Jennifer L. Whistler - US grants

Opioid receptors belong to the large superfamily of G-protein-coupled receptors, which as a class are of fundamental physiological importance as they mediate the physiological actions of the majority of known neurotransmitters and hormones. Opioid receptors are particularly interesting members of this receptor family as they are activated both by endogenously produced opioid peptides and by exogenously administered opiate drugs, which remain among the most effective analgesics known. A major limitation to long-term opiate use is the development of physiological tolerance, a profound decrease in analgesic effect. in addition, long term use of opiates causes physiological dependence, a requirement for continue administration of increasing doses of drug to prevent the development of opiate withdrawal symptoms. Receptor desensitization is thought to play a key role in the development of tolerance and dependence to opiate drugs. multiple mechanisms contribute to desensitization including the rapid internalization of receptors. The structurally homologous delta, mu and kappa opioid receptors differ substantially in their ability to undergo regulatory internalization when activated in the same cells by the same ligand. This type-specificity of receptor internalization provides a basis for understanding how closely homologous opioid receptors differ in their physiological regulation. Furthermore, unlike any other G protein-coupled receptor described to date. structurally distinct opiate drugs and native peptides, all of which activate receptor signaling, have opposing effects on receptor internalization. This agonist-selectivity of receptor internalization may provide important insight into why some opiate drugs produce less physiological tolerance and dependence.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The sorting of G protein-coupled receptors (GPCRs) is a fundamental mechanism that controls the regulation of receptors associated with serious neuropsychiatric disorders, including schizophrenia and depression. Our laboratory has recently shown that the cellular fate of receptors can be controlled by specific protein interaction of the C-termini of some GPCRs with GASP (GPCR-associated sorting protein) [Whistler, 2002 #1]. In particular, GPCRs that interact with GASP appear to be targeted to the degradative pathway and those receptors that do not interact with GASP recycle. Intriguingly, GASP binds to the C-termini of D2 receptors and not D1 receptors. Furthermore, in preliminary experiments we have shown that the D2 receptor is targeted for degradation unlike the recycling D1 receptor. D2 receptor antagonists are one of the primary therapeutic treatments for schizophrenia, implicating aberrant dopamine signaling in this disease. We hypothesize that the dopamine signaling pathway may be altered in the disease state as a consequence of the D2 receptor being targeted to the degradative pathway. We have designed and made dominant negative proteins that are able to inhibit GASP function both in vitro and in vivo and have developed an experimentally favorable in vitro cell model system and in vivo techniques suitable for testing this hypothesis. These studies will determine whether GPCR sorting can be manipulated both in vitro and in vivo. This may provide a new spectrum of therapeutic targets for the treatment of schizophrenia and depression. [unreadable] [unreadable]

During the previous funding period of this grant we made two conceptually distinct but important observations. First, we found that the endocytic and post-endocytic trafficking of opioid receptors is critically important for the development of tolerance and dependence to opioid drugs. Second, we demonstrated that opioid receptors heterodimerize to form a unique target for a heterodimer-selective agonist. Here we will follow up on both of these observations by examining how receptor pharmacology and trafficking are altered by coexpression/heterodimerization of the opioid receptors with a focus on heterodimers containing the delta opioid receptor (DOR). Two distinct pharmacologically-defined subtypes of DOR have been described, although only a single gene has been identified. Although there are many explanations for this finding, one possibility is that co-expression or heterodimerization of the DOR with other opioid receptors alters the pharmacology to produce unique opioid receptor subtypes. We will examine how DOR subtype pharmacology is altered by deletion of individual opioid receptor types. In addition, we propose that the constellation of opioid receptor subtypes or heterodimers is altered following chronic morphine administration and the development of tolerance and dependence, possibly as a consequence of alterations in receptor trafficking. Changes in the distribution of opioid receptor subtypes or heterodimers could provide novel targets for the prevention or treatment of opioid tolerance and dependence. In addition, it is possible that certain opioid receptor subtypes or heterodimers are expressed in a tissue-selective manner, a gender-specific manner, or in a disease state-specific manner (for example in chronic pain states or following prolonged use of opiate drugs). Thus targeting these subtypes or heterodimers could provide improved therapeutic utility.

