Allyn C. Howlett - US grants

The psychoactive properties of cannabinoids have been described in man and investigated in a variety of animal models. Association of these effects with neurotransmitter systems has been investigated with limited success. In vitro studies have failed to elucidate the mechanism of cannabinoid action at the neuroneal level. Some investigators have concluded that the lipid bilayer of the neuronal membrane is the primary site of action of the canabinoids, and that it is unnecessary to invoke the existence of a specific cannabinoid receptor. An argument against this is that the only physiological action of cannabinoids that "partial anesthetics" produce is the catalepsy response. Furthermore, the relevance of these membrane effects to the psychoactivity of cannabinoids is questionable due to the equal efficacy and similar potency of nonpsychoactive compounds. Adenylate cyclase is one plasma membrane enzyme system in which cannabinoid effects have been noted. However, it is difficult to interpret studies of hormone-regulated cyclic AMP accumulation in brain because of the variety of cell types and the multiple neurotransmitter receptors putatively associated with adenylate cyclase in brain. Study of this interaction at the level of a neuronal cell is necessary to provide a functional basis with which to search for a specific cannabinoid receptor, if one exists. The specific aims of this study are to evaluate properties of cannabinold inhibition of adenylate cyclase in enuroblastoma membrane preparations and to characterize the cannabinoid effects intact neuroblastoma cells.

This study examines the neuronal response to neuromodulators that interact with the adenylate cyclase system of the cell. Neuroblastoma cells in culture have been shown to respond to Alpha-adrenergic and muscarinic agents by a decreased synthesis of cyclic AMP. The mechanism of the adenylate cyclase inhibition in this and other cell types has not been fully elucidated. Other systems, muscarinic and Alpha-adrenergic hormones alter cell membrane phospholipids in such a way as to promote Ca++ mobilization and arachidonic acid release as intermediary functions mediating the tissue specific response. Recent findings in my laboratory are consistent with a mechanism of phospholipid metabolism as a concurrent response to the inhibition of adenylate cyclase in neuroblastoma cells. It is the purpose of the proposed studies to examine the relationship between adenylate cyclase inhibition and the membrane phospholipid events leading to arachidonic acid release and metabolism in neuroblastoma cells. It is expected that these experiments will advance our understanding of neurotransmitter and hormone actions in the nervous system.

Delta 9-Tetrahydrocannabinol (Delta 9-THC) is the primary psychoactive compound in marihuana extracts. The mechanism of action of Delta 9-THC at the cellular level is unclear. The long- term objective of the proposed research is to determine how neurons respond to Delta 9-THC. The methodology unique to this project is the use of cloned neuroblastoma cells (N18TG2) in culture as a model system for neurons. Delta 9-THC inhibits adenylate cyclase in these cells via a receptor-regulated G protein. The specific aims of this proposal are to: 1) characterize structure-activity relationships in neuroblastoma membranes using cannabimimetic analgetic compounds; 2) determine if cyclic AMP is altered by cannabimimetic drugs in target sites in the rat brain; 3) develop a ligand binding assay to characterize the receptor in neuroblastoma and brain membranes; and 4) determine if cannabimimetic drugs cause significant effects on adenylate cyclase by altering membrane fluidity. Cannabimetic drugs are potentially useful as anti-glaucoma, analgesic, anti-emetic or anti-convulsant agents. Further, Delta 9-THC is one of the most popular drugs of abuse (smoked marihuana). In spite of this, very little is known concerning the way these drugs alter neurons in the brain. The proposed studies will elucidate at least one mechanism of action of cannabimimetic compounds (inhibition of adenylate cyclase), and may uncover other mechanisms which may or may not be receptor-mediated. Further investigations can define the complex interaction of neurons that respond to Delta 9-THC with other neurons in the brain.

