Randy A. Hall - US grants

The beta1-adrenergic receptor (beta1AR) is a G protein-coupled receptor that mediates many of the physiological effects of adrenaline and noradrenaline. Little is known, however, about the molecular mechanisms of beta1AR targeting and regulation in cells. This project aims to study the interaction between the beta1tAR and MAGI-2, a previously-unknown intracellular beta1AR binding partner that associates with the beta1AR carboxyl-terminus via a high-affinity PDZ domain-mediated interaction. The functional significance of the beta1AR/MAGI-2 association is completely unknown. MAGI-2 may be either a scaffolding protein important for subcellular localization of the beta1AR, a regulatory protein that modulates receptor G protein coupling, or a signaling intermediate that couples the beta1AR to diverse intracellular signaling pathways. These possibilities will be explored with a combination of in vitro and cellular experiments. The in vitro experiments will determine the specificity of the beta1AR/MAGI-2 interaction and the potential ability of MAGI-2 to facilitate the formation of complexes between the beta1AR and other proteins. The cellular experiments will focus on the ability of MAGI-2 to regulate the subcellular distribution of the beta1AR and/or to alter beta1AR signaling to effectors such as adenylyl cyclase and MAP kinase. Furthermore, immunostaining of MAGI-2 and beta1AR in primary neuronal cultures and in brain sections will be examined in order to determine how well the two proteins co-localize in native tissues. These studies will provide mechanistic insight into localization and regulation of the beta1AR, a receptor that is a common target for therapeutics used in the treatment of heart disease and other disorders.

Stimulation of the beta2-adrenergic receptor (beta2AR) by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by beta2AR activation of intracellular G proteins. However, it has recently been found that beta2AR regulation of cellular Na+/H+ exchange in some cells involves agonist-promoted coupling of the beta2AR to an intracellular protein called the Na+/H+ exchanger regulatory factor (NHERF). The mechanisms and potential generality of this NHERF-mediated signaling by the beta2AR are unknown. This project aims to elucidate the molecular mechanisms by which the beta2AR can regulate Na+/H+ exchange via association with NHERF, and also aims to find out whether the beta2AR can regulate physiological processes other than Na+/H+ exchange in a NHERF-mediated fashion. Since NHERF seems to act as either an allosteric regulatory protein or adaptor protein, the ability of the beta2AR to regulate the set of intracellular proteins bound by NHERF will be examined. The ability of the beta2AR to regulate the activity of another NHERF binding partner, the platelet-derived growth factor receptor, will also be studied, as will the capacity of NHERF to alter cell growth and proliferation in a beta2AR-regulated fashion. The phosphorylation of NHERF by G protein-coupled receptor kinase 6A, and possibly by other kinases, will also be examined, since an understanding of the regulation of NHERF by phosphorylation may be required for an understanding of NHERF-mediated signaling by the beta2AR. These studies will provide insight into hovel signaling pathways activated by the beta2AR, a receptor that is a common target for therapeutics used in the treatment of hypertension, heart disease and other disorders.

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian brain. GABA exerts its physiological actions in the brain via the activation of two distinct types of receptor: GABA-A receptors, which are ligand-gated ion channels, and GABA-B receptors, which are G proteincoupled receptors. GABA-A and GABA-B receptors are known to exhibit forms of cross-talk and mutual regulation for which no mechanism has been defined. This project aims to study the importance of a novel and direct interaction found between the GABA-BR1 receptor and the gamma2 subunit of the GABA-A receptor. This physical association may provide a mechanism to allow for direct cross-talk between GABA-A and GABA-B receptors. The structural determinants and physiological significance of this interaction, however, are completely unknown at the present time. The specific regions of GABA-BR1 and the gamma2 subunit of the GABA-A receptor involved in mediating their interaction will be elucidated using a mutagenesis approach in combination with both co-immunoprecipitation and fusion protein pull-down studies. The effects of GABA-A receptor association on GABA-B receptor pharmacology will be studied in ligand binding assays, and GABA-A receptor modulation of GABA-B receptor signaling and internalization will also be analyzed. Furthermore, GABA-B receptor regulation of GABA-A receptor pharmacology, channel activity and phosphorylation will be examined, with an emphasis on determining the functional importance of the direct interaction between GABA-BR1 and the GABA-A receptor gamma2 subunit. These studies will shed new light on the regulation of cellular responses to GABA and the molecular basis for cross-talk between GABA-A and GABA-B receptors. Such information is critical for a comprehensive understanding of pharmaceuticals acting on GABA receptors. GABA-A receptors are the targets for such commonly prescribed therapeutic drugs as benzodiazepines and barbiturates, while the more recently-identified GABA-B receptors represent excellent potential targets for novel therapeutic drugs aimed at treating disorders such as schizophrenia, epilepsy, anxiety, chronic pain and depression.

