Ping-Yee Law - US grants

In order to answer one of the many questions on the multiple opioid receptors, i.e. whether or not these receptors represent different gene products, different splicing of the same gene, or post- transnational modification of the products, it is the goal of the current proposal to clone for the kappa-opioid receptor. Kappa- opioid receptor will be cloned from either the guinea pig cerebellum or human placenta library by the cDNA expression method. The library will be enriched in kappa-opioid receptor clones with 3 sets of probes: (a) restriction enzyme fragments of the mu- and delta-opioid receptor clones; (b) subtraction probes synthesized from hybridizing mRNAs from tissues containing low level of kappa- opioid receptor from that containing high level of kappa-opioid receptor, i.e. chronic kappa agonist (U50-488) or antagonist (MR2266) treatment will be used to alter the kappa-opioid receptor level and hence mRNA levels; and (c) the oligodeoxynucleotides sequences of the putative transmembrane regions V, VI and VII of the cloned 8-adrenergic receptor. The cDNA clones which hybridize with the first two sets of probes or with all three sets of probes will be expressed in eukaryotes previously devoid of opioid receptor activities. Clones which induced kappa-opioid receptor binding activity will be subcloned into Sp6 vectors for sense and anti-sense RNA synthesis. The RNAs thus synthesized will be injected into frog oocytes and the ability of kappa agonist to regulate the Ca+2 channels will be used to substantiate the identity of the clones. Antibodies will be developed against the deduced peptide sequence and will be used to immunoprecipitate ligand-kappa-opioid receptor complex and/or used to inhibit the kappa-opioid receptor binding activities in brain membranes. Such antibodies will be used also in immunocytochemical analysis of kappa-opioid receptor distribution and compared with the reported distribution of putative kappa binding sites. The identity of the kappa-opioid receptor clone will be substantiated further by the isolation and sequencing of the 125I-B-endorphin-receptor complexes when labelling was carried out in the presence of mu- and delta- opioid ligands. The gene structure and the nucleotide sequence of the kappa-opioid receptor clone will be determined and compared with that of mu- and delta-opioid receptor clones. Deletion and nucleotide insertion mutation studies will be carried out to investigate the structural requirement for kappa-opioid receptor activities.

The manner in which neuronal cells can distinguish the activation of second messenger systems by several receptor types is unclear. We surmise that instead of distinguishing the differences in the magnitude of activation or compartmentation of the second messengers, neuronal cells could distinguish the activation of a specific receptor by integrating the network of multiple effector systems which are simultaneously or sequentially activated by the occupancy of the receptor. If this is the case, then a single receptor type which transduces its signal via the family of G- proteins will interact with multiple G-proteins. We will test this hypothesis by investigating the identities of the G-proteins which can interact with delta-opioid receptor in neuroblastoma x glioma NG108-15 cells, and those which can interact with mu- and delta-opioid receptor in human neuroblastoma SH-SY-5Y. The identification of these G-proteins and the subsequent effectors which can be regulated by these G-proteins will also enable us to better understand the cellular adaptational processes during chronic agonist treatment. The identities of the G-proteins in these two clonal cell lines which can interact with the opioid receptors will be determined initially by utilizing the ability of cholera toxin (CTX) to catalyse the ADP- ribosylation of the G-proteins other than G8 or transducins when they are coupled to the receptor. Therefore, CTX catalysed ADP-ribosylation with 32P-NAD+ will be carried out in the presence of various concentrations of selective opioid agonists and antagonists. Different G-proteins will be separated by urea gradient/SDS PAGE and their initial identities determined by aligning the proteins which are being ADP-ribosylated with the positive bands obtained from western analysis of the blots using specific Galpha antisera. The ability of various treatment which have been reported to alter opioid receptor function, e.g. pertussis toxin treatment, chronic agonist treatment or differentiation, to alter the CTX catalysed ADP- ribosylation will be determined. Because of the homology among various Galphas, the final identities of the G-proteins will be demonstrated by isolation of the Galphas, sequence analysis of the proteolytic fragments and the nucleotide sequence of the isolated cDNA clones. From the deduced peptide sequences, specific polyclonal antibodies to the Galphas will be developed. The ability of these antibodies to modulate the cellular responses to opioid receptor will be determined. This will be carried out by first measuring the alteration in the second messengers level in single cells in the presence of opioid agonists. Affinity purified antibodies of the specific G-proteins will be introduced into the cells and the ability of the antibodies to attenuate the agonist responses will be measured. The G-proteins and their subsequent effectors which can be activated by the delta-opioid receptor will be compared with those which can be activated by mu-opioid receptor. This spectrum of G-proteins will also be compared with that of muscarinic and alpha2-adrenergic receptors. The question of how these two neuronal cell lines could distinguish the activation of these receptors will then be addressed.