The objectives of this project are to study the cannabinoid receptor- effector interactions at cellular and molecular levels in an effort to define the requirements for agonist and antagonist binding and production of a response. The first site of action of a drug is at the level of the receptor at the cell surface. If the ligand is an agonist, this ligand/receptor interaction will result in the induction or stabilization of a conformation of the receptor protein such that a productive interaction with an effector protein can take place. The adenylate cydase enzyme complex is the effector system coupled to the cannabinoid receptor. The inhibition of adenylate cyclase by cannabinoid drugs woldd modify the neuronal response as a result of a change in the phosphorylation state of critical regulatory enzymes and proteins. Subsequent alterations in the behavior of the neuron are manifest in the intact animal as one or more of the typical responses observed after administration of a cannabimimetic drug, e.g. cognitive and memory dysfunction, analgesia, changes in endocrine functions, or hypothermia and hypokinesia (immobility) observed in rodents. The specific aims are as follows: 1: To study the Structure-Activity Relationships of a series of cannabinoid compounds for their ability to bind to the cannabinoid receptor and to evoke the response of inhibition of adenylate cyclase. 2: To examine three irreversibly binding ligands for their actions at the cannabinoid receptor including binding and ability to evoke a persistenit response or an antagonistic action. 3: To evaluate three potential Positron Emission Tomography (PET) scanning ligands for their actions at the camabinoid receptor. The knowledge obtained from this study is essential 1) for the rational design of new therapeutic-entities, such as cannabinoid analgetic agents, 2) for the design and synthesis of analogs having agonist or antagonist activity at the cannabinoid receptor, and 3) to provide the basis for defining cannabinoid receptor subtypes which may ultimately be shown to be responsible for certain of the multiple biological effects of cannabinoid drugs.

DESCRIPTION (provided by applicant): The CB1 cannabinoid receptor is a target for pharmaceutical drug design for neuroprotection in stroke, muscle relaxation in spastic disorders, analgesia, metabolic energy balance, and substance abuse disorders. However, unwanted side effects, such as cognitive and memory dysfunction, emotional disorders, and sedation, have curtailed development of CB1 ligands for therapeutic purposes. CB1 receptors regulate signal transduction pathways that modulate neuronal functions intrinsic to neuroprotection, synaptic plasticity, and neurotransmitter release. The working hypothesis is that CB1 receptor agonist and antagonist ligands exert their influence on the juxtamembrane C-terminal domain to initiate key signal transduction pathways in neuronal cells;and that this signaling can be modulated by palmitoylation, phosphorylation, and protein association at the juxtamembrane C-terminal domain. The proposed research will elucidate mechanisms by which CB1 ligands trigger receptor-Gi/o protein activation of signal transduction via the effectors adenylyl cyclase and mitogen activated protein kinase (MAPK), leading to phosphorylation pathways regulated by protein kinase A (PKA) and ERK1/2. The aims are to: 1. Characterize the signal transduction dependence on the juxtamembrane C-terminal domain. The cAMP/PKA and ERK responses to CB1 agonists having diverse structure and efficacy will be investigated in CB1 (L7.60(404)I,F) mutants stably expressed in HEK293 fibroblast and SH-SY5Y neuronal cells. 2. Characterize the effects of palmitoylation at the juxtamembrane C-terminal on CB1 receptor function by determining agonist-evoked responses in a C7.71(415)S CB1 mutant stably expressed in HEK293 and SH- SY5Y cells. 3. Characterize the effects of phosphorylation at the juxtamembrane C-terminal Ser/Thr on CB1 receptor function by determining agonist-evoked responses in phosphorylation-deficient (S401A, S410A, S414A) and phosphorylation-mimics (S401D, S410D, S414D) CB1 mutants stably expressed in SH-SY5Y cells. 4. Characterize the regulation of signal transduction via GASP1 interaction with the juxtamembrane C-terminal H8 domain of the CB1 receptor by examining the influence of juxtamembrane C-terminal disruption (L7.60(404)I,F), palmitoylation, and phosphorylation on CB1-GASP1a protein associations will be examined. The results of the proposed research will advance our understanding of biochemical mechanisms by which CB1 orthosteric ligands activate neuronal signaling responses. This understanding is critical to the design of drugs targeting the beneficial clinical responses with minimal undesirable effects. PUBLIC HEALTH RELEVANCE: The CB1 cannabinoid receptor is a target for pharmaceutical drug design for neuroprotection in stroke, muscle relaxation in spastic disorders, analgesia, metabolic energy balance, and substance abuse disorders. Unwanted side effects (cognitive and memory dysfunction, emotional disorders, and sedation) have curtailed development of CB1 ligands for therapeutic purposes. The proposed research will elucidate biochemical mechanisms by which ligands regulate CB1 receptors at a domain that is key for initiation of cellular signaling processes. This information is critical for the design of drugs that can stimulate the therapeutic responses but diminish the undesirable effects. Development of legitimate CB1 medicines can dispel myths associated with the notion of "medical marihuana".

