Eric L. Barker - US grants

DESCRIPTION: (Applicant's Abstract) The broad, long-term objective of this R21 application is to advance the overall understanding of molecular mechanisms regulating endogenous cannabinoid signaling by a putative endocannabinoid uptake system. The endogenous cannabinoids such as anandamide are fatty acid-derived molecules whose metabolism and signal termination appear to rely upon transport back into releasing cells for subsequent breakdown by an intracellular amidohydrolase enzyme. Thus, the uptake process would play a critical role in the dynamic regulation of endogenous cannabimimetic activity. The Specific Aims are (1) To isolate and identify the protein(s) responsible for the uptake of endogenous cannabinoids, and (2) To characterize the transport activity associated with anandamide uptake. This project has direct health-relatedness as the endogenous cannabinoid system has been implicated in modulating various physiological and pathological processes including psychosis, motor movement, inflammation, blood pressure, and pain. Based upon data indicating functional similarities between anandamide transport and the uptake of long-chain fatty acids, the Research Design is to explore the potential contributions of various fatty acid transporters in endocannabinoid transport using heterologous expression systems. In addition, the pharmacologic profile of endocannabinoid uptake in native and transfected cells will be determined, and the tissue distribution of the identified anandamide transporter investigated to better understand the role of transport proteins in endocannabinoid signaling. The Methods to be used include molecular cloning techniques, characterization of transport proteins using radiolabeled substrate uptake assays, determinations of intracellular accumulation of endocannabinoid substrates using mass spectrometry, and in situ hybridization to reveal transporter distribution. These studies will provide a basis for future studies examining the molecular mechanisms associated with both structure/function and regulation of endogenous cannabinoid transport.

The serotonin transporter (SERT) is the molecular target for most antidepressant drugs as well as many drugs of abuse. Antidepressants such as paroxetine, fluoxetine, and imipramine bind to the transporter and inhibit serotonin uptake, thereby prolonging the extracellular lifespan of the neurotransmitter. The abused psychostimulants cocaine and amphetamine also bind to the transporter, but have distinct pharmacologic effects leading to abuse potential and possible neurotoxicity. Despite the cloning of SERT in the early 1990's, very little information is available regarding the molecular and cellular neurobiology surrounding the function of this transport protein. This project uses a multidisciplinary approach aimed at significantly advancing knowledge regarding the molecular pharmacology of psychostimulants at SERTs. The studies will use multiple techniques including expression and characterization of recombinant transporters in mammalian cells, electrophysiology, immunoblotting, the formation of chimeric proteins, and site- directed mutagenesis to identify amino acids involved with specific transporter functions including antagonist and substrate binding. In addition, computer-assisted structure-activity studies of cocaine and amphetamine derivatives will be coupled with mutagenesis in an attempt to identify direct ligand- transporter interactions. Therefore, the specific aims of this project are 1) to identify transporter domains and residues involved in recognition of SERT antagonists such as cocaine and 2) to determine the contributions of specific SERT domains to substrate properties such as translocation, efflux, and channel- like activity. The proposed strategies will provide critical new information linking protein structure to functional properties of the transporter and an enhanced understanding of the molecular mechanisms of action for psychotherapeutic and abused drugs.

DESCRIPTION (provided by applicant): The broad, long-term objective of this R21 CEBRA application is to advance the overall understanding of molecular mechanisms regulating endogenous cannabinoid signaling by identifying proteins responsible for endocannabinoid uptake. The endogenous cannabinoids such as anandamide are fatty acid-derived molecules whose metabolism and signal termination appear to rely upon transport back into releasing cells for subsequent breakdown by an intracellular amidohydrolase enzyme. Thus, the uptake process is thought to play a critical role in the dynamic regulation of endogenous cannabimimetic activity. The Specific Aim of this proposal is to identify proteins involved with the uptake and intracellular transport of the endocannabinoid anandamide. This project has direct health-relatedness as the endogenous cannabinoid system has been implicated in modulating various physiological and pathological processes including mood, anxiety, psychosis, motor movement, inflammation, blood pressure, and pain. Recent evidence suggests that "lipid raft-" or caveolae-related endocytosis may be involved with internalization and cellular transport of anandamide. Thus, a carrier protein for anandamide would be predicted to reside in this membrane microdomain. By developing novel probes and using cutting-edge proteomic methods, putative anandamide binding proteins will be identified. The role of these proteins in anandamide uptake will be explored using modern molecular, biochemical, and pharmacological approaches. These studies will provide a basis for future studies examining the molecular mechanisms associated with both structure/function and regulation of endogenous cannabinoid transport.

