Kenneth P. Mackie - US grants

The potent psychoactive properties of the cannabinoids and the identification and cloning of a cannabinoid receptor suggest they mimic endogenous compounds which modulate neural signals for mood, memory, movement and pain. Preliminary studies (below) have found that cannabinoids, potently and reversibly, inhibit N-type calcium channels via a Pertussis toxin-sensitive mechanism. The first part of the proposed study will extend these observations to acutely dissociated CNS neurons from regions rich in cannabinoid receptor with the aim to fully dissect the signal transduction pathway between the receptor and calcium channel. The second part proposes to develop polyclonal antibodies against the cannabinoid receptor using synthetic peptides based on the cDNA sequence of the cloned receptor. These antibodies will be used for anatomical, developmental, and biochemical characterization of the receptor. We anticipate that by utilizing two complementary techniques, one electrophysiological and the other immunological, we will substantially add to our knowledge of the cellular actions of the cannabinoids. This insight will be useful as it will both increase our understanding of a novel neurotransmitter system with marked behavioral effects, and will also allow a more rational assessment of the use of cannabinoids as therapeutic agents.

This is an application for a NIDA RSDA award. The primary focus of my research is to understand the mechanisms underlying the acute and chronic behavioral effects of cannabinoids (the principal psychoactive constituents of marijuana). My work is focused on the function and regulation of the neuronal cannabinoid receptor (CB1). Cannabinoids, acting at the CB1 receptor, are potent modulators of ion channel function. Specifically, they inhibit and Q-type voltage-dependent calcium channels and activate inwardly rectifying potassium channels. Consistent with these actions, cannabinoids decrease neuronal excitability and neurotransmission from CB1-expressing neurons. It is likely that these phenomena lead to the behavioral effects of cannabinoids. Over the next five years, we will continue our studies at the molecular and genetic level, emphasizing the following specific aims: * Is tolerance to cannabinoids in animals due to the phosphorylation and/or internalization of CB1 receptors? * Is the endogenous cannabinoid system necessary for the development of opiate tolerance? * What is the role of CB1 receptor dimerization in CB1 signaling and desensitization? * Are there CB1 and CB2 agonists that cause little or no desensitization of CB1 or CB2 receptor signaling? * Which domains of the potassium M current channel are important for its modulation by GPCR's? Accomplishing these goals will significantly advance our understanding of the cellular actions of cannabinoids. To accomplish these ambitious goals I have assembled a group of collaborators and consultants whose expertise complements that currently in place in my laboratory. Three facts underscore the importance of having a solid understanding of the CB1 receptor function at the molecular level: (1) Marijuana use is increasing and is a significant social issue. (2) Cannabinoids have therapeutic potential and we need a firm understanding of their cellular and chronic effects to understand the implications of their chronic use. (3) We know little about the physiological role of endogenous cannabinoids in healthy or diseased brain. Funding of this proposal will permit the investigator sufficient research time to accomplish the goal of elucidating the neuronal basis of cannabinoid action.

DESCRIPTION (Applicant's Abstract): Cannabinoids, the principal psychoactive constituents of marijuana, have profound effects on mood, memory, movement, and nocioception. Acting via the neuronal cannabinoid receptor (CB1), these compounds are potent modulators of ion channel function. Specifically, they inhibit N- and Q- type voltage-dependent calcium channels and activate inwardly-rectifying potassium channels. These actions suggest that cannabinoids may elicit their behavioral effects by decreasing neuronal excitability and neurotransmitter release from CB1- expressing neurons. During the next five years we propose to study in detail the molecular events involved in cannabinoids inhibition of neurotransmitter release and neuronal excitability, the modulation of these responses by protein kinase C, and the early molecular events associated with the development of tolerance to cannabinoids. These studies involve three major aims: 1. Defining the extent and mechanisms of inhibition of neurotransmitter release by cannabinoids in neuronal cultures using electrophysiological and imaging techniques. 2. Delineating the role of protein kinase C (PKC) in cannabinoid signaling. Recently we have found that ion channel modulation by cannabinoids is strongly disrupted by protein kinase C activation and that CB1 receptor is a substrate for PKC. We will determine if CB1 phosphorylation occurs in situ, if nurotransmitter activation of PKC disrupts CB1 signaling, and if PKC activation prevents cannabinoid-mediated inhibition of neurotransmitter release. 3. Identifying the molecular events associated with the development of cannabinoid tolerance. These studies will significantly advance our understanding of the cellular actions of cannabinoids. There are three reasons why this is imnportant: (1) Interest in marijuana is increasing and is a significant social issue (witness the ballot initatives in a number of states); (2) cannabinoids have therapeutic potential and we need a firm appreciation of their cellular physiology to exploit this potential; (3) we know little about the physiological role of endogenous cannabinoids in health or diseased brain.