Delta9-Tetrahydrocannabinol (THC) is the primary CNS-active component of marijuana or hashish (Cannabis sativa). Potent tricyclic and bicyclic cannabinoid analogs developed at Pfizer Central Research were utilized to elucidate a cellular mechanism of action for delta9-THC. We determined that cannabimimetic compounds interact with a cannabinoid receptor on neuronal cells which regulates the cyclic AMP second messenger system. We developed a radioligand binding assay using the potent bicyclic cannabinoid agonist [3H]CP-55940, and have used it to biochemically and pharmacologically characterize the cannabinoid receptor. The goal of this project is to study cannabinoid receptor-ligand interactions at the cellular and molecular levels. This work will continue through the following specific aims to: 1.Continue characterization of novel affinity ligands and positron emission tomography (PET) scanning ligands; 2.Identify a cannabinoid-like binding activity present in the CNS, and characterize and purify the factor(s) responsible for this activity; and 3.Develop an immunoassay for the cannabinoid-like binding factor(s). It is expected that the determination and characterization of an endogenous cannabinoid-like binding factor(s) for the cannabinoid receptor will allow the development of new structure-activity relationship studies based on this novel molecule. This knowledge could lead to the development of potentially useful antagonists for the cannabinoid receptor. In addition, receptor subtypes for the endogenous cannabinoid-like binding factor could be sought. These studies will advance our understanding of the role of cannabinoceptive neurons in analgesia, cognition and memory, and motor behaviors.

Delta9-Tetrahydrocannabinol (THC) is the primary CNS-active component of marijuana or hashish (Cannabis sativa). Potent tricyclic and bicyclic cannabinoid analogs developed at Pfizer Central Research were utilized to elucidate a cellular mechanism of action for delta9-THC. We determined that cannabimimetic compounds interact with a cannabinoid receptor on neuronal cells which regulates the cyclic AMP second messenger system. We developed a radioligand binding assay using the potent bicyclic cannabinoid agonist [3H]CP-55940, and have used it to biochemically and pharmacologically characterize the cannabinoid receptor. The goal of this project is to study cannabinoid receptor-ligand interactions at the cellular and molecular levels. This work will continue through the following specific aims to: 1.Continue characterization of novel affinity ligands and positron emission tomography (PET) scanning ligands; 2.Identify a cannabinoid-like binding activity present in the CNS, and characterize and purify the factor(s) responsible for this activity; and 3.Develop an immunoassay for the cannabinoid-like binding factor(s). It is expected that the determination and characterization of an endogenous cannabinoid-like binding factor(s) for the cannabinoid receptor will allow the development of new structure-activity relationship studies based on this novel molecule. This knowledge could lead to the development of potentially useful antagonists for the cannabinoid receptor. In addition, receptor subtypes for the endogenous cannabinoid-like binding factor could be sought. These studies will advance our understanding of the role of cannabinoceptive neurons in analgesia, cognition and memory, and motor behaviors.