DESCRIPTION (provided by applicant): The broad long-term objective of this proposal is to characterize possible mechanisms involved in the initiation of biosynthesis/release of the endocannabinoid anandamide. Anandamide has been shown to display the same physiological effects as plant derived cannabinoids by acting as an agonist at the brain and peripheral cannabinoid receptors (CB1 and CB2 respectively). A better understanding of the mechanisms that initiate the synthesis/release of anandamide may reveal new drug targets that provide therapeutic benefits in multiple conditions. Interestingly, anandamide has been recently shown to exhibit endovanilloid activity by activating the Ca2+-permeable vanilloid receptor (VR1), a member of the TRP family of receptors. The proposed studies examine the cellular localization of VR1 receptors and their potential link to anandamide synthesis/release. The hypothesis to be explored is that VR1 receptors are found in the lipid raft/caveolae domains of the plasma membrane and that their activation by noxious stimuli, and possibly anandamide itself, acts to stimulate the synthesis/release of anandamide from caveolae. The Specific Aims are (1) To determine if the VR1 receptor is localized to a specific domain of the plasma membrane and (2) To determine if VR1 receptor activation will stimulate the synthesis/release of anandamide from caveolae. Several TRP channels have been shown to localize to the caveolin-rich domains of cells, and evidence suggests that the precursors for anandamide are enriched in caveolin-rich membranes. The Research Design will seek to show that VR1 receptors are localized in the caveolae domains of the plasma membrane using subcellular fractionation techniques, Western blot analysis, and immunofluorescence. A novel use of the fluorescent calcium biosensor, yellow cameleon proteins will be exploited as a molecular marker of VR1-induced Ca 2. level increases in caveolae/lipid raft domains in living cells. The ability of VR1 to stimulate the synthesis/release of anandamide in a Ca2*-dependent fashion will be measured by quantification of anandamide accumulated in either the subcellular fractions or the assay buffer following VR1 stimulation. The use of the emerging technology of fluorescent biosensors to explore yet unidentified mechanisms associated with endocannabinoid biosynthesis will provide new opportunities to understand the cellular biology of the system modified by cannabinoids.

DESCRIPTION (provided by applicant): The broad, long-term objectives of this proposal are to develop new metabolomic methodologies to identify pathways involved with metabolism and biosynthesis of endogenous cannabinoid signaling molecules such as anandamide and 2-arachidonylglycerol (2-AG). Ultimately, these lipidomic strategies can be employed in in vivo or in vitro systems to better understand the profile of endogenous cannabinoids produced under a variety of stimuli. The endogenous cannabinoids are a family of lipid signaling molecules that are typically amides or esters derived from long-chain fatty acids. These compounds have neuromodulatory activity and appear to play a role in multiple physiological processes. The two major endocannabinoids identified in the brain are anandamide (N-arachidonyl- ethanolamide) and 2-AG. The proposed studies are designed to refine the approaches used to study production and recycling of anandamide as well as other putative endogenous cannabinoids. The specific metabolic and biosynthetic pathways linked to certain calcium channels as well as neurotransmitter receptor activation will be explored. The Specific Aims of this project are: 1) To develop and apply specific metabolite fingerprinting and profiling methodology for identifying the lipidomic profile for endogenous cannabinoids, and 2) To determine stimulus-dependent differences in the release of endogenous cannabinoids. The studies will use molecular, biochemical, analytical (thin layer chromatography, mass spectrometry), and lipidomic/ metabolomic approaches. These innovative comparative lipidomic studies will examine in detail the recycling products derived from metabolized anandamide. A better understanding of the mechanisms of endogenous cannabinoid production and release will provide new insight into the system that is acted upon by marijuana. The Public Relevance: This project has direct relevance to human health because the endocannabinoid system is implicated in modulation of memory, mood, cognition, pain, fever, and the immune system. As such, pharmacological manipulation of this system has potentially therapeutic effects in such conditions as convulsions, glaucoma, movement disorders, anxiety, obesity, and multiple sclerosis. Understanding the molecular basis for controlling the production of the endogenous cannabinoids is critical for future drug discovery efforts targeting this system.

DESCRIPTION (provided by applicant): There is currently an urgent need to identify risk factors that increase vulnerability to develop co-morbid alcoholism and post-traumatic stress disorder (PTSD) in order to devise and implement appropriate prevention and treatment strategies for these disorders. The endocannabinoid system (ECS) modulates anxiety-related and alcohol drinking behaviors and has been identified as a promising target for pharmacotherapies to treat anxiety disorders and alcoholism. For this R21 project, a unique animal model that represents increased genetic risk to develop co-morbid alcoholism and PTSD in humans will be used to study the role of the ECS in influencing fear-related behavior in mice that differ in genetic propensity toward alcohol preference. In Specific Aim 1, male and female mice selectively bred for high (HAP) and low (LAP) alcohol preference will be used to determine whether brain region specific levels of the endocannabinoids, anandamide (AEA) and sn-2 arachidonylglycerol (2-AG), are associated with genetic propensity toward alcohol drinking and fear-related behavior. In Specific Aim 2, it will be determined whether drugs that target the ECS reduce fear-related behavior and whether these effects depend on genetic predisposition toward alcohol preference. The secondary goal of Specific Aim 2 is to determine whether EC brain levels are correlated with observed drug effects on the expression of fear-related behavior. The results of this project will provide exciting new preclinical data on the role of the ECS in modulating fear-related behavior in a unique animal model for co- morbid alcoholism and PTSD. Results from this project may facilitate rapid development of novel pharmacological strategies that target the ECS to treat individuals with co-morbid alcoholism and PTSD. The results may also help identify pharmacotherapy or pharmacoprevention approaches that are particularly effective in people who are at increased genetic risk for both alcoholism and PTSD. PUBLIC HEALTH RELEVANCE: The endocannabinoid system (ECS) modulates anxiety-related and alcohol drinking behaviors and has been identified as a promising target for pharmacotherapies to treat anxiety disorders and alcoholism. The goal of this project is to use a use a unique animal model that represents increased genetic risk to develop co-morbid alcoholism and PTSD in humans to explore the role of the ECS in regulating genetic differences in anxiety- related behavior. The project will also determine whether drugs that target the ECS may represent effective pharmacotherapies to treat individuals with co-morbid alcoholism and PTSD.