In order to answer one of the many questions on the multiple opioid receptors, i.e. whether or not these receptors represent different gene products, different splicing of the same gene, or post- transnational modification of the products, it is the goal of the current proposal to clone for the kappa-opioid receptor. Kappa- opioid receptor will be cloned from either the guinea pig cerebellum or human placenta library by the cDNA expression method. The library will be enriched in kappa-opioid receptor clones with 3 sets of probes: (a) restriction enzyme fragments of the mu- and delta-opioid receptor clones; (b) subtraction probes synthesized from hybridizing mRNAs from tissues containing low level of kappa- opioid receptor from that containing high level of kappa-opioid receptor, i.e. chronic kappa agonist (U50-488) or antagonist (MR2266) treatment will be used to alter the kappa-opioid receptor level and hence mRNA levels; and (c) the oligodeoxynucleotides sequences of the putative transmembrane regions V, VI and VII of the cloned 8-adrenergic receptor. The cDNA clones which hybridize with the first two sets of probes or with all three sets of probes will be expressed in eukaryotes previously devoid of opioid receptor activities. Clones which induced kappa-opioid receptor binding activity will be subcloned into Sp6 vectors for sense and anti-sense RNA synthesis. The RNAs thus synthesized will be injected into frog oocytes and the ability of kappa agonist to regulate the Ca+2 channels will be used to substantiate the identity of the clones. Antibodies will be developed against the deduced peptide sequence and will be used to immunoprecipitate ligand-kappa-opioid receptor complex and/or used to inhibit the kappa-opioid receptor binding activities in brain membranes. Such antibodies will be used also in immunocytochemical analysis of kappa-opioid receptor distribution and compared with the reported distribution of putative kappa binding sites. The identity of the kappa-opioid receptor clone will be substantiated further by the isolation and sequencing of the 125I-B-endorphin-receptor complexes when labelling was carried out in the presence of mu- and delta- opioid ligands. The gene structure and the nucleotide sequence of the kappa-opioid receptor clone will be determined and compared with that of mu- and delta-opioid receptor clones. Deletion and nucleotide insertion mutation studies will be carried out to investigate the structural requirement for kappa-opioid receptor activities.

It remains our overall objective to elucidate the mechanism of how neuronal cells could distinguish activation of multiple G-proteins coupled receptors when they appear to couple to same effector systems. Our hypothesis has been that these receptors could activate multiple G-proteins and cellular distinction lies in ability of neuronal cells to effector systems which are being activated. We tested this hypothesis by investigating delta- opioid receptor coupling to G- proteins in several neuroblastoma cells. By carrying experiments using GTP photoaffinity labels, and the ability of agonist to promote cholera toxin catalysed ADP-ribosylation of cognate arginine moiety in Galphas, we observed that delta-opioid receptor could interact with multiple G-proteins which are pertussis toxin (PTX) substrates in NG108-15, NS20Y and N1E115 cells. To our surprise, the affinity of agonist to promote these interaction is similar for all G-proteins. By taking advantage of different level of expression of cloned receptor in transfected CHO cells, we could demonstrate that receptor- G-protein interaction is a function of receptor density while ability of agonist to inhibit adenylate cyclase is not. These data, together with others, suggested to us that there are cellular proteins other than heterotrimeric G-proteins which are involved in opioid receptor signal transduction. Therefore, in current proposal, we will continue our efforts in identifying the G-proteins involved in opioid receptor signal transduction. We will now examine probable interaction between opioid receptor and G-proteins which are not PTX substrates. We will utilize all the recently cloned opioid receptors, mu-, kappa- and delta-opioid receptor, to establish cell lines in which either homogeneous population of receptor is expressed or a combination of these receptors will be expressed. Cell lines such as AtT20, GH3 and other cells which possess multiple effector