Synthetic derivatives of delta/9-THC are known to have certain therapeutic effects, including relief from pain, diminished nausea and vomiting due to cancer chemotherapies, and decreased ocular pressure in glaucoma. However, to date no therapeutic cannabinoid has been developed that is devoid of psychoactive effects such as sedation, loss of short-term memory, and disorientation. In addition, no high-affinity antagonist exists with which important biochemical and pharmacological data concerning the cannabinoid receptor could be obtained. To address the cannabinoid receptor as a potential site for therapeutic pharmacological intervention, additional information concerning cannabinoid receptor- ligand interactions is required. The specific aims of this Biochemistry section of the Program Project are: 1. Characterization of covalent affinity ligands: A) Characterize the binding of novel cannabinoid ligands to wild-type cannabinoid receptors in baculovirus-infected Sf9 cell membranes and, B) Determine the site(s) of receptor-ligand interaction in wild-type receptors by analysis of covalently labeled proteolyzed receptor peptides. 2. Characterization of cannabinoid receptor chimeras: A) Characterize the binding of cannabinoid ligands to cannabinoid receptor chimeras expressed in Sf9 membranes. B) Characterize the functional properties of wild-type and chimeric receptors when reconstituted with G proteins in defined phospholipid vesicles.

DESCRIPTION: (Applicant's Abstract) Central nervous system (CNS) responses to delta-9-tetrahydrocannabinol (delta-9-THC) in Cannabis sativa and synthetic cannabimimetic drugs include the therapeutically beneficial effects of analgesia, attenuation of the nausea and vomiting in cancer chemotherapy, appetite stimulation in wasting syndromes, and reduction of intestinal motility. Untoward side effects accompanying these therapeutic responses include alterations in cognition and memory, dysphoria/euphoria, and sedation. Potential therapeutic applications can be found for both CB1 receptor agonist and inverse agonist ligands. It is expected that the CB2 receptor will play a major role in therapeutic manipulation of the immune response. In order to develop therapeutic agents that exhibit selectivity for one subtype over another, it is necessary to understand how agonist ligands interact with the CB1 and CB2 receptors to bind with high affinity and evoke a response. The proposed studies directly examine this interaction, and define mechanisms for inverse agonists that can reverse the effects of active precoupled receptors. Computer-Aided Drug Design (CADD) approaches will be applied (1) to elucidate the structural prerequisites for binding and activity of ligands for the CB1 and CB2 cannabinoid receptors, and (2) to design potent and selective ligands for these receptors. The specific aims are to test the following hypotheses: 1. CB1 cannabinoid receptor agonists of multiple structural classes (cannabinoid, aminoalkylindole (AAI), and eicosanoid) can bind to common pharmacophoric sites in the CB1 receptor to evoke a response. 2. Agonist ligands of multiple structural classes (cannabinoid, AAI, and eicosanoid) can bind to common pharmacophoric sites on the CB2 receptor. A subset of these pharmacophoric points will differ from those of the CB1 receptor. 3. CB1 receptor antagonists can compete with cannabinoid agonists for binding interactions with the CB1 receptor but are incompetent to induce a conformational transition in the receptor. 4. A population of CB1 cannabinoid receptors exist in a state that can be stabilized by inverse agonist binding (R or R) Determinants of that conformation can be found from the SAR of inverse agonist ligands.

