Nephi Stella - US grants

Delta9-tetrahydrocannabinol (THC) is best known as a psychotropic agent. Yet, this agent also produces multiple non- psychotropic effects; in particular reducing immunologic competence. These effects raise the possibility that cannabinoids regulate inflammations occurring within the CNS. The work proposed here addresses the role of cannabinoids in the regulation of microglial cell activation. Microglial cells are immune cells that reside in the CNS and become activated in diseases such as multiple sclerosis and HIV encephalopathy. Activated microglial cells release toxins and cytokines that lead to remodeling of affected tissue. By analogy with peripheral macrophages, we hypothesize that the engagement of cannabinoid receptors on microglial cells tempers their activation process. This hypothesis is supported by the observation that cannabinoids inhibit CNS inflammation associated with experimental allergic encephalomyelitis, an animal model of multiple sclerosis. In addition, several small clinical studies indicate that patients with multiple sclerosis experience relief from symptoms with marijuana use. We propose to identify and characterize the cannabinoid signaling system in microglial cells. The specific aims are: 1: What types of cannabinoid receptors do resting or activated microglial cells express? 2: How are endocannabinoids generated by microglial cells? 3: Do microglial cells inactivated endocannabinoids? Understanding how cannabinoids interact with microglial cells is of particular interest because patients with multiple sclerosis and HIV encephalopathy often use these drugs for medicinal purposes, and because recent evidence implicates cannabinoid signaling pathways in the symptomatology of these diseases. Finally, because cannabinoids are immunoactive, characterization of the cannabinoid signaling system in microglial cells might lead to new pharmaceutical targets that could specifically temper inflammation of the CNS.