[unreadable] DESCRIPTION (provided by applicant): Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. Glutamatergic signaling between neurons is critically regulated by neighboring astrocytes, which surround most synaptic junctions and express specific glutamate transporters that control glutamate concentrations by removing glutamate from the synaptic cleft. Astrocytes also express glutamate receptors, specifically the metabotropic glutamate receptor subtypes mGluR3 and mGluR5, which are activated by glutamate and known to regulate glutamate transporter activity. Whereas much is known about the regulation and sorting of glutamate receptors in neurons, very little is known about the potential for specialized regulation of glutamate receptors and transporters in astrocytes. The mGluR subtypes 3 and 5, as well as the astrocytic glutamate transporter EAAT1, all possess large intracellular carboxyl-termini (CT) that play key roles in the control of their activity. Since these CTs terminate in consensus motifs for potential association with a class of conserved protein-protein interaction domains known as PDZ domains, we screened a custom-made PDZ domain proteomic array and found that the CTs of mGluR3, mGluR5 and EAAT1 all exhibit robust and specific interactions with the PDZ domains of the multifunctional scaffold protein NHERF-2. These interactions were confirmed in a cellular context, and immunohistochemical studies revealed that NHERF-2 is abundantly expressed in astrocytes in the brain. We hypothesize that NHERF-2 is a central regulator of the activity and localization of mGluRs and EAAT1 in astrocytes, and may facilitate mutual regulation between mGluRs and glutamate transporters. We will test this idea by examining NHERF-2 regulation of mGluR3, mGluR5 and EAAT1 functional activity and cross-talk, as well as by exploring the possibility that NHERF-2 may control mGluR and EAAT1 localization in vivo by performing immunohistochemical analyses on brain tissue from wild-type versus NHERF-2 knockout mice. These studies are of significant clinical importance because metabotropic glutamate receptors and glutamate transporters are considered to be excellent potential therapeutic targets in the treatment of stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, schizophrenia, and other brain disorders. PUBLIC HEALTH RELEVANCE Astrocytes and neurons are intermingled throughout the brain, and astrocytes possess receptors that can sense neuronal activity as well as transporters that can regulate the levels of synaptic neurotransmitters. Astrocytic receptors and transporters are often clustered near synapses and known to be highly regulated, but the mechanisms underlying this targeting and regulation are largely unknown. The work described in this application will explore the mechanisms by which astrocytic receptors and transporters, specifically those that respond to the neurotransmitter glutamate, are targeted and regulated. These studies are of significant clinical importance because astrocytic glutamate receptors and transporters are considered to be excellent potential therapeutic targets in the treatment of stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, schizophrenia, and other brain disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Abnormalities in the development of the cerebral cortex can lead to a range of distinct neurodevelopmental disorders, including autism and Asperger's syndrome. Fresh insights into the dysregulation of cortical development associated with complex polygenetic disorders such as autism can be gained from studies on simpler monogenetic disorders that also are rooted in abnormal development of the cerebral cortex. One such disorder is bilateral frontoparietal polymicrogyria (BFPP), a condition in which patients exhibit disordered cortical connectivity resulting in profound cognitive abnormalities. BFPP is an autosomal recessive syndrome that results from mutations in the orphan receptor GPR56, which is a member of the poorly-understood adhesion family of G protein-coupled receptors. GPR56 possesses an extremely large extracellular amino-terminus (NT) that has homology to adhesion proteins and has been shown to be cleaved during receptor processing but yet remain associated with the seven-transmembrane (7TM) region of the receptor. However, the fundamental mechanisms of the activation and regulation of GPR56 signaling are presently unknown. Our preliminary studies have shown that expression of wild-type GPR56 in heterologous cells results in strong activation of Rho and b-catenin/Tcf signaling, and that truncation of the large GPR56-NT results in a receptor that is trafficked efficiently to the plasma membrane but no longer activates the aforementioned signaling pathways. We hypothesize that interaction of the GPR56-NT with an adhesion partner promotes receptor signaling, and will explore this idea by elucidating the structural determinants of the GPR56-NT that control receptor activity. We furthermore hypothesize that GPR56 signaling is highly regulated by cytoplasmic kinases, arrestins and PDZ scaffolds, and will explore these possibilities using a combination of phosphorylation assays, imaging studies, co-immunoprecipitation experiments, and siRNA knockdown approaches in both transfected cells and cultured cortical neurons. Knowledge gained from these studies will provide fresh insights into the role of GPR56 in human disease and also into the fundamental mechanisms that regulate the development of the cerebral cortex. PUBLIC HEALTH RELEVANCE: Abnormalities in the development of the cerebral cortex can lead to a range of distinct neurodevelopmental disorders, including autism and Asperger's syndrome. To shed light on the factors that contribute to such diseases, we propose to study the fundamental properties of the orphan receptor GPR56, which is believed to play a key role in cortical development since mutations in GPR56 cause a rare genetic disorder in which the development of the cerebral cortex is grossly distorted. Knowledge gained from these studies focused on the normal function of GPR56 will provide fresh insights into the role of GPR56 in human disease and also into the fundamental mechanisms that regulate the development of the cerebral cortex.