[unreadable] DESCRIPTION (provided by applicant): Endogenous cannabinoids (endocannabinoids) and the CB1 cannabinoid receptor form a signaling system that plays a key role in several forms of short and long term neuronal plasticity. In addition, CB1 receptors are the primary target of cannabis, the most commonly used illicit drug in the US. Despite an increasing awareness of the importance of the cannabinoid signaling system in normal and pathological CNS function, the potential therapies targeting this system, and the social impact of cannabis use, our knowledge of how CB1 receptors and endocannabinoid production is regulated is quite modest. Understanding the control of the endocannabinoid system is vital to the rational assessment of therapies affecting this system, as well as to appreciating the impact of chronic activation of this system (as might happen therapeutically or by excessive cannabis use). In the proposed work we will address three specific aims: Is desensitization of cannabinoid inhibition of neurotransmission due to phosphorylation of the CB1 receptor at residues $426 and $430? Does regulated association of CB1 cannabinoid and D2 dopamine receptors underlie some of the interactions between dopamine and cannabinoid signaling? Which endocannabinoids are secreted by cultured neurons and from where are they released? By accomplishing these specific aims we will have a firm understanding of the molecular events associated with desensitization of CB1 receptor signaling, insight into the interactions between the cannabinoid and dopamine signaling pathways, knowledge of the endocannabinoids released by neurons and the domains of the neuron important in endocannabinoid release. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): CB2 cannabinoid receptors: new vistas is an original and unique conference whose aim is to bring together established scientists, post-docs, and graduate students from academia and industry who are interested in the biology of the CB2 cannabinoid receptor system. This meeting will be held in Banff, Alberta, Canada from May 31 to June 3, 2007. Our understanding of the biological role of CB2 receptors has lagged considerably behind that of CB1 receptors. However, ongoing work from a number of labs, both academic and industrial, suggests that CB2 receptors play an important role in many chronic inflammatory diseases. On one hand the pathogenesis and progression of these diseases may well be affected by cannabis use. Alternatively, manipulation of CB2 receptors and/or the endogenous cannabinoids activating them may be therapeutically beneficial. CB2 receptors have been implicated in processes and diseases as diverse as chronic pain, hepatic fibrosis, osteoporosis, gastrointestinal motility, atherosclerosis, and neuroinflammation. In addition to advances in understanding the role of CB2 receptors in pathological states, progress has been made in the unraveling CB2 receptor signaling and numerous novel CB2 ligands have been developed. An unintended consequence of the rapid development of the CB2 field is that these advances have occurred in disparate disciplines, which for a variety of reasons have a limited opportunity to communicate and share findings. The goal of this meeting is to bring together these diverse groups, present the most recent findings in the CB2 field, and to determine the most important and rewarding areas for future investigation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is an application for a NIDA senior scientist award. The primary goal of my research is to understand the mechanisms underlying the acute and chronic effects of cannabinoids (the principal psychoactive constituents of cannabis). My studies are focused on the function and regulation of the CB1 (neuronal) and CB2 ("peripheral") cannabinoid receptors. Cannabinoids, acting at CB1 receptors are potent modulators of ion channel function. Consistent with these actions, efficacious cannabinoids decrease neuronal excitability and inhibit neurotransmitter release. Interestingly, endogenous cannabinoids acting via CB1 receptors inhibit neurotransmission and mediate several forms of short and long-term neuronal plasticity-in part through non-ion channel signaling pathways. CB2 receptors, whose activation is devoid of apparent psychoactivity, have recently been shown to be involved in processes as diverse as analgesia, bone growth, and atherosclerosis, and are receiving intense scrutiny as potential therapeutic targets. Over the next five years we will continue our studies at the molecular and genetic level, emphasizing the following specific aims. Is behavioral tolerance to cannabinoids due to CB1 receptor phosphorylation? Is it possible to identify CB1 and CB2 agonists that cause little desensitization? What role does dimerization have in the signaling of CB1 and CB2 receptors? What are the determinants of the speed of CB1 receptor signaling? What is the role of GPR55, a third cannabinoid receptor, in cannabinoid action? Accomplishing these goals will significantly advance our understanding of the cellular actions of cannabinoids. To accomplish these ambitious goals I have assembled a group of collaborators and consultants whose expertise complements that currently in place in my laboratory. Three facts underscore the importance of having a solid understanding of cannabinoid receptor function at the molecular level. 1. Chronic cannabis use is a significant social issue. 2. We need a firm understanding of the cellular effects of cannabinoid receptor activation to interpret the possible benefits of their therapeutic manipulation. 3. We know little about the physiological role of endogenous cannabinoids in healthy or diseased tissues.

Absence of pain sensation; Absence of sensibility to pain; Adipocytes; Adipose Cell; Agonist; Analgesic Agents; Analgesic Drugs; Analgesic Preparation; Analgesics; Animal Model; Animal Models and Related Studies; Anodynes; Antinociceptive Agents; Antinociceptive Drugs; Assay; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Autoregulation; Behavior; Bioassay; Biologic Assays; Biological Assay; Bone Growth; CB1 Receptor; CB2 Receptor; Cannabinoids; Cannabinoids, Endogenous; Cannabis; Cell Communication and Signaling; Cell Signaling; Cells; Cellular biology; Characteristics; Chemistry; Chronic Disease; Chronic Illness; Class; DNA Molecular Biology; Deep; Depth; Development; Dimerization; Drugs; Drugs, Illicit; EC 2.7.2-; Endocannabinoids; Event; Extracellular Signal-Regulated Kinases; Fat Cells; Feels no pain; G Protein-Complex Receptor; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GPCR Signaling; Grant; Hemp Plant; Hepatic Cells; Hepatic Parenchymal Cell; Hepatocyte; Homeostasis; Illicit Drugs; Image; Intracellular Communication and Signaling; Investigators; Knowledge; Lead; Ligands; Lipocytes; Liver Cells; Localized; MAP kinase; MAPK; Marihuana; Mature Lipocyte; Mature fat cell; Mediating; Medication; Memory; Metabolic; Mitogen-Activated Protein Kinases; Molecular; Molecular Biology; Names; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurons; No sensitivity to pain; Numbers; Pb element; Pharmaceutic Preparations; Pharmaceutical Preparations; Physiological Homeostasis; Play; Principal Investigator; Process; Programs (PT); Programs [Publication Type]; Protein Dimerization; Rate; Receptor Activation; Receptor Protein; Receptor Signaling; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Regulation; Research Personnel; Research Resources; Researchers; Resources; Role; Science of Chemistry; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Specificity; Symptoms; Testing; Therapeutic; Work; analgesia; atheromatosis; atherosclerotic vascular disease; biological signal transduction; cannabinoid receptor; cell biology; chronic disease/disorder; chronic disorder; craving; desensitization; drug/agent; heavy metal Pb; heavy metal lead; imaging; insight; interdisciplinary approach; interest; macrophage; model organism; neuroinflammation; neuronal; programs; receptor; social role