DESCRIPTION (provided by applicant): Treatment of Co-Occurring Alcohol Use Disorders and Depression/Anxiety Disorders highlight the need, recognized by the NIAAA, to identify new targets/drugs for the treatment of alcoholism and its co- morbidities. Naltrexone a non-selective opioid receptor antagonist is one of the few therapeutics currently used in the treatment of alcoholism, validating the opioid receptor system as a relevant target for alcohol abuse. However, naltrexone shows highly variable eficacy in treatment seeking alcoholics and is plagued by side effects and consequent lack of compliance of use. We propose that the non-selective nature of naltrexone may be contributing to the variable efficacy and/or the side effects that limit compliance. Here, we propose to examine specifically the role of the delta opioid receptor (DOR) subtypes, DOR1 and DOR2 in alcohol related behaviors. We are particularly intrigued by the DOR as a target because it is involved in both alcohol consumption and anxiety, which is often co-morbid with alcohol abuse and is a key risk factor for relapse. In our preliminary studies we have found that drugs that target the DOR1 and DOR2 subtypes of opioid receptor have opposing effects on ethanol consumption. In addition, we have found that an antagonist at DOR2 and an agonist at DOR1 can act synergistically to reduce ethanol consumption clearly indicating that these two receptor subtypes are distinct targets with opposing actions. We also have preliminary evidence that the DOR1 may be a heterodimer of the DOR and the mu opioid receptor (MOR). In this proposal, we will examine which commercially available DOR ligands are effective for the reduction of ethanol consumption and ethanol withdrawal induced anxiety. We will also examine whether prolonged ethanol exposure alters the potency and/or efficacy of the DOR ligands, in particular the subtype selective ligands. As a third goal, we will determine whether the effects of any of the DOR drugs require expression of the other opioid receptors, in particular the MOR which could indicate that they target a DOR/MOR heterodimer to exert their effects. Together, these studies may validate the DOR as a target for alcohol abuse disorders, indentify the pharmacological profile of the most ideal ligands for alcohol abuse disorders, and perhaps provide in vivo relevance for the MOR/DOR heterodimer as a target.

Contact PD/PI: Whistler, Jennifer Summary. Alleviating anxiety and depression is pivotal for reducing the risk of relapse in alcoholics. Currently available anxiolytic and anti-depressant treatments have limited utility in alcohol use disorders (AUDs) due to their reduced efficacy in alcohol dependent subjects. Consequently there is a great need to identify new targets for intervention in anxiety and depression, in particular for use in treatment-seeking alcoholics to manage these comorbidities. Neuropeptide S (NPS) and its receptor (NPSR) comprise a recently de-orphanized G protein- coupled receptor (GPCR) system that has been implicated in stress and anxiety. Activation of the NPSR produces anxiolytic and anti-depressant effects without sedation. In humans, there are two allelic variants of the NPSR that occur at almost equal frequency: I107, the wild type allele, and N107, a mutant variant. Importantly, genome wide analysis studies have demonstrated that variation at this locus is linked to risk of anxiety disorders, as well as anxiety sensitivity in the context of stressful environmental states. In addition, very recently, the mutation at this locus has been linked to AUDs. These data suggest that variation in the NPSR could influence risk for AUDs, in particular risk for anxiety and depression in abstinence during treatment. We have recently reported that NPS can reduce ethanol consumption at doses that are not innately either rewarding or aversive. Importantly we have also found that the anxiolytic and anti-depressant effects of NPS are maintained in mice that have been consuming ethanol, unlike classic anxiolytic and antidepressants. Together these data suggest that the NPSR could be a viable target for intervention in AUDs. Here we propose to generate mice with the mutation allele of the NPSR. We will then use these mice to examine whether allelic variation in the NPSR alters risk for AUDs by affecting ethanol consumption, ethanol reinforcement, baseline anxiety and/or depression as well as anxiety and depression during withdrawal. Taken together, the studies here will further validate the NPSR for intervention in AUDs, and could also provide novel insight as to whether variation at this locus could contribute to genetic risk for AUDs. Project Summary/Abstract Page 6