DESCRIPTION: Applicant's Abstract This R13 application is being submitted by the International Cannabinoid Research Society (ICRS), a not-for-profit scientific professional society incorporated in the United States, for funding of travel award support for participants to attend the ICRS-sponsored annual Symposia on the Cannabinoids. Consistent with the ICRS goal of recruiting and retaining young scientists into careers in drug abuse research, these awards will be made by the ICRS Executive Committee to predoctoral and postdoctoral students and young investigators who demonstrate financial need and promising research careers. The ICRS also recognizes the need to encourage research careers by individuals from racial and ethnic minority groups. Efforts will be made to increase the pool of researchers from these populations with the use of travel awards to the ICRS Symposia. The meeting format for this international conference is that ICRS members, students and nonmembers (estimated at 100-150), present either oral or poster presentations over the course of the 3 day conference. Research topics to be covered include biochemical, physiological, pharmacological, behavioral, and therapeutic aspects of cannabinoid drugs and endogenous compounds. Annual funding of $25,000 is requested for a total of five years of support for this international conference ($125,000). In the first year of requested support, the ICRS 2000 Symposium on the Cannabinoids will be a 3-day conference to be held at Hunt Valley Inn in Baltimore, MD, June 22-24, 2000. Subsequent years are being planned with the following venue: June, 2001 in Madrid, Spain; July, 2002 in San Francisco, CA; June, 2003 in east coast US; June, 2004 in Sorrento, Italy. Funding (80%) is requested to provide travel, the room and board package and registration fees for United States citizens and permanent residents to participate in the annual Symposium on the Cannabinoids. Additional support (20%) is requested for conference abstract publication, supplies, audiovisual equipment rental and operation, and communications. Appropriate representation of women has been assured in all aspects of planning, organization and implementation of the annual conferences.

DESCRIPTION: (Applicant's Abstract) North Carolina Central University is responding to the NIH-National Institute on Drug Abuse and Office of Research on Minority Health initiative entitled HBCU Research Scientist Award. The proposed NCCU drug abuse research program in collaboration with Wake Forest University School of Medicine, will focus on molecular mechanisms of drug-cell interaction, seeking to understand the molecular correlates of addictive behavior. The program will be housed in the new Biomedical/Biotechnology Research Institute. Working with NIDA and ORMH, NCCU will address the program goals and support the growth and development of drug abuse research by: assembling a panel of scientists external to NCCU that could serve as a drug abuse research advisory committee: identifying and recruiting an experienced individual generally recognized as an accomplished drug abuse research scientist: establishing formal linkages with scientists at the drug abuse research center located at Wake Forest University School Medicine, located approximately 110 miles from NCCU: cost effective assembly and training of a high quality team of scientists to conduct drug abuse research at NCCU: and serving as a catalyst to enhance public awareness, particularly among the minority community, of the behavioral and public health consequences of drug addiction.

[unreadable] DESCRIPTION (provided by applicant): This is a Research Scientist Award (RSA) for Dr. Allyn C. Howlett, whose academic experience includes a B.S. cum laude in Biochemistry from Pennsylvania State University, a Ph.D. in Pharmacology and Toxicology from Rutgers University, post-doctoral work with Alfred G. Gilman at the University of Virginia, and 20 years on the faculty at Saint Louis University School of Medicine. Dr. Howlett is a tenured Professor in the Department of Biology and Director of the Neuroscience/Drug Abuse Research Program at the Julius L. Chambers Biomedical/Biotechnology Research Institute at North Carolina Central University. Dr. Howlett's research effort is concentrated on the pharmacological, biochemical and cellular regulation of the CB1 receptor. Her intention is to maintain an active research laboratory in which students, fellows, and visiting scientists can exchange ideas and share expertise. Toward this goal, Dr. Howlett is seeking this RSA in order to allow ample research time to conduct her research, mentor students and fellows, and to maintain research collaborations. The RSA will also foster continued professional development through participation in research meetings and short courses, and communication with other research scientists. Dr. Howlett's research projects have been described by two NIDA-funded R01 grants. "Cannabinoid Receptor Structure-Activity Relationships" examines the receptor interactions with ligands of various chemical classes (cannabinoid, amincialkylindole, eicosanoid and arylpyrazole) in an effort to define the requirements for high affinity ligand-receptor association and efficacy. This project involves close collaboration with synthetic chemists and computational chemists. "Cannabinoid Receptors in Neuronal Cells and Brain" addresses the signaling mechanisms by which the CB1 receptor evokes its response through selected G protein subtypes and their ensuing signal transduction pathways [adenylyl cyclase, mitogen-activated protein kinase (MAPK) and other kinases, Ca2+ mobilization and nitricoxide synthase (NOS) activation]. The project also examines the mechanisms by which the cell regulates signal transduction through the reversible post-translational modifications of palmitoylation and phosphorylation.