systems for probable opioid receptor regulation will be used in these studies. We will also use Xenopus oocytes as expression system for studying opioid receptor-G-protein interactions, using CFTR, a cAMP sensitive chloride channel as a reporter for opioid receptor activity. G-proteins which interact with these receptors will be identified with 32P-alpha- azidoanilido GTP, subsequent separation with urea/SDS PAGE and Galpha- specific antibodies. Effect of one receptor activation on ability of other receptor to interact with G-proteins will be addressed. We will correlate specific G-proteins which are involved in selective opioid receptor actions by use of Galpha specific antisense oligodexoynucleotides so as to eliminate opioid responses and to use Galpha mutated to eliminate PTX-sensitivity so as to retain opioid receptor activity in cells treated with PTX. The opioid receptor responses to be measured will be agonist regulation of intracellular cAMP level, IP3 level, Ca+2 level and overall cell proliferation. The sites on the receptor which interact and might infer G-protein selectivity will be identified by receptor mutagenesis studies. Involvement of other cellular proteins will be identified by receptor mutagenesis studies. Involvement of other cellular proteins will be identified by examining the role of bg-subunits of heterotrimeric G- proteins, smg or GAP-like proteins. The presence of these cellular proteins will be examined in immunoaffinity purified agonist-opioid receptor complexes or complexes generated with constitutively active opioid receptor mutants. The role of these cellular proteins and others in opioid receptor signal transduction will be determined.

Opioid activity is a function of its receptor steady state level. How neuronal cells regulate the opioid receptor level could be the key in understanding the cellular mechanism of opioid tolerance. Prior to the cloning of the three opioid receptor types, reagents such as receptor specific antibodies are not available for such studies. With the development of receptor specific antibodies and epitope tagged opioid receptor, we and other laboratories have demonstrated that delta-opioid receptor internalized via the clathrin-coated vesicles and trafficked to the endosomes and later to lysosomes for degradation. This iternary of opioid receptor is similar to that observed with other G protein-coupled receptors (GPCR). The exact cellular components involved and the signals that triggered the opioid receptor internalization remain elusive. Therefore, in the proposed studies, we will determine the beta-opioid receptor domains that are involved in the receptor internalization and down-regulation, and the cellular proteins that participate in the trafficking of delta-opioid receptor. The approaches we will use is to construct delta-opioid receptor mutants, i.e., truncation mutations to locate the general area followed by site-directed mutagenesis to pin-point the amino acids involved. Since earlier reports with GPCR and delta-opioid receptor suggested involvement of protein kinases in receptor internalization and down-regulation, initial focus will be on the Ser and Thr within the intracellular domains of the receptor that can be phosphorylated. However, the random mutation approach will also be used to identify any motifs or amino acids that might be involved and are not predicted by our current understanding of GPCR. IN order to accomplish this goal, a green-fluorescent protein-delta receptor conjugate will de developed. The receptor domains identified to be involved in receptor down-regulation and desensitization will be used to isolate the cellular proteins that participate in the receptor trafficking. The comparison of the immunoprecipitated complexes between wild-type and mutant receptors with antibodies against proteins that are known to participate in protein trafficking, e.g., G proteins, the roles of these proteins in receptor internalization and down-regulation will be identified. These information in combination with the isolation of receptor complexes in the Tet-on system in which the receptor levels can be induced will allow us to deduce and isolate other cellular proteins that are involved in this processes. In addition, we will continue to explore the mechanism in which the opioid receptor mRNA level can be regulated by second messengers and the role of the mRNA levels on the steady state level of receptor protein.