Project Summary Neuropeptide S (NPS) and its receptor (NPSR) comprise a recently deorphanized G protein-coupled receptor system that has been implicated in stress and anxiety (1, 2). Moreover, there is gathering evidence for a role of this receptor in addiction. For example, the NPSR has been shown to affect ethanol consumption, morphine place preference, and cue induced relapse to cocaine. Humans express two common allelic variants of the NPSR, N107 and I107 (the primordial allele expressed in mice). NPS shows enhanced activity at variant I107 despite no change in agonist affinity leaving a mystery as to how this change is affected. We have found that the trafficking profiles of the N107 and I107 alleles of the NPSR variants differ. In particular, we have found that, not only is NPS more potent on the I107 variant, but that this receptor is recycled after endocytosis while the N107 variant is degraded. Consequently, we predict that the anxiolytic properties of NPS will be enhanced when the I107 allele is expressed. By extension, if the anxiolytic properties of the endogenous NPS/NPSR system modulate drug use/abuse or relapse, one would expect the N107 allele to increase risk of drug seeking and/or relapse. Because N107 is a common allelic variant (expressed in ~40% of humans) it is therefore important to determine whether this locus is risk factor for drug abuse. While condition place preference (CPP) and drug self-administration are often used as paradigms for modeling ?addiction? in rodents, it is clear from several studies in rat that the transition to compulsive drug seeking can be dissociated from both reward and consumption, suggesting that neither of these models are good surrogates for ?addiction?. Because of our interests in the role of genetic variation in drug abuse liability, we have developed a novel model of the transition from controlled to compulsive opioid drug use in mice that recapitulates many features of human addiction, including persistent drug seeking despite adverse consequences and a decreased preference for alternative rewards. Here we propose to use this model together with knock-in mice expressing the N107 allele of the NPSR and their wild type littermates to examine whether variation at this locus alters risk for progression to addiction like behavior and/or relapse to drug seeking.  

Summary Currently we are faced with a significant conundrum with regard to the opioid crises. On the one hand, we have no viable alternative for treating severe post-surgical and chronic pain. On the other hand, people taking these drugs for legitimate purposes are transitioning at an alarming rate to opioid overuse and abuse. While the dramatic increase in opioid abuse has coincided with the much-reported increase in opioid prescriptions, it is still true that only a fraction of patients who are prescribed opioids for pain transition to abuse. If we could identify those pain patients most at risk prior to opioid treatment, we could potentially stem the tide of opioid abuse. A number of compelling studies have recently shown that alterations in the gut microbiome can influence a plethora of central nervous systems disorders, leading to widespread acceptance of the concept of a gut-microbiome- brain axis. We find it compelling that, coincident with the increase in opioid prescriptions, there has also been a large increase in the routine and often unnecessary use of oral antibiotics?a trend that that has altered the gut microbiome of an entire generation. Not surprisingly, given the widespread expression of opioid receptors in the gut, opioids have been shown to alter the gut microbiome. Furthermore, alterations in the microbiome have been shown to alter opioid analgesia and reward, suggesting that opioids influence the gut-brain axis. However, to date, no studies have examined whether the gut microbiome influences abuse liability. Here we will capitalize on both behavioral variability in a mouse model of compulsive drug seeking and relapse, and mouse microbiome variability, both innate and in response to morphine, to determine whether abuse liability and the microbiome co- vary (Aim 1). We will also examine whether the microbiome from an ?addict? mouse or ?non-addict? mouse can predispose to or protect subjects from compulsive drug seeking or relapse (Aim 2). This project is a dual Principal Investigator proposal that capitalizes on the complementary expertise of two experts in their respective fields, one a molecular microbiologist the other an opioid pharmacologist.