DESCRIPTION (provided by the applicant): The NCCU-WFU Biomedical Sciences Bridge Partnership is a Bridges to the Future initiative for the training of M.S. students in the Department of Biology at North Carolina Central University (NCCU) to continue in their Ph.D. training at the Department of Physiology and Pharmacology at Wake Forest University (WFU). Our goal to create and maintain a "research environment" at NCCU will be achieved by maintaining strong research programs through NCCU's J.L. Chambers Biomedical/ Biotechnology Research Institute (JLC-BBRI), by increasing awareness of the NCCU-WFU Bridge Partnership program among Ph.D.-directed candidates, selecting graduate students with potential for success, and nurturing them through laboratory research and research-related activities. Our goal to foster the "research thinking process" among graduate students will be achieved by promoting level Ill cognitive skills into coursework. Our goal to bridge students into an advanced position when entering the Ph.D. phase of their training, yet diminishing the social disadvantage, will be implemented by developing faculty research collaborations between institutions so that students can work on aspects of the project at both institutions, and by fostering joint programs between students at NCCU and WFU. The NCCU-WFU Biomedical Sciences Bridge Partnership proposes to take advantage of the unique research environment of the JLC-BBRI on the NCCU campus by making available to underrepresented minority students the dual environments of an active biomedical research facility and the HBCU community. To achieve these goals, we are requesting support for 3 graduate assistantships, tuition, health insurance, computers and travel to an annual scientific meeting for 3 M.S. students in year one, to increase to 6 students in year three. We are also requesting 3 years of support for a program administrator and office assistant at 25% effort each, and support supplies and travel cost related to the administration of this program. Support of students via the NCCU-WFU Biomedical Sciences Bridge Partnership will solidify the existing effective inter-institutional partnership between NCCU and WFU that will improve the quantity, but more importantly, the quality, of the next generation of underrepresented minority scientists in the U.S.

[unreadable] DESCRIPTION (provided by applicant): This is the first competitive continuation of a Bridge to the Ph.D. graduate training partnership in the biomedical sciences between North Carolina Central University (NCCU) M.S. and Wake Forest University (WFU) Ph.D. programs. In the first cycle of funding, we built the program at NCCU to support four to six M.S. students in Biology (particularly in cardiovascular and neuropharmacology research), and have successfully sent our first Bridge M.S. in Biology graduate to the WFU Physiology and Pharmacology Ph.D. program. We now propose a goal to expand the disciplines of study at both partner institutions and to develop mechanisms to foster a smoother transition between the two phases of academic training for our students. Aim 1 proposes to expand the program at NCCU to embrace students participating in the expanding number of research disciplines that are being developed at this institution. This will be accomplished as NCCU formally develops an internal minor in biochemistry within the Chemistry M.S. program, and develops advanced graduate coursework in cancer biology, bioinformatics and bioprocessing. Aim 2 proposes to expand the participating programs at WFU. This will be accomplished with the inclusion of the well established Ph.D. programs in Biochemistry, Cancer Biology, Neuroscience, and Molecular and Cellular Pathobiology. Aim 3 proposes to strengthen transition to the Ph.D. phase for students. This will be accomplished by fostering student participation in NCCU and professional society programs in which student research communications skills are developed, by incorporating research rotation modules at WFU for students during the completion of their M.S. phase, and by continuing to develop research collaborations between the faculty at the two institutions that can facilitate student interactions with researchers at the Ph.D. institution prior to entering the Ph.D. phase of the training. In the next cycle of funding, we expect to be able to support six M.S. students through two to three years of M.S. graduate research assistantships with tuition, send two Ph.D.-directed undergraduate students per year to WFU for an intensive summer research experience, and support travel for each student to a professional society meeting to present their work. [unreadable] [unreadable]