DESCRIPTION (provided by applicant): The dynamic cellular control of G protein-coupled receptor (GPCR) trafficking impacts many functional aspects of the protein. Accumulating evidences have suggested the endocytosis and intracellular trafficking of GPCR not only are critical in the desensitization and resensitization of the receptor but also these processes participate in generating alternative signals. The trafficking of the opioid receptors is also under stringent cellular control, and appears to have a role in the cellular responses to the chronic opioid agonist treatment. Previous reports from our laboratory and others have indicated that mu and delta-opioid receptors, though structurally homologous, have different intracellular trafficking patterns. The agonist-induced internalized mu-opioid receptor can be resensitized and recycled back to the cell surface, while the delta-opioid receptor is directed to the lysosomal degradation pathway. Receptor chimeras studies suggest that the carboxyl tail domains of these two receptors are responsible for such trafficking patterns. However, as demonstrated in our Preliminary Data, carboxyl tail domains participate but not sufficient in directing the trafficking of the opioid receptors in a neuronal cell model, neuroblastoma Neuro2A (N2A) cells. An interaction between a dileucine motif within the 3 rd intracellular loop of the delta-opioid receptor with the carboxyl tail is needed for directing the receptors to lysosomes. Thus, we hypothesize that in addition to the linear receptor sequence, multiple three-dimensional sequences generated by covalent modifications of the receptors such as phosphorylation and ubiquitination are involved in directing the opioid receptor traffic. The scaffolding of the proteins in the endocytic pathways with the modified receptors will determine the direction of the receptor traffic. Hence, in the current studies, we propose to identify: (a) the linear receptor sequences that participate in the intracellular trafficking of the opioid receptor; (b) the role of ubiquitination and phosphorylation on generating the three-dimensional sequence in the intracellular trafficking of the receptor; and (c) the tmns-endocytic proteins that are critical in the recycling and lysosomal trafficking of the receptor. Truncation, deletion, single amino acid and random mutational analyses of the mu and delta-opioid receptor sequences will be carried out to delineate the primary sequences involved. Yeast two-hybrid screens and proteomic approaches using mass spectrometry will be used to identify the trans-endocytic proteins involved. Dominant negative mutants of these proteins will be used to demonstrate their roles in receptor trafficking. By establishing the itineraries of the/,- and 0-opioid receptors, and by identifying the endocytic protein complexes in various pathways in N2A cells, we will be able to illuminate the dynamic nature of the cellular control and elucidate the significance of the receptor trafficking in the opioid agonist function in neurons.

DESCRIPTION (provided by applicant): During the last 5 years, PI was able to utilize the current K05 award to develop two new NIDA funded projects in his laboratory, and was able to spend a semester leave in Dr. Chris Evans'laboratory at UCLA to explore the use zebrafish as an alternative model for studying the molecular mechanism of opiate tolerance and dependence, a life-long career goal of PI. This was made possible because of the relief from his administrative and teaching commitments at University of Minnesota due to the K05 award. Therefore, the objective of this K05 award application remains to be a mechanism allowing PI to continue his successful program of taking periodic leaves of absence from University of Minnesota and spend time in his collaborator's laboratory in the pursuit of new or alternative approaches and technologies in studying the molecular mechanism of tolerance and dependence. It is clear from the on-going projects in PI's laboratory that opioid receptor signals via the formation of receptorsomes. The scaffolding of cellular proteins with opioid receptor within the microdomains greatly affect the receptor signaling. By recruiting different proteins to the receptor vicinity, e.g., beta-arrestin, Src, RGS and AGS, the magnitude and duration of signals could be modulated. PI has initiated studies to identify the cellular proteins that could modulate opioid receptor activities. Using yeast two-hybrid screens of mouse brain library, protein candidates, such as the FK506 binding protein FKBP12 that specifically interacts with the carboxyl tail domain of MOR could modulate the agonist-induced intracellular Ca2+ movement. However, the use of a specific receptor domain limits the identification of proteins that require multiple domains or tertiary receptor structure for binding. Thus we will continue our on-going projects to determine the components of opioid receptorsomes by the use of proteomic approaches and yeast two-hybrid screens using whole receptor protein. Over-expression of these proteins with adenoviruses, or the knockdown of these proteins levels in neuroblastoma N2A cells by siRNA will be carried out to determine their effects on two the effectors regulated by opioid receptors, i.e., adenylyl cyclase and intracellular Ca2+ homeostasis. The alteration of these proteins levels in primary hippocampal cultures enriched in neurons expressing endogenous MOR will be carried out also. The inducible siRNA vector will be developed and used in the temporal knockdown of the proteins involved in the receptorsomes formation. Genetic alteration of the proteins levels will be carried out in mice and other models to test the effects of these proteins in chronic opiate drug actions.