DESCRIPTION (provided by applicant): The orphan G protein-coupled receptor GPR56 is highly-expressed in neural stem cells (NSCs), and mutations in GPR56 cause disordered brain development in humans. The key role that GPR56 plays in NSC function makes it an attractive target for therapeutics that might be capable of highly-selective NSC modulation. However, little is currently known about the fundamental properties of GPR56. The specific targeting of GPR56 by therapeutics will require a more comprehensive understanding of the molecular mechanisms controlling receptor activation, regulation and localization. We will therefore study the mechanism of activation for GPR56, examining in particular the issue of whether shedding of the N-terminus (NT) is involved in receptor activation. We will also study the factors controlling GPR56-NT shedding, as well as whether receptor activation can be influenced by NT-binding peptides. Furthermore, we will assess the regulation of GPR56 activity by cytoplasmic binding partners of the receptor that have been identified in preliminary work, including beta-arrestins and PDZ scaffolds. Finally, we will study the factors controlling GPR56 localization in NSCs and other cell types, with a particular emphasis on determining the importance of receptor targeting to cilia. In addition to providing insights about the fundamental properties of GPR56, these studies will lay the groundwork for targeting GPR56 as a means of selectively modulating NSC function in the treatment of various neurodevelopmental and neurodegenerative diseases.

DESCRIPTION (provided by applicant): GPR37 and GPR37L1 are orphan G protein-coupled receptors that are most abundantly expressed in the central nervous system. We assessed potential activation of GPR37 and GPR37L1 by a handful of orphan neuropeptides and found that both receptors can be activated in transfected non-neuronal cells by prosaptide, which is a 14-amino-acid peptide derived from the precursor protein prosaposin. Prosaptide is known to exert neuroprotective actions in certain areas of the brain, such as the substantia nigra, via unidentified G protein-coupled receptors, and also known to exert beneficial effects in vivo on relieving neuropathic pain and promoting remyelination in animal models of nerve damage. The overall goals of the experiments described in this exploratory R21 application are to test whether GPR37 and/or GPR37L1 mediate the ability of prosaptide to stimulate survival signaling in primary neurons and promote the survival of dopaminergic neurons in vivo. The signaling studies will focus on primary cultures of dopaminergic neurons from the ventral mesencephalon and cortical neurons, which preferentially express GPR37 vs. GPR37L1, respectively. Cultures will be prepared from wild-type mice as well as from mice that are null for expression of GPR37 (GPR37-KO) or GPR37L1 (GPR37L1-KO). The ability of prosaptide to stimulate phospho-ERK and phospho-Akt in these neurons will then be assessed in cultures prepared from the WT vs. KO mice. In addition to these experiments studying the effects of prosaptide on survival signaling, we will also challenge the neurons with insults (such as trophic factor withdrawal or treatment with the toxin MPP+) in the absence and presence of varying doses of prosaptide to assess the effects of the peptide on neuronal survival. In parallel with the experiments on cultured neurons, the whole-animal studies will assess the neuroprotective effects of prosaptide on dopaminergic neurons in the substantia nigra following treatment with the dopaminergic neurotoxin MPTP. We will employ 2 different MPTP regimens (both acute and chronic) to assess the role of GPR37 and GPR37L1 in the neuroprotective effects of prosaptide in vivo. These MPTP toxicity studies will be performed in parallel with WT, GPR37-KO and GPR37L1-KO mice. If the work described in this proposal can establish GPR37 and/or GPR37L1 as the receptor(s) that mediate the neuroprotective actions of prosaptide in primary neuronal cultures and in vivo, then future studies can focus on the discovery of small molecule agonists and/or positive allosteric modulators of these receptors that might serve as novel therapeutics in the treatment of Parkinson's Disease, stroke, neuropathic pain and myelination disorders.