DESCRIPTION (provided by applicant): The study of pro- and anti-nociceptive molecules produced by the body may yield important targets for improved treatments of pain. A recent and promising avenue of research has been the elucidation of the biological effects of a family of lipid signalling molecules referred to as fatty acid amides. The proposed experiments focus on the characterization of N-acyldopamines, a novel group of fatty acid amides that may serve as signalling ligands for the vanilloid and/or cannabinoid receptors. Since activation of cannabinoid receptors typically suppresses pain, while activation of vanilloid receptors (e.g. by capsaicin) typically heightens sensitivity to pain, N-acyldopamines may serve endogenously to facilitate or dampen pain. We hypothesize that the actions of these molecules depend on the locations at which they are formed and the state of the cellular environment. Hence, we plan to investigate the occurrence of these compounds at various sites within the nervous system and the effect of various physiological and pathophysiological conditions such as inflammation on their formation and bioactivity. In addition to examining cannabinoid and vanilloid mechanisms, we plan to examine oxygenated metabolites of NADA that we observed in vivo. The proposal aims to elucidate the biology and function of endogenous N-acyldopamines and their oxygenated metabolites in order to enhance our understanding of this emerging class of molecules, which may be important to the perception and modulation of pain. A better understanding of this system may yield novel targets for drug development aimed at treating pain and/or inflammation.

DESCRIPTION (provided by applicant): This is an R21 application in response to PAR-08-216, "Developmental Pharmacology". Inhibitors that slow the degradation of the endocannabinoid anandamide show great promise for treating anxiety, depression, and pain, and are in clinical trials for the latter two indications. These inhibitors increase anandamide levels by blocking its hydrolysis by fatty acid amide hydrolase (FAAH) leading to enhanced endocannabinoid signaling, primarily through CB1 cannabinoid receptors. The acute side effects of FAAH inhibitors are well delineated and relatively mild. However, FAAH inhibitors are being developed for chronic use and the long-term effects of these drugs, particularly on the brain as it develops, are poorly studied and not well understood. Endocannabinoids such as anandamide play key roles in numerous physiological processes throughout the body. One of these processes is neurodevelopment where endocannabinoids modulate neurogenesis, neuronal migration, and axonal pathfinding. Genetic or pharmacological disruption of endocannabinoid signaling during neurodevelopment alters CNS development. The proposed studies will use mice and a multidisciplinary approach to determine if therapeutically active doses of FAAH inhibitors raise embryonic brain levels of anandamide and related N-acyl amides to levels sufficient to impair neurodevelopment and later CNS function (behavior and neuronal excitability) by addressing two specific aims: Aim 1. Will therapeutic dosing of FAAH inhibitors suppress embryonic brain FAAH activity leading to abnormal neurodevelopment? We will use mass spectrometry to determine if therapeutic doses of FAAH inhibitors increase anandamide and related N-acyl amides in developing brain. We will then determine if these FAAH inhibitors affect neurogenesis, neuronal migration, or axonal pathfinding. The involvement of cannabinoid receptors in these processes will be evaluated using pharmacological or knockout approaches. Aim 2. Will therapeutic dosing of FAAH inhibitors during the perinatal period lead to sustained impairment of behavior and synaptic function/plasticity during adulthood? Mice will be treated through the perinatal period with FAAH inhibitors identified in the first specific aim. These mice will then undergo behavioral testing as adults in a gender-specific fashion for anxiety, drug preference, spatial learning, and fear conditioning. In addition we will examine synaptic plasticity and excitation/inhibition balance during adulthood. By completing these two aims we will gain insight into the neurodevelopmental effects of FAAH blockade and will be provided with valuable data on the potential consequences of FAAH inhibition during pregnancy. PUBLIC HEALTH RELEVANCE: Inhibitors of the breakdown of the endogenous cannabinoid, anandamide, are undergoing clinical trials for pain and depression. We have found that perturbation of endocannabinoid signaling, including inhibition of anandamide breakdown, leads to derangements in neurodevelopment. In the proposed work we will determine if therapeutic doses of anandamide degradation inhibitors detrimentally affect neurodevelopment.

DESCRIPTION (provided by applicant): CB1 cannabinoid receptors mediate the psychoactivity of cannabis, the most commonly abused illicit drug, and are also attractive therapeutic targets. Sustained CB1 receptor activation, as happens with heavy cannabis use, or if CB1 agonists are used therapeutically on a regular basis, leads to the development of behavioral tolerance - that is, more drug is required to produce the same effect. Earlier studies by us and others in simple preparations (e.g., cell culture models) indentified two serines in the C-terminus of the CB1 receptor whose phosphorylation desensitized CB1 signaling. To test the hypothesis that phosphorylation of these residues was necessary for the development of behavioral tolerance in an animal, we made a knockin mouse mutating these two residues to alanine (S426A/S430A). In preliminary experiments we have found that these mice, compared to wildtype mice are more sensitive to ?9tetrahydrocannabinol (THC), develop tolerance to repeated administration of THC more slowly and also recover from tolerance more rapidly. In the proposed work we will use these mice as a novel model system to address significant questions relating to the development and expression of tolerance to CB1 receptor agonists by completing three specific aims: 1. What is the role of CB1 receptor serines 426 and 430 during the development of tolerance to cannabinoids? By using our S426A/S430A "knockin" mouse we will determine the role of phosphorylation of these two serines in the acute response to cannabinoids, in the development of cannabinoid tolerance and dependence, and in the severity of precipitated withdrawal. 2. How does preventing phosphorylation of serines 426 and 430 affect CB1 signaling during tolerance? In these experiments we will use biochemical and electrophysiological approaches (GTP?S autoradiography, MAP kinase activation, and whole cell patch clamp of cultured neurons and in slices) to assess the "biochemical" signature of CB1 tolerance and its modification in the S426A/S430A mouse. 3. What is the mechanism of "delayed" tolerance? In our preliminary studies we found that while tolerance was slowed in the S426A/S430A knockin mice, it still developed. This delayed tolerance is likely to be important in the adaptive responses to chronic CB1 receptor stimulation. In this aim we will take a "discovery" approach to identify potential mechanisms underlying delayed tolerance. We will use microarrays to determine the transcriptional changes that take place as tolerance to THC develops in wildtype and S426A/S430A knockin mice. We will also use mass spectrometry to test the hypothesis that delayed tolerance is due to additional CB1 receptor phosphorylation. Tolerance in the clinical setting is significant and often difficult to manage problem. The completion of these studies will be helpful both for managing tolerance to CB1 agonists as it occurs in the therapeutic setting as well as for understanding the consequences of sustained and high intensity recreational cannabis use. PUBLIC HEALTH RELEVANCE: Repeated use of cannabis, socially or therapeutically, leads to chronic adaptations in neurons where more drug is required to produce the same effect. This proposal tests the hypothesis that phosphorylation of two serines of the CB1 cannabinoid receptor is responsible for both tolerance and dependence that develop during chronic cannabis use. If these phosphorylation events underlie the adaptive changes that occur in neurons during cannabis use this will help us to better understand the implications of heavy cannabis use as well as sustained use of medical marijuana.