DESCRIPTION (provided by applicant): Opioids have been used very successfully for the treatment of moderate to severe acute and chronic pain. Unfortunately, their uses have been associated with many troublesome side effects such as nausea, constipation, respiratory depression, sedation, pruritus, tolerance and dependence development. Many approaches have been used to alleviate these side effects without diminishing the analgesic effects, with variable success. Notable approaches have been the design of receptor selective ligands that would activate one of the three cloned opioid receptors and co-administration of pharmaceutical agents to block the opioid side effects. Although these approaches and others are viable ones, one of the reasons for their limited success is the high amino acid sequence homology among the cloned receptors. Such homology has slowed the design and development of an opioid drug that will target and activate a specific opioid receptor. In the current studies, we propose to use an alternative approach, i.e., to engineer mutant opioid receptors for pain management. Our approach is based on an accidentally discovered receptor mutation, S4.45(196)L in the u-opioid receptor (MOR), that resulted in the ability of opioid alkaloid antagonists to activate the mutant receptor, both in vitro and in vivo. We hypothesize that, if such a mutant receptor could be delivered to and expressed in the nociceptive neurons, then activation of the mutant receptors by antagonists should produce analgesic responses without eliciting the tolerance responses during chronic treatment with the antagonists. Our overall goal is to develop such receptor mutants as therapeutic agents for pain management. Thus, in the current proposal, we will (1) determine the molecular bases for the activation of receptor mutants by opioid antagonists;(2) validate and refine the receptor model for S4.54 mutation so as to engineer a mutant MOR in which naloxone and naltrexone behave like full agonists;and (3) develop a double stranded adenoassociated virus (dsAAV) for the delivery of the mutant receptor at specific sites of the pain pathway and evaluate the antagonist and agonist efficacies in eliciting antinociceptive responses. We will examine both the acute and chronic responses to opioid agonists and antagonists after dsAAV injection. By developing the receptor model and demonstrating the structural bases for the antagonist activities via receptor mutagenesis studies and generation of mutant mouse lines, we could engineer a mutant receptor in which opioid antagonists behave like full agonists. Since dsAAV has been used successfully in gene therapy, the eventual delivery by dsAAV and expression of the mutant receptor at the nociceptive neurons that normally express MOR should result in analgesic responses after systemic administration of opioid antagonists without tolerance development. Such a mutant opioid receptor gene therapy approach could be a new paradigm for the eventual treatment of chronic pain. PUBLIC HEALTH RELEVANCE: In the treatment of moderate to severe pain, morphine remains the drug of choice. However, the many side-effects of the drugs, notably tolerance and dependence development in prolonged treatment, have reduced the desirability in the use of this opioid analgesic for pain management. It has been the Holy Grail of pharmacologists and pharmaceutical chemists to develop drug molecules or treatment paradigms that elicit the pain relief effects without any side effects. With the discovery of a mutant u-opioid receptor (MOR) that could be activated by opioid antagonists without altering the agonists'properties, we hypothesize that such a mutant receptor could be developed into therapeutic agents for the purpose of pain management. We have demonstrated the feasibility of such approach by "knocking-in" this mutation, S4.54(196)A, into MOR, and generating a mouse line in which opioid antagonists, naloxone and naltrexone produced antinociceptive responses. However, this mutation only resulted in partial agonistic properties observed with these two opioid antagonists. Therefore, in the proposed studies, we will investigate the molecular bases for such antagonistic activities using modeling and mutational analysis. Additional receptor mutations will be identified in order to convert the antagonists into full agonists. We will develop a gene therapy vehicle, initially using dsAAV2 virus, to deliver the mutant receptors into various regions of the pain pathway and to examine the feasibility of using opioid antagonists as antinociceptive agents. The activation of the exogenously introduced mutant MOR, and the inactivation of the endogenous opioid receptors by the antagonists, will provide a unique opportunity to develop a pain treatment paradigm without possible development of tolerance and dependence.