[unreadable] DESCRIPTION (provided by applicant): Endogenous cannabinoid ligands (endocannabinoids, eCBs) are produced by cells and activate cannabinoid receptors, the molecular target for marijuana's bioactive ingredient A9-tetrahydrocannabinol. Several eCBs have been indentified, including the prototypical eCB anandamide and the most abundant eCB in brain 2-arachidonoylglycerol. Yet the scientific literature is full of description of additional eCB candidates, the biosynthesis and inactivation of which remains to be elucidated. Thus it is currently particularly difficult to elucidate the involvement of a specific eCB in a given biological function because we do not know which proteins control their levels. Analysis of the yeast genome showed that many potential players in eCB biosynthesis and inactivation are conserved between yeast and higher eukaryotes. Recently we were able to unambiguously identify and quantify eCBs in Saccharomyces cerevisiae by using gas chromatography / mass spectrometry (GC/MS). An exciting feature of this result is that yeast provides an unbiased genetic tool to identify modifiers (some of which have human homologs) of biological functions, in our case eCB biosynthesis and inactivation. This R21 proposal has two specific aims: 1) identify genes that control eCB levels in yeast 2) determine if human homoloqs of these yeast genes control eCB biosynthesis and inactivation in a human neuronal cell line. Results obtained with this R21 grant will provide the basis for an RO1 aimed on understanding the genetic and molecular basis of mammalian eCB production and inactivation, a pivotal question in the field of drug of abuse research. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Synthetic and plant-derived cannabinoids hold promises for the development of medicines aimed a relieving pain and anxiety. Unfortunately, their broad action on cannabinoid receptors limits their use as they also induce severe adverse effects, such as memory impairment and psychotropic effects. Cannabinoid receptors are normally engaged by endocannabinoids, which are produced and hydrolyzed by cells. A more promising avenue to develop cannabinoid-based therapeutics devoid of adverse effects is to inhibit endocannabinoid hydrolysis, thus only locally increasing endocannabinoid levels and allied cannabinoids receptor activation. Endocannabinoid are inactivated by two well-characterized enzymes: FAAH and MGL. Specific and potent inhibitors of these enzymes have been developed. We have recently identified a novel enzymatic activity which we refer to as MGL2, as it hydrolyzes the most abundant endocannabinoid 2-arachidonoyl glycerol (2-AG). We have also identified several compounds capable of inhibiting MGL2. The work that we propose in this CEBRA grant aims at: 1. Purifying MGL2 and clone its cDNA. 2. Further develop MGL2 inhibitors. Our long-term goal is to develop pharmacological and genetic tools capable of selectively inhibiting endocannabinoid hydrolysis to favor the therapeutic properties of endocannabinoids, while avoiding the adverse effects produced by cannabinoid drugs. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cannabis is best known as a psychotropic drug of abuse; yet this plant has medicinal properties. Some patients with multiple sclerosis (MS) use marijuana in an attempt to relieve their symptoms and several animal studies have confirmed the analgesic properties of cannabinoid compounds. Evidence now shows that CB2 receptors are expressed in the CMS and CB2 agonists may actually be used as therapeutics. Indeed, when given to mice developing experimental autoimmune encephalomyelitis (EAE), CB2 receptor agonists prevent microglia activation, T-cell infiltration and favor remyelination. While very promising, these results require further testing and characterization. In the prior funded cycle, we discovered that microglia produce and inactivate the most abundant endocannabinoid (eCB), 2-arachidonoylglycerol (2-AG), and express CB2 receptors, which regulate their migration. Given that activated microglia determine the onset and maintenance of EAE, we propose that activation of CB2 receptors by 2-AG regulates microglial cell recruitment toward lesion sites. Because activated microglia express high levels of CB2 receptors, they represent a valuable target for the development of specific therapies designed to control neuroinflammation. We will test our hypotheses by addressing three aims. To what extent are CB2 receptors required for microglial cell migration? How do cytokines affect eCB signaling in microglia? Do CB2 agonists and antagonists control microglia cell migration in vivo and EAE pathogenesis? Completion of these aims will establish the mechanisms of action and pre-clinical efficacy for the use of compounds acting through CB2 receptors as therapy for MS. In doing so, we will also gain a better understanding of the molecular mechanism controlling eCB signaling in the CMS. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cannabinoids are the principal psychoactive component of cannabis, the most commonly used illicit drug in the United States. Going beyond the abuse issues there is also great interest in the possible medicinal use of cannabis and cannabinoids. Furthermore, manipulation of the endogenous cannabinoid system shows great therapeutic promise. While it is known that cannabinoids induce biological effects by engaging CB1 and CB2 receptors, strong experimental evidence suggests that additional cannabinoid receptors exist. We have recently confirmed that the orphan receptor GPR55 constitutes one such novel cannabinoid receptor. Specifically, we found that A9THC, JWH-015 (a synthetic cannabinoid) and methanandamide (a stable endocannabinoid analog) activate phospholipase C, increase intracellular calcium and stimulate p38 MAP kinase phosphorylation in GPR55-expressing HEK293 cells. GPR55 is expressed at high levels throughout the brain, immune system, adipose tissue and testis, as well as in neurons, astrocytes and microglial cells in culture, as assessed by RT-PCR. In this application we describe a plan to fully characterize GPR55 distribution and signaling, and determine its place in mediating the effects of plant-derived, synthetic and endogenous cannabinoids. We will do this by completing the following specific aims: 1. Determining the tissue and cellular distribution of GPR55. 2. Identifying the best pharmacological tools to study GPR55 and elucidating its signaling pathways. 3. Ascertaining how GPR55 regulates neuronal, microglial, and adipocyte functions. The completion of these specific aims will allow us to evaluate the role of GPR55 as an additional cannabinoid receptor, and will provide the necessary pharmacological and molecular tools required to determine its involvement in mediating cannabimimetic effects. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Compounds that target endocannabinoid (eCB) signaling in the brain represent powerful pharmacological tools to probe the basic biological function of this signaling system in healthy and diseased brain and may represent novel therapeutic venues to treat neurological diseases such as Huntington's disease (HD). During the funding cycle of this RO1, our laboratory studied the function of ABHD6 in neurons and glia, as well as the therapeutic potential of ABHD6 inhibitors in R6/2 mice, an early-onset mouse model of HD. We leveraged functional proteomics and shRNA technology and identified ABHD6 as a candidate enzyme for 2-AG hydrolysis in brain. We then developed new ABHD6 inhibitors and showed that ABHD6 activity tightly controls the levels and efficacy of 2-AG at cannabinoid receptors. Together, these studies demonstrated that ABHD6 belongs to the eCB signaling system. Recently we found that in vivo ABHD6 inhibition greatly reduces seizure incidence and attenuates hippocampal neuropathology in R6/2 mice. In this grant, we will test the following hypothesis: The novel enzyme, ABHD6, controls both the level and efficacy of 2-arachidonoylglycerol (2-AG) at cannabinoid CB1 receptors. ABHD6 inhibitors reduce seizure activity in two HD mouse models (R6/2 and HDQ200) and may represent a novel class of therapeutics to treat seizures in general. To test this hypothesis, we propose 3 Aims: AIM 1: Development, validation, and mechanism of action of 3rd generation ABHD6 inhibitors that exhibit superior in vivo selectivity and efficacy. AIM 2: Determine to what extent ABHD6 in vivo inhibition and genetic deletion reduces seizure incidence in HD and chemically-induced seizures mouse models. AIM 3: Why do HD mice seize, and how does ABHD6 prevent this process? Thus we will characterize newly optimized inhibitors of ABHD6, an eCB-hydrolyzing enzyme that we identified during the previous funding period. Coupled to genetic approaches, we will use ABHD6 inhibitors to determine the molecular and cellular details of how this enzyme controls seizure incidence in HD mice models. Completion of the studies outlined above will provide a comprehensive understanding of the role of ABHD6 in healthy and HD mouse brain within the context of epileptic activity. Our long-term goal is to increase our understanding of th role played by ABHD6 in healthy and diseased brain, and help develop novel therapeutics that lack the potential for abuse and adverse effects produced by classic cannabinoid agonists.