DESCRIPTION (provided by applicant): Modern drug discovery and development require the training of scientists who understand the molecular, physiological and quantitative basis of drug action and specificity, and who can apply modern technologies and concepts to the development of novel therapeutic strategies. This multidisciplinary doctoral training program in the Pharmacological Sciences is designed to help meet that demand by preparing students for biomedical research careers in schools of medicine, dentistry and pharmacy, in research institutes, and in governmental or industrial laboratories. The most important component of training is laboratory research, first as a series of research rotations, then in the dissertation laboratory. This training is complemented by a core course that integrates the theoretical and experimental foundations of modern biological sciences; core courses in pharmacology that emphasize quantitative analysis of drug action, pharmacokinetics, drug disposition, biostatistics and experimental design; advanced courses in specialty areas; seminar courses and journal clubs. Emphasis throughout is placed on development and refinement of communication and analytical skills. The 48 training faculty represent 17 basic science and clinical departments at Emory providing a wealth of diverse research training opportunities. Research foci in the program include Neurological Diseases and Therapy, Cancer Pharmacology, Cardiovascular Pharmacology, Chemical Biology and Drug Discovery and Novel Therapeutic Modalities. Cell Signaling, Systems and Integrative Pharmacology, and Toxicology are crosscutting themes. This Program currently supports six students each year, who are selected mainly from a pool of approximately 20-30 eligible students in the first three years of the Molecular and Systems Pharmacology (MSP) Program. Six slots are requested in this renewal. Graduates will have acquired broad familiarity with pharmacology, knowledge in depth in the area of dissertation research, and the technical, communicative and analytical skills necessary to pursue an independent research career. Students graduate an average of 5.9 years after matriculation. The research conducted by the trainees in this program will advance our knowledge of disease processes and contribute to development of novel and improved therapeutic strategies that will benefit the health of our citizens. By preparing young scientists to contribute to and lead the nation's efforts in these areas, this training program will help to ensure that our ability to imprve the nation's health remains strong in the future.

DESCRIPTION (provided by applicant): Stroke is the third leading cause of death in the USA and the leading cause of long- term disability. Unfortunately, there are few available pharmacotherapies capable of limiting damage following a stroke. For this reason, novel targets for post-stroke therapies are desperately needed. We recently identified the secreted factor prosaposin and its active fragment prosaptide as ligands for the brain-expressed G protein-coupled receptors GPR37 and GPR37L1. Interestingly, prosaptide has been extensively studied for two decades as a peptide that improves recovery in rodent models of stroke. However, it is not known at present if prosaptide exerts these protective effects in vivo via stimulation of GPR37 and/or GPR37L1. Moreover, nothing is known about the signaling pathways downstream of GPR37 and GPR37L1 that might be relevant to the protective actions of prosaposin and prosaptide. In this project, we will study the protective actions of prosaptide on primary cortical astrocytes, a cell type that expresses both GPR37 & GPR37L1, and elucidate the signaling pathways downstream of these receptors that mediate the pro-survival effects of prosaptide treatment following oxygen/glucose deprivation. We will also seek to understand the mechanisms by which the protective signaling pathways downstream of GPR37 & GPR37L1 are regulated, since nothing is currently known about this topic. Furthermore, we will perform parallel studies in vivo in which we will study damage induced by focal cerebral ischemia in wild- type mice as well as mice lacking expression of GPR37 and/or GPR37L1 in order to assess the importance of these receptors and their downstream signaling pathways in mitigating damage following a stroke and mediating the protective actions of prosaptide. These studies will provide insights into the potential utility of GPR37 & GPR37L1 as novel targets for the treatment of stroke and also shed light on the signaling pathways downstream of these receptors that are relevant to their protective effects.

