addiction and possibly neuropsychiatric and movement disorders, such as schizophrenia and Parkinsonism.

DESCRIPTION (provided by applicant): Cannabinoid CB1 receptors mediate the CNS effects of delta9-tetrahydrocannabinol (THC), the main psychoactive constituent in marijuana, and the endogenous cannabinoids. This system plays a role in drug abuse, and has therapeutic potential for treatment of nausea, appetite disorders, muscle spasticity and chronic pain, neurodegenerative diseases and mood disorders. However, marijuana/THC have side effects, including sedation, impairment of coordination and short-term memory, and abuse liability, which limits their utility. A better understanding of mechanisms that regulate CB1 receptors could lead to improved strategies to target this system, including the potential to selectively target receptor interaction with regulatory proteins. The proposed project will investigate the function of cannabinoid receptor-interacting protein 1a (CRIP1a) using a null mouse model. CB1 receptors are G-protein-coupled receptors that inhibit synaptic neurotransmission. Our published and preliminary data in cell models co-expressing CRIP1a with CB1 receptors indicate that CRIP1a inhibits both basal (constitutive) and agonist-induced G-protein activation by CB1 receptors without affecting receptor expression levels. We propose to investigate the effects of genetic deletion of CRIP1a on biological phenotype, including weight gain, body temperature, motor activity and coordination, anxiety and pain sensitivity. We will then examine effects of CRIP1a deletion on the pharmacological actions of THC in vivo, and on CB1 receptor expression and activation of G-proteins in CNS regions associated with cannabinoid effects. CRIP1a is postulated to negatively regulate CB1 receptors, so we will test the hypothesis that CRIP1a null mice will display a phenotype that could: 1) mimic the administration of THC (weight gain, hypothermia, anxiolysis, hypolocomotion, reduced coordination, antinociception) and/or 2) enhance the pharmacodynamic potency or efficacy of THC. We further will test the hypothesis that these effects will be associated with enhancement of CB1 receptor-mediated G-protein activity in specific CNS regions, and predict there will be CNS region-specific differences in the effects of CRIP1a deletion corresponding to different effects on behavior and in vivo sensitivity to THC.

DESCRIPTION (provided by applicant): This is a competing renewal application to continue our preclinical research on expression, neurobiology and treatment of pain-related behavioral depression. Pain is a significant clinical challenge that is often associated with clinically relevnt depression of behavior and mood. Moreover, relief of pain-related depression is a common goal of treatment in both human and veterinary medicine. Our research is founded on the proposition that research on pain-related depression could provide new basic-science insights on mechanisms that mediate affective dimensions of pain and new strategies for pain treatment. Our data so far suggest a role for dysregulated mesocorticolimbic dopamine (DA) signaling in nucleus accumbens (NAc) and prefrontal cortex (PFC) as a mediator of pain-depressed behavior, and studies proposed in this application would pursue hypotheses related to this mechanism. Specifically, during the current project period, we developed and validated a new behavioral assay of acute and chronic pain-depressed behavior in rats. We then used this procedure to achieve the following research goals: (1) evaluation of >40 drugs from multiple drug classes to confirm that our procedure is both sensitive to known analgesics and selective for analgesics vs. non- analgesics; (2) correlation of pain-related depression of behavior and pain-related depression of DA release in NAc; and (3) discovery that pain states also increase PFC expression of brain-derived neurotrophic factor, a protein implicated in depression consequent to non-pain stressors. In this competing renewal application, we propose to extend on this work in a series of three specific aims. Aim 1 will test the hypothesis that DA agonists wil compensate for pain effects and produce analgesia in behavioral assays of pain- depressed behavior. We propose to evaluate analgesic effects of D1, D2 and D3 DA receptor agonists in assays of acute and chronic pain-depressed behavior. Our hypothesis predicts that agonists at one or more DA receptor subtype will be effective. Effects of indirect DA agonists will also be examined for comparison. Aim 2 will test the hypothesis that pain states modulate downstream mediators of DA signaling in NAc and PFC. We propose to assess pain effects on PFC DA release to complement our microdialysis studies in NAc. In addition, we propose to test consequences of pain-altered DA release by evaluating (a) density of the DA transporter and of D1, D2 and D3 receptors, and (b) expression of two complementary and physiologically relevant indicators of DA tone ( FosB and phosphorylation of protein kinase B). We predict compensatory changes in response to pain-related decreases in DA. Aim 3 will test the hypothesis that pain states will also modulate signaling mediated by BDNF in NAc and PFC. Increased expression of BDNF within the mesocorticolimbic system has been implicated in the development of depressive behavior. We predict that pain states will augment BDNF signaling, and that direct overexpression or knockdown of BDNF signaling within this system with viral vectors will modulate expression of pain-depressed behavior.

PROJECT SUMMARY (See instructions): The primary mission of this core will be to provide services, facilities and training to facilitate the study of receptors or related small molecule targets, such as transporters, with a goal of enhancing research in drug abuse and related co-morbidities. Services will include measurement of receptor binding, receptor localization, receptor signaling (with a primary focus on GPCRs, although not limited to GPCRs), receptor trafficking and receptor-protein interaction. The core will also perform moderate-throughput ligand screening to determine small molecular structure-activity relationships at particular target sites. The core will also provide relevant tissue preparative functions, such as tissue sectioning and primary cell culture development, for core studies. Finally the core will provide relevant training for PIs, students or post-doctoral fellows.

? DESCRIPTION (provided by applicant): Chronic pain diminishes the quality of life for millions of patients, but currently used classes of analgesics possess varied efficacy and are associated with a variety of untoward side effects. Thus, novel targets to treat chronic pain and development of new drugs that have better efficacy and/or fewer side effects than existing pharmacotherapies are greatly needed. A particularly promising target is the sphingosine-1-phosphate (S1P) receptor system, which mediates CNS neuromodulatory functions. FTY720-phosphate, the active metabolite of FTY720 (FTY; fingolimod), approved by the FDA for treatment of relapsing multiple sclerosis, acts as an agonist at four of the five S1P receptors (S1P1, 3, 4, 5). Interestingly, studies have demonstrated that FTY and other S1P receptor (S1PR) agonists produce antinociception in acute thermal rodent pain models and these effects are blocked by central administration of an S1P1-selective antagonist. Moreover, FTY reverses hyperalgesic states in rodent neuropathic pain models. However, it is unclear whether S1P1 or other S1PR subtypes mediate these effects and their site(s) of action. Thus, the overarching hypothesis of this application is that the S1P1 receptor represents a novel and promising target for the treatment of neuropathic pain. Here, we will test whether S1P1 receptors in the CNS mediate anti-hyperalgesic effects in a mouse neuropathic pain model, using a combination of pharmacological and gene targeting approaches. Therefore, the Specific Aims are to: 1) Determine the role of S1P1Rs in alleviation of neuropathic pain by S1PR ligands; 2) Determine the role of FTY-induced S1PR adaptation in FTY-mediated reversal of neuropathic pain; and 3) Determine the role of S1P and S1P1 receptors in spinal glia in CCI-induced neuropathic pain and its reversal by FTY. The studies proposed herein will establish whether FTY and selective S1PR ligands reverse pain-related behavior in the mouse CCI neuropathic pain model, whether S1P1 receptors in the nervous system mediate these actions and the specific cell types involved in the response. In order to be useful in treating chronic pain, the drug must retain its effectiveness during prolonged treatment. Thus, evidence supporting a role of S1P1 in specific cell types to reduce neuropathic pain without tolerance or motor impairment will provide proof of principle that S1P1 receptors are a viable target to treat neuropathic pain and possibly other chronic pain-related disorders.