DESCRIPTION (provided by applicant): This is a NIDA CEBRA R21 application to determine if the deleterious effects of organophosphates on neurodevelopment are due to inhibition of endocannabinoid degradation. Organophosphates are effective and widely used pesticides that have improved human health and crop yields. However, one concerning chronic toxicity of organophosphates is their deleterious effect on neurodevelopment, which can occur independent of acetylcholinesterase (AChE) inhibition. In addition to AChE, organophosphates inhibit other esterases, including fatty acid amino hydrolase (FAAH) and monoacylglycerol lipase (MGL). FAAH and MGL are the two most important enzymes for the degradation of endocannabinoids. Significantly, inhibition of FAAH and MGL occurs at organophosphate concentrations that can be achieved in vivo. How might organophosphates perturb neurodevelopment? Emerging evidence has established that the endocannabinoid system plays a central role in brain development including in the proliferation of neural progenitors, neuronal migration and neural circuit formation. We have found that pharmacological blockade of endocannabinoid signaling and degradation disrupts these processes. In the proposed work we will complete two specific aims to determine if organophosphate inhibition of endocannabinoid degradation leads to abnormalities in neurodevelopment and later behavior: Aim 1. Does perinatal organophosphate exposure inhibits eCB degradation in the developing brain to cause abnormal neurodevelopment? Aim 2. Will perinatal organophosphate treatment produce behavioral changes in adult animals? If so, are these changes mediated by CB1 signaling during development? Successful completion of these aims will enable us to determine if inhibition of eCB degradation and enhanced cannabinoid receptor signaling underlie the adverse neurodevelopmental effects of organophosphates. Furthermore they will help us understand the role of FAAH and MGL in orchestrating the complex task of assembling the nervous system. Finally, they will tell us if perturbation of MGL and FAAH function during development predisposes to later behavioral abnormalities and susceptibility to drug use. PUBLIC HEALTH RELEVANCE: Commonly used organophosphate pesticides can cause abnormalities in nervous system development. This proposal will test the hypothesis that organophosphate pesticides impair degradation of endogenous cannabinoids in the fetal and newborn brain and that this leads to anatomical and behavioral deficits in later life. The results of this study could have significant public health benefits for children exposed in utero to organophosphate pesticides.

DESCRIPTION (provided by applicant): This is a proposal to investigate if CB2 cannabinoid receptor agonist functional selectivity can be exploited to identify more specific and effective CB2 receptor agonists for the treatment of chronic pain. Functional selectivity (also known as biased agonism and ligand-directed trafficking) is an increasingly appreciated property of receptor signaling. When a functionally selective agonist binds to a receptor capable of activating multiple signaling pathways, only a subset of those pathways are activated, or the rank order potency of activating specific pathways varies among agonists. Most G protein-coupled receptors (GPCRs) engage multiple signaling pathways, offering many opportunities for functional selectivity. Functional selectivity has substantial therapeutic implications. Properly applied, it may allow activation of specific pathways that are beneficial for treating a specific condition, while avoiding activation of pathways that may contribute to unwanted side effects. Thus, functional selectivity adds another dimension beyond potency, subtype selectivity and intrinsic efficacy to GPCR agonists-a dimension that may be exploited for therapeutic benefit. In preliminary studies examining CB2 receptor signaling we identified striking functional selectivity in several classes of CB2 agonists. For example, members of the aminoalkylindole family of CB2 agonists activated several CB2 signaling pathways, but failed to inhibit voltage dependent calcium channels or internalize CB2 receptors. This degree of functional selectivity is important for both mechanistic and therapeutic reasons. Mechanistically, it offers us the possibility of identifying the signaling pathways underlying CB2-mediated analgesia in specific pain states. Therapeutically, it offers us the possibility of designing drugs that may be efficacious for pain based upon their functional selectivities, while lessening the possibility of undesired effects. Ths proposal will evaluate the therapeutic potential of functional selectivity of CB2 agonists through two specific aims. The first specific aim will complete the characterization of the signaling of currently available CB2 agonists, including CB2 agonists that have failed clinically, using a battery of biochemical and cell-based functional assays. The second specific aim will take the three compounds with the most striking functional selectivity in the in vitro assays and examine their efficacy in preclinical models of neuropathic (paclitaxel and spinal nerve ligation) and inflammatory (complete Freund's adjuvant) pain using CB1 receptor knockout mice. Efficacy of these functional selective CB2 agonists will be compared to a balanced-CB2 agonist (CP55,940) that activates all tested signaling pathways to a similar extent (see Table 1 in Research Strategy). Completion of these specific aims will give us a comprehensive understanding on how functional selectivity contributes to the therapeutic efficacy of CB2 ligands and also provide a rich pharmacological characterization of CB2 signaling that will be crucial for the future evaluation of CB2 agonists for other therapeutic indications.