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): The goal of the proposed Postdoctoral Research, Instruction, and Mentorship Experiences (PRIME) IRACDA program is to develop highly skilled biomedical scientists to teach the next generation of clinical researchers and allied health professionals, as well as to promote research competencies of under-represented minority undergraduate and graduate scholars in the health professions. Wake Forest University School of Medicine (WFSM) and Winston-Salem State University (WSSU), an HBCU with longstanding commitment to biomedical and allied health training programs will train PRIME scholars by integrating traditional mentored postdoctoral research at WFSM, regular in-depth professional development programming at WFSM, WFU and WSSU, and a broad variety of teaching assignments in pre-professional and allied health professional (with a focus on Physical Therapy) courses at WSSU. PRIME will aid in recruitment, retention, and development of more underrepresented minority clinical scientists by immersing PRIME scholars, their WFU mentors, and undergraduates in the MARC U*STAR and MBRS RISE programs at WSSU in a rich collaborative learning environment. Additionally, PRIME will strengthen existing collaborations in research and teaching between WFSM and WSSU. To meet these goals, the PRIME Program aims are to: 1. Train scholars in research with a faculty member in WFU's programs in Integrative Physiology and Pharmacology, Neuroscience, Molecular Medicine and Translational Sciences, or Molecular Pathology. 2. Train scholars in mentored teaching experiences at WSSU for the entire three year training, including tutoring, lecturing, laboratory design and development, guiding students through robotics simulations, leading case-based learning (PBL), and open-source digital teaching tools. 3. Introduce PRIME scholars to current pedagogical techniques and educational philosophy through a semester-long course, and short workshops from the WFU Teaching and Learning Center and others. 4. Facilitate mentoring skills by pairing PRIME scholars with WFSM faculty to oversee the research training of WSSU MARC U*STAR and MBRS-RISE undergraduates, and PREP postbac students. 5. Train PRIME scholars in translational research practices (WSSU DPT) and grant writing (WFSM). 6. Train PRIME scholars to become leaders in Responsible Conduct of Research (RCR) education programs. We will recruit 3 PRIME scholars each year for 3 years (a total of 9) from a national pool of racial and ethnically diverse individuals who hold the Ph.D., M.D. or comparable degree. Outcomes of the program will be documented as academic progress in both research and teaching (presentations, publications, meritorious activities), as well as benefits to partner WSSU (improved outcome measures for students~ sustained course modules digitally captured for plug-and-play use). Strategic Evaluations, Inc. is the external evaluation team.