Summary The newly discovered enzyme, ?/?-hydrolase domain 6 (ABHD6), controls the amount of 2- arachidonoylglycerol (2-AG), the most abundant endocannabinoid (eCB) in the brain. We recently found that in vivo ABHD6 inhibition decreases seizure incidence in several mouse models of epilepsy, and that this therapeutic response does not undergo tolerance nor does it produce overt side effects. These results suggest that ABHD6 inhibition might represent a novel anti-epileptic therapeutic approach. In this R21 grant proposal, we will test the therapeutic efficacy of a novel inhibitor of ABHD6, KT-182, in Scn1a+/- mice, a preclinical mouse model of Dravet Syndrome that accurately mimics this devastating intractable epilepsy that begins in infancy. Our hypothesis is that ABHD6 inhibition will significantly reduce seizure severity in Scn1a+/- mice without producing overt side effect and tolerance. To test this hypothesis, we will address three aims: 1. Effect of ABHD6 inhibition on thermally-induced seizures 2. Effect of ABHD6 inhibition on spontaneous seizures and premature death 3. Effect of ABHD6 inhibition on EEG and neuronal excitability The results gathered through this R21 grant will provide a solid foundation for future research aimed at developing ABHD6 inhibitors as new therapeutics for the treatment of Dravet Syndrome. This research will also increase our basic understanding of the molecular mechanisms by which endogenous 2-AG signalling controls synaptic transmission and seizure incidence.

Microtubule targeting agents (MTAs) are commonly prescribed to successfully treat many types of cancers; yet their use for the treatment of brain cancers, such as GBM, has been hindered by their inability to penetrate the brain in therapeutically-relevant doses. Recent evidence shows that GBMs are particularly sensitive to disruptions of microtubule (MT) functions and die by apoptosis, providing a rational to develop brain-penetrant MTAs. The laboratory of Dr. Nephi Stella at the University of Washington developed a new series of MTAs (ST compounds) and showed that these compounds destabilize MT through a different MOA from currently known MTAs. These MTAs kill GBM cells by triggering apoptosis both in vitro and in vivo. The work outline in this R21 grant proposal will provide foundational results on the novel MOA of ST compounds, as well as proof-of- concept in vivo efficacy results in preclinical model systems of PD-PGM, both of which will help develop this therapeutic approach for the treatment of GBM. Our aims are Aim 1: Determine the MOA of ST compounds on tubulin and MT dynamics, as well as their ability to cross cell membranes Aim 2: Determine the therapeutic efficacy and mechanism of ST compounds in PD-GBM xenograft model Our long-term goal is to help better understand and develop novel therapeutics to treat devastating cancers such as GBM.

Summary Recent evidence shows that the potent brain stimulatory effects of amphetamine are controlled by the endocannabinoid (eCB) signaling system. Our laboratory established that the enzyme, ?/?-hydrolase domain 6 (ABHD6), represent a novel molecular component of the eCB signaling system. Indeed, this post-synaptic enzyme controls the activity-dependent production of 2-arachidonoylglycerol (2-AG, the most abundant eCB in the brain), and as such controls the levels and efficacy of 2-AG at cannabinoid CB1 receptors (CB1R). We recently evaluated the involvement of ABHD6 in the locomotor responses stimulated by amphetamine in mice and found that its pharmacological inhibition and genetic deletion exerts a profound enhancing effect on the acute amphetamine-stimulated locomotor activity through a CB1R-dependent mechanism. In this R21 grant, we propose to identify brain regions involved in the ABHD6-dependent control of psychostimulants using several recently developed tools, including a brain-penetrant selective inhibitor of ABHD6 (KT-182 and MJN193), a Cre-dependent ABHD6 mouse line (ABHD6lox/lox), a specific antibody for ABHD6, and a CRISPR/Cas9 Slc6a3 (DAT) knockout model of hyperactivity. There are two main questions that will be addressed by this proposal. First, where in the brain is the interaction between ABHD6 and amphetamine occurring? Second, does ABHD6 alone, or in combination with low dose amphetamine, result in paradoxical calming response measured in a mouse model of attention deficit hyperactivity disorder (ADHD)-like phenotypes. Our aims are: 1: Identify brain regions involved in the ABHD6-dependent control of psychostimulants. 2: Establish the dose-dependent effects of ABHD6 inhibition on the paradoxical calming effects of psychostimulants in an animal model of hyperactivity. The completion of these studies will provide foundational results on the molecular mechanism by which ABHD6 regulates psychostimulant behavior in mice. A better understanding of this novel molecular interaction should help optimize the therapeutic use of psychostimulants while reducing their addiction and toxicity profile.