RESEARCH & RELATED - OTHER PROJECT INFORMATION - PROJECT SUMMARY/ABSTRACT CB2 cannabinoid receptor agonists show intriguing therapeutic promise in diverse preclinical models. However, the lack of detailed information about the signal transduction mechanisms underlying the observed in vivo efficacies has thwarted translational efforts aimed at developing CB2-based therapeutics. We have recently described striking functional selectivity (whereby different agonists activate distinct networks of signaling pathways) among different CB2 agonists. We hypothesize that the inability to move CB2 agonists forward from preclinical development to novel therapies is in part due to an insufficient appreciation of the functional selectivity of this class of drugs. We contend that understanding of the mechanism of CB2 agonist functional selectivity is a prerequisite for rationally designing CB2 agonists exhibiting functional selectivity for specific classical CB2 agonist signaling pathways. We will test the hypothesis that selective activation of a restricted set of signaling pathways will produce unique therapeutic benefits by evaluating the effects of novel, functionally selective CB2 agonists in four different preclinical models. This project works iteratively with Core B, and Projects 1 & 2 to systematically evaluate functional selectivity of the novel CB2 agonists synthesized in Project 1 (in vitro assays) and determine if CB2 agonist functional selectivity can be exploited to develop more efficacious CB2 agonists for the treatment of chronic pain, inflammation, and reward (in vivo assays). We will test our central hypothesis and accomplish these objectives by completing two specific aims: 1. To characterize the in vitro functional selectivity of rationally designed CB2 agonists from distinct chemical classes. The compounds showing the most distinct profiles of functional selectivity will then be advanced to the second specific aim. 2. To identify the optimal functional selectivity profile of CB2 agonists for suppressing neuropathic pain, inflammation, and reward in vivo. The most promising compounds identified in Aim 1 will be evaluated in Aim 2 for efficacy with acute and chronic (as appropriate) treatment. Completion of the first aim will map CB2 agonist chemotypes associated to specific patterns of functional selectivity. The second aim will identify signaling networks required to suppress neuropathic pain, inflammation, or reward in our preclinical models, which may or may not involve overlapping functional selectivity profiles. (This approach will also identify the signaling pathways that are not necessary (i.e. dispensable) for a particular behavioral outcome, which is important information to obtain and will be a helpful guide for future medicinal chemistry efforts.) Together, the knowledge gained from these aims will allow us and others to develop novel CB2 ligands with focused pharmacological efficacy and possibly reduced side-effects for treating indications where CB2 agonists are likely to prove beneficial.

? DESCRIPTION (provided by applicant): Schizophrenia is a common and debilitating psychiatric disease, often presenting late in the second decade of life. The etiology of schizophrenia is multifactorial, with both genetic and environmental components increasing an individual's risk to develop schizophrenia. Accumulating evidence suggests that schizophrenia develops following the accumulation of progressive insults to the developing nervous system-that is, schizophrenia is a neurodevelopmental disease. Thus, to reduce the societal and individual burden of schizophrenia it is important to identify, understand the mechanisms, and to limit these insults. Work over the last two decades has identified early, heavy adolescent Cannabis use as a risk factor for developing schizophrenia. As the prefrontal cortex is maturing during adolescence, and deficits in prefrontal cortex function are prominent in schizophrenia, it is logical to hypothesize that Cannabis adversely impacts the developing adolescent prefrontal cortex. The primary psychoactive component of Cannabis is delta-9-tetrahydrocannabinol (THC). While THC modulates synaptic transmission, it also affects neurodevelopment. In a recent study on the effect of THC on adolescent eye blink conditioning in rats, we made the intriguing observation that low dose adolescent THC impaired the acquisition of eye blink conditioning, and also activated cerebellar microglia. Interestingly, both the impaired eye blink conditioning and microglial activation were absent when THC was co- administered with cannabidiol (CBD), a bioactive, but not overtly psychoactive occasional component of cannabis. In pilot experiments we have replicated the finding that adolescent, low-dose THC activates microglia in the prefrontal cortex via CB1 cannabinoid receptors and increases IL-6 mRNA. The increase in IL- 6 was prevented by concurrent cannabidiol. These results lead us to propose the following innovative hypotheses: (1) THC activates microglial cells in the adolescent prefrontal cortex, which may lead to impaired synaptic pruning, with long lasting effects on synaptic structure. (2) CBD counteracts the effects of THC and serves a protective role. We will evaluate these hypotheses through a series of targeted experiments aimed at elucidating the role and extent of THC activation of microglial cells and their impact on prefrontal cortex development. This will be accomplished by completing two specific aims: 1. Does adolescent THC permanently alter the mPFC transcriptome? 2. Will cannabidiol suppress neuroinflammation produced by THC? Completing these specific aims will define the extent, duration, and consequences of microglial activation in the prefrontal cortex following adolescent THC administration. This will lay the groundwork for future studies that will examine the implications of THC-induced microglial activation on PFC neuronal network activity and PFC-mediated behaviors.

? DESCRIPTION (provided by applicant): Cancer chemotherapy frequently causes a painful peripheral neuropathy that is dose-limiting and can be irreversible. Gabapentin is clinically used to treat diverse forms of neuropathic pain. However, the mechanism(s) responsible for its antinociceptive effects remain poorly understood. Unpublished work from our groups suggests that gabapentin suppresses neuropathic pain induced by the chemotherapeutic agent paclitaxel in rodents through interactions with CB2 cannabinoid receptors. At the cellular level, gabapentin selectively increases ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) to recruit ?-arrestin to CB2 receptors. These observations suggest a previously unrecognized interaction between CB2 receptors, ?- arrestin/ERK1/2 signaling and gabapentin-induced antinociception. We postulate that gabapentin analgesic efficacy is due (at least in part) to a CB2-specific mechanism that involves increased ?-arrestin signaling in microglia or neurons. We will thoroughly test this hypothesis by completing three Specific Aims: Aim 1 will characterize the impact of gabapentin on CB2 receptor signaling using transfected cells lines, cell lines natively expressing CB2 receptors and primary cultures of CB2-expressing cells. In addition, potential allosteric interactions between gabapentin and CB2 will be probed. Aim 2 will use conditional deletion of CB2 from neurons, microglia, and astrocytes to determine which cell type(s) express the CB2 receptors mediating gabapentin antinociception during the development and maintenance phases of paclitaxel neuropathy. Since CB2 agonists efficaciously relieve paclitaxel-induced allodynia and hyperalgesia, this approach will also be used to determine the cell type(s) mediating antinociception elicited by direct acting CB2-agonists. Aim 3 will extend the findings of the first two aims to determine if gabapentin efficacy is also CB2-mediated in other nerve injury and inflammatory pain models. The relevant cell type(s) will be determined using conditional deletion of CB2 as warranted and as described in the second specific aim. This aim will also investigate the mechanism of direct-acting CB2 agonists in these pain models using the above conditional deletion approach. Our research team combines expertise in (1) CB2 receptor binding, signaling, trafficking, and regulation, (2) cannabinoid pharmacology and antinociceptive mechanisms, and (3) mouse preclinical models of pain. Our studies suggest a highly novel and previously unrecognized intersection between CB2 receptors, endocannabinoids, and arrestin signaling that underlies the therapeutic efficacy of gabapentin. Understanding the cross-talk between these pathways is critical both for elucidating the mechanism of action of gabapentin to exploit and optimize its therapeutic efficacy and for identifying novel therapeutic targets for drug development that lack unwanted side effects of conventional treatments. Lastly, our studies will also identify the cellular targets of CB2 agonist as they relieve a variety of pathological pain states.