? DESCRIPTION (provided by applicant): BAI2 is a G protein-coupled receptor that is expressed almost exclusively in the central nervous system. This receptor is highly expressed in astrocytes as well as in certain neuronal populations. A BAI2 mutation was recently identified by the NIH Undiagnosed Diseases Program as being associated with a novel neurological disorder. This disease-associated BAI2 mutation is a missense mutation that alters an amino acid in the receptor's cytoplasmic carboxyl-terminus, a region of BAI2 that is known to be important for controlling the receptor's signaling and regulation. In this project, we will assess the effects of the disease-associated mutation on the trafficking and signaling of BAI2 in order to determine how this mutation might be causing human disease and also gain insights into potential therapeutic options for patients harboring this mutation. Moreover, these studies will also shed significant light on the normal function of BAI2 and thereby set the stage for targeting this receptor pharmacologically in order to benefit the wider population of patients who express wild-type BAI2 but suffer from neurological or psychiatric disorders that might be treatable via modulation of BAI2 activity.

? DESCRIPTION (provided by applicant): GPR37L1 is a G protein-coupled receptor that is expressed almost exclusively in the central nervous system. This receptor is highly expressed in astrocytes as well as in oligodendrocytes and certain neuronal populations, and my laboratory recently identified GPR37L1 as a receptor for the secreted neuroprotective and glioprotective factor prosaposin. A GPR37L1 mutation was recently found to be associated with a novel inherited neurological disorder characterized by headaches and seizures starting at the onset of puberty, with the seizures growing more frequent and severe throughout the teen years and ultimately resulting in death by the late teens. The GPR37L1 mutation identified in the affected members of this family is a missense mutation that changes an amino acid in the receptor's third cytoplasmic loop, a region of most G protein-coupled receptors that is important for controlling their signaling and regulation. We will assess the effects of the mutation on the folding, trafficking and signaling of GPR37L1 in order to determine how this mutation might be causing human disease and how patients harboring this mutation might be treated. In addition to providing insights into the neurological disorder associated with the GPR37L1 mutation, these studies will also shed significant light on the normal function of GPR37L1 and thereby set the stage for targeting this receptor pharmacologically in order to generally benefit patients sufferin from stroke and/or other neurodegenerative conditions.

Project Summary Multiple sclerosis (MS) is characterized by progressive demyelination of axons in the central nervous system (CNS). One major approach to developing new MS treatments is the development of therapeutics that can act on myelin-producing oligodendrocytes to enhance the production and/or stability of myelin. Toward this end, it is important to identify potential drug targets that are selectively enriched in oligodendrocytes and capable of promoting myelin stability. GPR37 is a nervous system-specific G protein-coupled receptor that is highly expressed in oligodendrocytes. We performed proteomic analyses of brain tissue from GPR37 knockout (Gpr37-/-) mice and found that one of the most dramatically down-regulated proteins was the myelin- associated glycoprotein (MAG), which is a myelin-enriched protein known to regulate myelin stability. In parallel studies, we found that GPR37 and MAG form complexes in cells. Since MAG-deficient mice are known to exhibit significantly more severe demyelination in response to insults, we explored the Gpr37-/- mice in the cuprizone model of demyelination and found that these mice exhibit a much more dramatic loss of myelin than wild-type mice in response to cuprizone treatment. Given the striking similarity in phenotypes between Gpr37-/- and MAG-deficient mice, we hypothesize that the interaction between GPR37 and MAG exerts effects on oligodendrocyte physiology and myelination. This hypothesis will be tested by i) dissecting the structural determinants of the interaction between GPR37 & MAG and ii) studying mutual regulation between GPR37 and MAG in cultured oligodendrocytes from Gpr37-/- mice. These studies will shed light on the interaction and mutual regulation between the oligodendrocyte-enriched proteins GPR37 and MAG, which will lead to a greater understanding of both proteins in the regulation of oligodendrocyte physiology and myelination. Moreover, the studies proposed here will enhance our knowledge of the action of GPR37 in regulating myelination and thereby set the stage for the targeting of this receptor by novel therapeutics aimed at treating MS and other myelination disorders. ! !