The Molecular Cellular and Genetic Core (MCGC) is being reviving in the Center for the purpose of providing expertise in the areas of molecular and cell biology and assisting in the construction of various vectors used in the proposed studies within the Center. The MCGC will provide all the scientific components the primary cultures from multant mice needed for their studies. The MCGC also will maintain lines of mutant mice that are used by multiple components and are highly requested by the scientific community. By centralizing all these functions in MCGC will allow not only substantial cost saving but also the standardization of the reagents, vectors, cultures and animals in all the projects within the Center. Therefore, the specific aims of MCGC are: 1) To establish banks of reagents and cell models to be used by Center's investigators. The revived MCGC will resume the duties in maintaining the inventories of vectors, bacteria strains, libraries and cell lines generated by the Center investigators from Administrative Core. MCGC will be responsible for sending out any request for the reagents and cell models, and expand the inventories to include viral and vector constructs that are useful for the in vitro primary culture and in vivo animal studies. 2) To provide expertise in the areas of molecular and cell biology to Center's investigators. The MCGC will consult on the construction of the various mammalian expression vectors, or targeting vectors for the development of genetic altered animals. In addition, MCGC will assist in constructing the siRNA vector and viral vectors for delivery the siRNA ortransgenes to be used by the Center's investigators. 3) To maintain a bank of mutant mouse models for the Center's investigators. In order to eliminate the drain in the individual investigator's resources, MCGC will maintain breeding colonies of mouse models so as to fulfill the requests by the Center's investigators and external investigators. 4) To provide primary cultures from wild type and mutant mice for the studies of opioid receptor action. The use of primary cultures as models is a common theme among all 5 scientific components of this proposed Center. Especially, the use of primary neurons from mutant mouse models is critical for establishing the validating of the cell models'observations. Thus, the MCGC will initiate the program of supplying primary cultures to Center's investigators. This program not only could utilize the primary cultures obtained from mutant mice more efficiently, it could also standardize the quality of the primary among all investigators within the Center.

DESCRIPTION (provided by applicant): For years, many laboratories, including ours, have focused on elucidating the molecular and cellular basis for opiate tolerance and withdrawal. Regardless of the hypotheses, one constant is that chronic drug effects are initiated by receptor signaling. Since the ?-opioid receptor (OPRM1) is a member of the rhodopsin family of G protein-coupled receptors (GPCR), the model for GPCR desensitization involving the G protein-receptor kinase (GRK) and ?-arrestin was applied to the ?-opioid receptor (OPRM1) to account for the chronic drug effect. However, other signaling pathways, such as N-methyl-D-aspartate (NMDA) receptor, protein kinase C (PKC) and even ?-opioid receptor (OPRD1), have been implicated in morphine tolerance development. The involvement of multiple protein kinases in chronic morphine effect is best exemplified by our recent observations that both Src kinase and Raf-1 kinase participate in adenyly cyclase (AC) superactivation after chronic drug treatment. These observations and others have led us to propose the hypothesis that recruitment of protein kinases by the agonist-OPRM1 complex will determine the pathway selected for cellular adaptational processes, such as opiate tolerance and withdrawal. Thus, the goals of our proposed studies are to determine whether there is an agonist-selective mechanism (i.e., protein kinase-dependent) in opiate tolerance development and whether PKC is involved in the blunting of in vivo morphine and not other agonist actions. In addition, the roles of the Src/Raf-1 kinase signaling cascade and the phosphorylation of OPRM1 in AC superactivation will be elucidated. The significance of our in vitro observations will be validated with the proposed in vivo studies. By using the approach of viral delivery of the wild type or mutant OPRM1, wild type or phosphorylation minus mutant of the PTX-insensitive Gi/o ?-subunits, or siRNA constructs to regulate the protein kinases involved, into vlPAG area of OPRM1-/- mice or double knockout mice of OPRM1 and ?Arrestin2, we will address our hypothesis with 2 specific aims: (1) To demonstrate that PKC mediates the in vivo tolerance development to morphine; (2) To delineate the pathway involved in Src-mediated OPRM1-directed AC superactivation and linking AC superactivation to naloxone precipitated withdrawal signs. We anticipate that we will demonstrate that the agonist-selective pathway in opiate tolerance development, i.e., both ?Arr and PKC pathways, are involved in tolerance development and that, by controlling the Src kinase activity within the OPRM1 signaling complex, the phosphorylation of AC by Raf-1 activated by Src leads to some if not all of the withdrawal signs observed during naloxone-precipitated withdrawal in mice chronically treated with an opioid agonist. Our proposed studies will link the action of the various protein kinases recruited and activated by OPRM1 in the behavioral responses to the chronic drug treatment. PUBLIC HEALTH RELEVANCE: Even after decades of intensive research by many laboratories, including our own, the exact detailed molecular mechanism for morphine tolerance and withdrawal remains elusive - possibly because the many mechanisms and pathways involved are different among the various opioid agonists. Our proposed studies will investigate the detailed mechanism by which the protein kinases participate in chronic drug action. From our studies, we anticipate a better understanding of molecular mechanism for morphine tolerance and dependence, eventually leading to treatment paradigms.