Cannabis use is prevalent in the US population, and its prevalence is likely to increase due to ongoing legalization efforts. Thus, understanding the implications (both beneficial and harmful) of long-term cannabis use is imperative. While the long-term cognitive and psychiatric sequelae of cannabis use have been intensely investigated, few studies have investigated the metabolic effects of long-term cannabis use. Metabolism is a likely target for cannabis as several of the compounds abundant in cannabis (e.g., delta-9- tetrahydrocannabinol, THC) directly affect feeding behaviors and/or metabolism. Interestingly, despite the popular view that cannabis ingestion enhances consumption of calorically dense food, which might lead to increased obesity, the metabolic syndrome, and type II diabetes among chronic cannabis users, most epidemiological studies have found the opposite. Thus, chronic cannabis use is associated with decreased body mass index, decreased incidence of metabolic syndrome, and a decreased risk for developing type II diabetes. The mechanism for this is unclear?stimulation of CB1 cannabinoid receptors by THC would be expected to increase consumption of calorically rich foods and increase fat deposition, thus it is likely that THC may target other receptor(s) involved in metabolism and whose engagement by THC leads to more beneficial metabolic effects. The proposed work will address the hypothesis that activation of GPR119 beneficial metabolic consequences. GPR119 is a G protein-coupled receptor expressed in pancreatic beta cells as well as K and L enteroendocrine cells in the gastrointestinal tract. Its activation increases insulin secretion from the pancreas, secretion of the incretins GIP and GLP-1, as well as PYY, from the gut, and may protect beta cells from apoptosis. Cumulatively, GPR119 activation is expected to lead to a favorable metabolic profile, which has made GPR119 activation a target for drug development. In preliminary studies investigating GPR119 signaling, we made the surprising observation that THC is a GPR119 agonist. Further studies found that chronic administration of THC or a GPR119 agonist decreased weight in obese mice to the same extent. In the proposed R21 CEBRA we will complete two specific aims to better understand the consequences of THC signaling through GPR119. Aim 1. Fully characterize the signaling of THC and other cannabinoids at GPR119 using cell lines natively expressing GPR119. Aim 2. Determine if THC-induced weight loss in obese mice requires GPR119. Completion of these two specific aims will greatly advance our understanding of the potential role of GPR119 in mediating the apparent metabolic benefits of THC.  

Cannabis is the most frequently used illicit drug. Its widespread use in adolescents is associated with an increased risk for mental illness and is a major public health concern. For example, an increased rate of psychosis and an earlier onset of psychotic illness have been observed with heavy adolescent cannabis use. There is also a correlation between early cannabis use, the age at onset of psychotic disorders and the age at first hospitalization for 'pure' cannabis users. However, the pathophysiological mechanisms underlying the adverse effects of early cannabis use, such as perturbed social interactions, psychosis and addiction risk remain to be elucidated. A better understanding of these mechanisms is necessary to develop therapies & effective prevention programs, and to inform decisions on public policy, including cannabis legalization. Adolescence is a period of profound neurodevelopmental maturation, notably in the mesocortico-limbic system (MCS), an ensemble of interconnected structures involved in higher cognitive functions, emotions, reward and social behaviors. MCS development occurs at different rates in males and females. During adolescence, social play, an MCS-based behavior, guides the emergence and proper maturation of social interactions. Disruption of social play in the adolescent negatively affects adult behaviors. Impaired adolescent social play may also be a sign of emerging psychopathology. The main psychoactive ingredient of cannabis (i.e., THC) engages the abundant CB1 cannabinoid receptor (CB1R), competing with endogenous cannabinoids. CB1Rs are a core component of the endogenous cannabinoid system (ECS). The ECS is abundant throughout the MCS and modulates many neurodevelopmental, neuronal and synaptic processes, including adolescent social play. Recent discoveries from our laboratories fueled the concept that ECS dysfunction plays a key role in diverse neuropsychiatric diseases of environmental or genetic origin. Motivated by new, unpublished data, and using a rodent model, we propose a multi-disciplinary approach to establish the sex-specific development and distribution of ECS components in the MCS and how these affect the molecular, synaptic and behavioral consequences of adolescent cannabis (THC) exposure. Our 3 aims will: 1/ Characterize and compare the normal functional development of neuronal and synaptic responses in the MCS between male and female rats from pre-adolescence to adulthood. 2/ Establish the developmental patterns of expression and localization of key components of the ECS in the rat MCS in a sex and age-specific fashion. 3/ Determine the sex-specific functional (molecular, synaptic, and behavioral) consequences of THC exposure during critical periods of adolescence and adulthood. Together, the results of these studies will define new structural, molecular and functional synaptic substrates of the sex-specific effects of adolescent cannabis use on ECS function and behavior.