Project Summary Schizophrenia is a devastating psychiatric disorder that affects ~1% of the USA population. Over the past few decades, twin studies and other genetic analyses have established that approximately 80% of schizophrenia risk is due to heritability, but at present only a small number of genes have been definitively linked to this clearly polygenic disorder. Thus, the identification of additional genes that contribute to schizophrenia risk can provide new insights about the disease and also suggest potential new avenues for treatment. Recently, whole-exome analyses of an extensive set of multiplex schizophrenia families revealed several mutations in the brain angiogenesis inhibitor 1 (BAI1) to be associated with schizophrenia. BAI1 is a synaptic receptor known to regulate dendritic spines and synaptic plasticity, and the BAI1 mutations linked to schizophrenia in these analyses are all missense mutations with extremely high CADD scores. We will assess the effects of these schizophrenia-associated mutations on the trafficking and signaling activity of BAI1 in order to determine how these mutations may be contributing to the development of schizophrenia. The signaling studies will assess the effects of the mutations on both i) constitutive signaling activity by the receptor and ii) signaling stimulated by co-expression of BAI1 with phospholipid scramblases. In parallel studies, we will determine whether this scramblase-mediated potentiation of BAI1 signaling is due to externalization of phosphatidylserine, a previously reported ligand for the extracellular domains of BAI1. Thus, the proposed studies will shed new light on the fundamental biology of BAI1 while also assessing the functional effects of the schizophrenia-associated BAI1 mutations, with the goal of facilitating the eventual targeting of this receptor by small molecule therapeutics that may be useful new treatments for schizophrenia and other psychiatric disorders. ! !

Project Summary A significant fraction of epilepsy patients cannot be effectively treated by current medications. For this reason, new drug targets that can modulate seizure vulnerability are needed. G protein-coupled receptors (GPCRs) are outstanding drugs targets, and thus GPCRs that can influence seizure vulnerability are of special interest to study as potential new drug targets in the treatment of epilepsy. The GPCR known as GPR12 has been linked in genetic studies to epilepsy in humans. Moreover, recent data from our lab has shown that GPR12 forms a complex with the amyloid precursor protein (APP) and exerts striking control over APP expression in cultured cells. These two observations are potentially linked, as APP is known to strongly influence seizure vulnerability in vivo. We hypothesize that GPR12 regulates APP processing via physical association and/or receptor signaling, with this regulation contributing to GPR12 modulation of seizure vulnerability. These ideas will be tested in two aims: i) we will dissect the interaction between GPR12 and APP and determine how GPR12 controls APP expression, and ii) we will examine GPR12-mediated regulation of APP in vivo and also assess GPR12 modulation of seizure vulnerability in a mouse model. If deletion of GPR12 is found to result in both perturbed APP levels in vivo and altered seizure vulnerability, these findings would set the stage for future studies focused on determining how these two observations might be related. Moreover, such findings would also pave the way for the eventual targeting of GPR12 by drug-like small molecules to develop novel therapeutics for the treatment of epilepsy and other disorders. !

Project Summary Epilepsy and migraine have long been recognized as being linked in certain families, but the genetic basis of this comorbidity in not well understood. We and others have recently found evidence in human genetic studies that variation in GPR37L1 is a major contributor to epilepsy and migraine risk, especially in cases where epilepsy and migraine are comorbid. GPR37L1 is expressed predominantly in astrocytes, and thus studies on this receptor may lead to more general insights into how astrocyte dysfunction contributes to epilepsy and migraine. The goal of this project is to elucidate the cellular and mechanistic basis by which GPR37L1 regulates seizure and migraine susceptibility. We will assess the trafficking and signaling properties of disease-associated GPR37L1 variants in astrocytes and also address the mystery of why GPR37L1 signaling appears to be dependent on the astrocytic context. Furthermore, we will study the seizure vulnerability of knock-in mice harboring pathogenic human variants and also explore how astrocyte development in human cortical organoids is affected by pathogenic variants of GPR37L1. Additionally, we will assess mice lacking Gpr37L1 or expressing pathogenic Gpr37L1 variants for migraine-relevant phenotypes and evaluate these mutant mice for changes in cortical circuit excitability that might inform both the migraine and seizure phenotypes. These studies will provide novel insights into the fundamental biology of GPR37L1 and also pave the way for the development of novel GPR37L1-targeted therapeutics for the treatment of epilepsy, migraine and other diseases.