PROJECT 3 CONSEQUENCE OF NOVEL COCAINE PHARMACOTHERAPIES ON NEUROCHEMISTRY IN RODENT MODELS OF COCAINE DEPENDENCE Allyn Howlett, Director; Rong Chen and Steven Childers, Co-Investigators SUMMARY Cocaine addiction produces neuroadaptations in multiple neurotransmitter systems that perpetuate cocaine relapse. We hypothesize that neurochemical neuroadaptions resulting from cocaine self-administration will be modified by drug combinations that consists of an agonist pharmacotherapy with a cognitive remediator. Project 3 will delineate how these neuroadaptions can be treated with a cocaine-substitute (agonist) drug, phendimetrazine (active form is phenmetrazine (PM)), combined with Drug B candidates that serve as cognitive remediators, as identified by cognition testing in Project 1. We will examine the cellular and molecular mechanisms by which these combination medications modify brain neurochemistry to reduce cocaine self- administration and reverse cognitive deficits produced by cocaine. Aim 1 will determine the impact of PM in combination with Drug B cognitive remediators on neuroadaptations associated with chronic cocaine self- administration in rats, beginning with the mGluR2/3 agonist LY-379268. We will examine the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC) and other limbic areas for changes in: 1) gene expression determined by quantitative PCR; 2) membrane expression of receptors and transporters assessed by radioligand binding; 3) receptor function by GTP?S binding; 4) trafficking of receptors and transporters assayed by tissue fractionation and Western blotting; 5) signal transduction determined by phosphorylation of regulatory proteins. Aim 2 will determine the differential role of D2 autoreceptors and postsynaptic D2 receptors in NAc (using Intracranial lentivirus delivering siRNA constructs) in the mechanism of action of drug combination pharmacotherapies determined by Project 1. Experiments will determine: neuroadaptations of intra-VTA or intra-NAc knockdown of D2 receptors; how neuroadaptations of PM alone or in combination with drug B are modified by D2 autoreceptor or postsynaptic D2 receptor knockdown; how neuroadaptations of cocaine self-administration are modified by D2 autoreceptor or postsynaptic D2 receptor knockdown; and the efficacy of drug combinations on normalization of neuroadaptations in D2 knockdown animals self- administering cocaine. These neurochemical data will explain mechanisms for the development of addiction and cognitive deficits associated with cocaine self-administration, and will define how PM and cognition remediators function at the neuronal level. Since the animals used in Project 3 will be essentially the same as those used in Project 2, biochemical data from Project 3 can be directly compared with voltammetry, microdialysis, and neuronal activity data from Project 2. These results will provide pharmacodynamic evidence needed for rational design of clinical studies of novel drug combinations in addiction treatment.

Summary The CB1 receptor (CB1R) a therapeutic target for treatment of addictions, neurodegenerative disorders and pain management, but medicinal compounds based upon the CB1R have been limited. The functions of the CB1R to regulate neuronal processes in development, retrograde signaling in neurotransmission, and cellular mechanisms of neuroprotection are critical to brain function. Endocannabinoid ligands 2-arachidonoylglycerol and anandamide are the primary neuromodulators of synaptic activity, but the full understanding of how CB1R signaling can be regulated by associated proteins in specific cell types is just beginning to be appreciated. The Scientific Premise is that CRIP1a modulation of the CB1R can be understood at the structural and functional level such that drug design based on peptide or small molecule interventions can target the CRIP1a-CB1R interaction. Our recently published studies have demonstrated that CRIP1a reduces the density of cell surface CB1R, attenuates the agonist-dependent but not the constitutive internalization processes by competing with ?-arrestins for binding to C-terminal sites, and curtails the trafficking of newly- synthesized CB1R to the cell surface after prolonged WIN55212-2 but not CP55940. Other studies demonstrated that CRIP1a has a critical role in regulating CB1R cellular signaling by altering the preference for coupling from Gi3 & Go, which require the C-terminus for activation, to Gi1 & Gi2, which do not. In unpublished studies, we have determined the high resolution structure from X-ray crystallography, and found that CRIP1a is a member of the family of carriers for myristoylated or isoprenylated proteins. Based upon this major advance in knowledge of the structure and function of CRIP1a, we hypothesize that the function of CRIP1a is to interact with the CB1R?G-protein complex in ways that can be regulated by G-alpha and/or G-gamma subunit specificity, phosphorylation, and interaction with other regulatory proteins that are known to release cargo. We propose to investigate the CB1R associated proteins in the N18TG2 neuroblastoma cell model which endogenously expresses the CB1R and its associated proteins, as well as in in vitro experiments of purified recombinant proteins and peptides derived therefrom. The aims of this project are to investigate: the interaction of CRIP1a with the CB1R; the structural and functional interaction of CRIP1 with G- proteins; and the regulation of CRIP1a function by cargo-releasing proteins and phosphorylation. The results of the proposed investigation should prove to be transformative for the field by providing evidence that CB1R and associated CRIP1a interact to direct cellular signaling pathways. From this understanding, novel peptides and small molecules could be developed as therapeutic agents for neurological diseases in which both CB1R and CRIP1a co-exist in neurons.