Cannabis is the most frequently used illicit drug. Maternal perinatal cannabis use has been associated with a range of adverse neurodevelopmental consequences in the offspring. The underlying mechanism(s) remain incompletely understood, but are consistent with impaired cortical neuronal circuit formation. A coordinated program of transcriptional and physiological events governs the assembly of cortical circuits. The evolutionary conserved switch of gamma amino butyric acid (GABA) from an excitatory to an inhibitory neurotransmitter is crucial to the normal development of cortical circuits and associated behaviors. The switch is primarily driven by increased expression of a potassium/chloride co-transporter, KCC2, which extrudes chloride from the cell. In our preliminary experiments, we have found that administration of a synthetic cannabinoid or THC for the first 10 days after birth to lactating rat and mice dams suppresses KCC2 expression in the PFC at postnatal days 10-15, prolonging the time during which GABA excites PFC networks. Perinatal exposure also impaired prefrontal cortex synaptic plasticity and cognitive or social behaviors in the adult progeny of both sexes. The proposed work will follow up these exciting preliminary data to determine the immediate and long-lasting effects of the cannabinoid-induced delay in KCC2 expression by addressing three specific aims. Aim 1. Identify the early molecular, functional and behavioral consequences of exposing dams to THC ± CBD during lactation on the progeny of both sexes. These experiments will characterize the early consequences on neuronal circuits in the PFC, determine THC's mechanism (and possible antagonism by cannabidiol (CBD)) to delay KCC2 expression, examine the localization and levels of components of the PFC endocannabinoid system and measure ecologically-relevant pup behaviors (ultrasonic vocalizations and homing following maternal separation) after maternal exposure to cannabinoids. Aim 2. Determine the long-term consequences of THC ± CBD exposure during lactation. These experiments will determine if THC ± CBD exposure during lactation has enduring effects on synaptic plasticity in adolescent and adult, on levels or localization of PFC endocannabinoid components, on naturalistic social behaviors, and cognitive function. Aim 3. Strategies to ameliorate the long-term deleterious consequences of THC exposure during lactation. These experiments will test the hypothesis that enhancing endocannabinoid signaling (CB1 positive allosteric modulators or inhibitors of eCB degradation) will rescue the behavioral and physiological deficits that are a consequence of PCE. Completion of these experiments will reveal the underpinnings of the impact of perinatal THC exposure on neuronal functions and behavior and provide new therapeutic strategies to ameliorate associated behavioral deficits.

Cannabis is the most commonly used illicit drug during pregnancy and its use during this time is associated with an increased risk of adverse neurological and developmental outcomes in exposed children. This suggests that the most abundant active component of cannabis, ?9-tetrahydrocannabinol (THC), may affect nervous system development. While the cerebellum was once thought to be primarily involved in coordination of motor movement, several recent studies indicate that it is involved in a range of sophisticated behaviors, including cognition. In addition, cerebellar dysfunction is found in neurodevelopmental disorders including autism and schizophrenia. Thus, a better understanding of how environmental and genetic factors (both implicated in neurodevelopmental disorders) impact cerebellar development is clearly warranted. The proposed study will examine roles of the endogenous cannabinoid system on cerebellar development, and how THC affects this process. While THC can detrimentally impact cerebral cortical development, little is known on the role of endogenous cannabinoids and THC in cerebellar development. In preliminary studies using CB1 receptor knockout mice we find a strikingly abnormal foliation of the anterior lobe of the cerebellum in a sex-dependent fashion. Furthermore, perinatal low-dose THC recapitulates these findings. We hypothesize that proper granule cell CB1 function is necessary for the appropriate maturation of granule cells. As a corollary, we also hypothesize that disruption of this CB1 receptor signaling (for example, by THC) impairs cerebellar development, increasing an individual's risk for psychiatric disease. In the following R21 proposal we will test the hypothesis that CB1 receptors play a significant role in cerebellar development by completing the following three aims. Aim 1. Characterize expression and developmental requirements of CB receptors and associated components of the eCB signaling machinery during postnatal cerebellar development. Using well- characterized antibodies, expression of CB1 and the enzymes synthesizing and degrading endocannabinoids in the developing cerebellum will be localized in time and space. Aim 2. How does exposure to exogenous cannabinoids affect cerebellar development? Expanding on our preliminary results, the role of CB1 receptors in modulating cerebellar development and granule cell maturation following THC exposure will be investigated. Aim 3. Assess the role of CB signaling in the GC proliferation to differentiation switch. Following up on preliminary studies, we will determine if THC treatment affects the switch of granule cells from proliferation to differentiation, as a likely mechanism for THC impairment of cerebellar development. Together, the results of these studies will define the role of CB1 receptors in the cerebellar foliation in a sex- specific fashion and determine if perinatal cannabis (as modeled by THC administration) affects this process.

Despite significant limitations, opioids remain a mainstay for the treatment of severe acute and chronic pain. Two clinically significant limitations that accompany the long-term therapeutic use of opioids are: (1) The development of tolerance (requiring escalating doses of opioid to maintain the desired therapeutic benefit or leading to diminished benefit with constant dose) and (2) Physical dependence (withdrawal symptoms on cessation of opioid use, which are very unpleasant, and seeking their avoidance may increase the risk for opioid addiction). Currently, there are no practical approaches for decreasing opioid tolerance or suppressing the development of physical dependence. However, in exploring potential interactions between CB2 cannabinoid receptor (CB2R) and mu opioid receptor (MOR) signaling, we made the exciting discovery that certain CB2 agonists effectively prevented the development of tolerance to opioid-induced anti-allodynic efficacy in a murine neuropathic pain model, while also blunting the physical dependence that accompanies chronic morphine exposure. These findings, if they can be translated to humans, hold the promise to significantly improve the clinical use of opioids. On the path to translating these findings, we will complete three specific aims to better understand how CB2 and mu opioid receptor agonists interact to suppress opioid tolerance and dependence: Aim 1: Identify the CB2-expressing cells engaged by LY28282360 to prevent opioid tolerance. We will delineate the cell types responsible for the ability of LY2828360 to block development of morphine tolerance and identify site of action using both pharmacological manipulations and a conditional deletion approach. Separate studies will target primary afferent nociceptors vs. microglia. Aim 2: Define the conditions under which CB2 agonists suppress opioid-induced physical dependence. We will delineate the cell types responsible for the effects of LY2828360 on opioid dependence, as measured using naloxone precipitated opioid withdrawal, using both pharmacological manipulations and a conditional deletion approach. Separate studies will target primary afferent nociceptors vs. microglia. Aim 3: Mechanistic characterization of CB2R/MOR interaction. We will characterize CB2 ligands for their G protein/arrestin signaling bias as well as their kinetics of G protein activation. We will also determine if the slow activation of G protein signaling by LY2828360 and related CB2 agonists is due to the kinetics of receptor binding. Finally, if the results of Aims 1 or 2 suggest that MOR and CB2R are interacting in the same cell, we will characterize the differences in CB2R/MOR crosstalk between slowly and rapidly signaling CB2 agonists. Completion of these aims will fully characterize interactions between CB2 and opioid receptors in preclinical and cell-based models. These studies will help define the clinical settings where CB2 agonists may be useful in countering two major limitations on the use of opioids in treating chronic pain.