Microtubule targeting agents (MTAs) are commonly prescribed to treat many types of cancers; yet their use for the treatment of glioblastomas (GBM) is limited by their poor brain penetrance. We developed a new series of MTAs (ST-compounds) that destabilize microtubules (MT) and kill GBM through a novel mechanism of action (MOA). Our recent results show that ST-compounds pass the blood brain barrier (BBB) and exhibits in vivo therapeutic efficacy in a preclinical mouse model of GBM. This new R01 uses complementary expertise and approaches to study how the novel MOA of ST- compounds differs from known MTAs and its therapeutic efficacy in several preclinical mouse model of GBM. Specifically, it leverages several innovative technologies, including live-cell imaging and artificial learning algorithms, to better understand why GBM are particularly sensitive to the antitumor activity of ST-compounds. Experiments will be performed on patient-derived GBM (PD-GBM) in culture and include measure of real-time changes in GBM cell migration, cell division and cell cycle fate as fundamental readouts of tumorigenesis. In vivo experiments will be done on both genetic orthotopic mouse models of GBM and orthotopic mouse model of PD-GBM. Our aims are: 1: Differential impact of MTAs on the migration and mitosis of GBM in culture. 2: Differential impact of MTAs on the viability and fate of GBM in culture. 3: Therapeutic efficacy and mechanism of ST-401 in GBM models in vivo. Our immediate goal is to increase our understanding of the precise MOA by which MTAs regulate GBM tumorigenesis and how this interacts with standard care treatments. This work will help set a solid foundation for the development of a new class of MTAs for the safe treatment of patients diagnosed with GBM.

Summary Edibles that contain ?9-tetrahyrocannabinol (THC), the principal psychoactive ingredient produced by the cannabis plant, are becoming increasingly popular among adolescents, making examination of this method of use on the adolescent developing brain urgently needed. An exciting collaboration between the Stella and Land laboratories led to developing and validating a new model to study how oral self-administration of THC impacts adolescent rodent brain and ensuing behavioral impairment in adulthood. Thus, this model of voluntary oral consumption of THC-gelatin edibles achieves relevant blood THC levels, produces acute cannabimimetic effects and can easily be combined with other drugs, here cannabidiol (CBD). Adolescence is a critical period where maturation of neural systems is still occurring, particularly in the limbic system, and disruption of this maturation by drug use may lead to severe behavioral impairments in adulthood. Our preliminary results highlight one of the first successful self-administration models of THC in rodents, thereby enabling comparisons of the long-term consequences of voluntary THC on brain development and behavioral outcomes. Our new questions are to determine how long-term adolescent use of THC alone or in combination with CBD impacts 1) cannabimimetic response, which may provide insight as to the mechanism of 2) altered signaling within the mesocorticolimbic system, which may determine deficits in 3) adult motivated behavior and pain responses to opioids. Our aims are to determine: Aim 1: Establish optimal THC-THC/CBD consumption regimen by adolescent mice. Aim 2: Impact of THC and THC/CBD consumption during adolescence on adult mice behavioral responses to morphine. The completion of these studies, which utilize an innovative mouse model of voluntary oral consumption, will increase our mechanistic understanding of the impact of THC and THC/CBD use on adolescent brain development, and further determine how such perturbations influence motivated behavior and pain response to opioids.

Summary The most abundant endocannabinoids (eCB) in brain, 2-arachidonoyl glycerol (2-AG), is inactivated by two distinct enzymes: monoacylglycerol lipase (MAGL) and ????hydrolase domain-contain 6 (ABHD6). The selective inhibition of these enzymes increases the net levels of 2-AG in different neuronal compartments, presynaptic and postsynaptic, respectively. Accordingly, selective inhibition of these enzymes induces different spatiotemporal enhancement of 2-AG signaling and distinct or synergistic therapeutic benefits. We recently gathered results showing that ABHD6 hydrolyzes additional monoacylglycerol substrates than 2- AG and that ABHD6 inhibitors increase the net levels of 2-AG only in highly activity neurons, suggesting a novel molecular mechanism. To increase our mechanistic understanding of ABHD6 at the structural and atomic levels, we initiated an effort and have now successfully purified several functional ABHD6 protein constructs ready for structural analysis. Based on this premise, we propose the following two aims that will determine the: Aim 1: Molecular mechanisms of ABHD6 enzymatic activity. Aim 2: Structure function relationship of ABHD6. Completion of the work outline in this new R21 grant proposal will provide a comprehensive understanding of the role of ABHD6 in controlling eCB signaling in brain within the context of neurological diseases. Our long-term goal is to increase our understanding of the role played by ABHD6 in healthy and diseased brain and help develop novel therapeutics that lack the potential for abuse and adverse effects produced by classic cannabinoid agonists.