DESCRIPTION (provided by applicant): Although opioid drugs are effective for short-term pain relief, major obstacles remain with long-term use of these drugs. Specifically, the many side effects are associated with the drugs, such as increasing incidents of drug abuse leading to addiction, have hampered opioid drug usage. A probable mechanism for drug addiction involves the activation and alteration in the neural circuitry that normally is involved in pleasure, incentive motivation, and learning, during chronic drug exposure. In addition to dopaminergic inputs from the ventral tegmental area and substantia nigra to the nucleus accumbens and striatum, glutaminergic inputs from the prefrontal cortex, amygdala, and hippocampus also have important roles in chronic drug action. Since the hippocampus is the structure involved in the storage, consolidation, and retrieval of decorative, spatial, and long-term memory, understanding its roles in drug acquisition and relapse, as well as drug reward experiences, has gained importance. Both electrophysiological and morphological plasticity have been observed with the various hippocampal structures during the course of drug exposure. In addition, integration of newborn neurons to the existing circuit within the hippocampus may have pronounced effects on the drug experience. Considering that all addictive drugs have been shown to alter adult neurogenesis, elucidating the mechanism by which opioid drugs regulate adult neurogenesis, and identifying the specific aspect of the drug experience that adult neurogenesis participates in will have a significant impact in understanding the long-term use of opioid drugs. During the course of our studies on ?-opioid receptor (OPRM1) biased agonism, we observed that morphine and fentanyl, two highly prescribed opioids, regulate the microRNA-190 level differentially, leading to differences in NeuroD levels within primary hippocampal neuron cultures. Since NeuroD is the transcription factor involved in differentiation and maturation of neurons, we hypothesize that OPRM1, by controlling miR-190/NeuroD pathway activity, regulates adult neurogenesis in the hippocampus. We further hypothesize that, since morphine and fentanyl are both addictive, differential control of miR-190/NeuroD activity by these two agonists is not involved in the acquisition of addictive behavior, but rather in the consolidation and retrieval of the context memory associated with drug reward. Therefore, the proposed studies are designed: (A) to understand the molecular mechanism involved in morphine and fentanyl biased agonism so as to manipulate the outcomes of this biased agonism; (B) to establish that miR-190/NeuroD regulation is central to the agonists differential regulation of adult neurogenesis in the hippocampus; and (C) to link the regulation of NeuroD activities and neurogenesis with the extinction of conditioned place preference induced by opiate agonists. From these studies, we anticipate that, by manipulating the miR-190/NeuroD pathway activity, the extinction of the opioid drug reward experience and subsequent drug relapse, can be regulated, and a future treatment paradigm can be developed. PUBLIC HEALTH RELEVANCE: With the recent increase in prescriptions written, leading to increased assess to opioids, elucidation of the mechanism in which long-term use of opioid medication leads to addiction and relapse, is critical. Upon completion, our studies will provide important information on whether, by regulating the various aspects of the differentiation and maturation of newborn neurons in the brain, the drug experience can be modulated. This information will be essential in the future development of eventual treatment paradigms for opioid addiction and relapse.