This proposal requests support for the second renewal of a highly successful, integrative pre-doctoral training program in the neuroscience of drug abuse at Indiana University Bloomington. Despite substantial advances in understanding drug addiction within specific levels of analysis (e.g., behavioral, clinical, and molecular), the problem of drug abuse will not be solved by focusing on a single level of analysis. If the next generation of researchers is to make meaningful progress, they must be well-rounded scientists with an appreciation that drug abuse is a multi-faceted problem, while possessing the flexibility to respond to and incorporate rapidly evolving technologies that will enable them to understand mechanisms and develop treatments for drug abuse. To prepare trainees for success in the next decade and beyond, our program emphasizes a team-driven, inter- disciplinary approach based on the translational model. Our program is successful because it brings together 12 core faculty members who are committed to integrative training and have a long history of collaboration on questions integral to drug abuse research. They include senior and junior investigators, molecular neurobiologists, cognitive neuroscientists, epidemiologists, and clinical scientists. They come from several departments in the College of Arts and Sciences and the School of Public Health, and all have joint appointments in the campus-wide Program in Neuroscience. Working together in state-of-the-art facilities, this group has access to a pool of highly talented trainees motivated to pursue careers in drug abuse research. Our training program develops trainees by emphasizing three key components: integrative course work, translational research training, and professional skills development. Course work covers basic neuro- and psychopharmacology, provides an integrative view of biobehavioral processes in substance use disorders, and brings a translational perspective to theoretical and empirical knowledge. Research is guided by a mentor in molecular, systems, cognitive, or clinical neuroscience closely integrated with a co-mentor representing a complimentary level of analysis. This integrative approach is reinforced through discussion groups, attendance at colloquia, and participation at national meetings. Instruction in ethical scientific behavior includes formal course work and campus workshops as well as specialized instruction led by a core faculty member who has many years of experience leading seminars on ethical issues unique to substance use research. Trainees also learn to develop skills in grant writing, manuscript preparation, teaching, and community outreach and organize an annual program retreat with outside experts in drug abuse. In short, our program relies on a combination of course work and research training aimed at integrating and translating bench and bedside approaches to produce scientists well prepared for productive and transformative careers in drug abuse research.

PROJECT SUMMARY/ABSTRACT Chronic pain ? often arising from musculoskeletal injury, neurological dysfunction, cancer, or autoimmune disorders ? affects ~100 million Americans. Overreliance on opioid analgesics has resulted in a national public health crisis in which opioid overdoses have claimed over 47,000 lives in 2017 and are now the leading cause of avoidable deaths in the nation. The Cannabis plant has analgesic and anti?inflammatory properties owing to its rich content of cannabinoids, terpenes, lignans, and flavonoids. However, research on the biological effects and molecular mechanisms of the numerous bioactive phytochemicals ? alone or in combination (entourage effect) ? has been limited. We have assembled a complementary and interdisciplinary team that combines expertises in molecular and cellular signaling, ion channel biology, natural products chemistry, and molecular pharmacology as well as all aspects of endocannabinoid biology. Our preliminary high? throughput screening (HTS) bioassays have identified several cannabinoids that inhibit calcium signaling in immune cells and may therefore reduce inflammation and the associated pain. Results from the work proposed here will identify the anti?inflammatory molecules contained in Cannabis sativa and characterize the mechanisms of action they engage. We hypothesize that specific phytochemicals in Cannabis suppress cellular Ca2+ signaling and subsequent release of pro? inflammatory cytokines in immunocytes that contribute to inflammatory pain. We further hypothesize that combinations of Cannabis phytochemicals synergistically inhibit certain ion channels and G protein?coupled receptors involved in immunocyte Ca2+ signaling and cytokine release, thereby ameliorating inflammatory pain. We propose to perform pharmacological profiling of individual and entourage effects of Cannabis phytochemicals on Ca2+ signaling in 5 specific pro?inflammatory human immune cells (Aim 1A). We will determine the cellular and molecular Ca2+ mobilizing mechanisms engaged by active Cannabis phytochemicals in these immune cells (Aim 1B); and profile Cannabis phytochemicals on established molecular targets of nociceptive, inflammatory and neuropathic pain (specific TRP channels and G?proteins) using heterologous expression systems, HTS bioassays and single cell electrophysiology (Aim 1C). In Aim 2 will assess analgesic properties of active cannabinoids and combinations using in vivo mouse models of inflammatory and neuropathic pain. Here, we will first determine in vitro ?Absorption, Distribution, Metabolism, and Excretion? (ADME) properties (Aim 2A) and in vivo pharmacokinetics (Aim 2B) of said cannabinoids. We will then assess the most favorable cannabinoid(s) in Complete Freund's Adjuvant (CFA)?induced inflammation and paclitaxel?mediated toxic neuropathic pain (Aim 2C). Together, these studies will create a comprehensive and mechanistic knowledge base about the efficacy, potency and suitability of Cannabis?derived phytochemicals as anti?inflammatory analgesics and may contribute to ameliorating the current opioid epidemic.