Stephen J. Moss - US grants

GABAB receptors are the major sites of slow synaptic inhibition in the brain. Changes in GABAB receptor function play key roles in epilepsy, nociception, depression, addiction, mental retardation and neuroprotection. The number of GABAB receptors expressed on the cell surface of neurons is dependent on the formation of heterodimers between GABABR1 and R2 subunits. Recent studies have demonstrated that once at the cell surface GABAB receptors are stable entities, which do not undergo agonist-induced internalization. Therefore primary determinants of the efficacy of slow synaptic inhibition mediated by GABAB receptors will be the production of receptor heterodimers, the trafficking of assembled heterodimers to the cell surface, and their coupling to the appropriate effectors. Here we hypothesize that GABAB receptor activity is regulated by the activity of AMP-dependent protein kinase (AMPK),which phosphorylates serine residues withinboth receptor subunits. Phosphorylation increases GABAB receptor cell surface expression levels by enhancing receptor trafficking and assembly in the secretory pathway. In addition phosphorylation by AMPK enhances receptor effector coupling by reducing desensitization. Therefore AMPK activity provides a dynamic mechanism for regulating the efficacy of GABAB receptor signaling. We will use a combination of cell biological, biochemical and electrophysiological approaches to carry out three independent but related specific aims: 1. To test the hypothesis that GABAB receptor are dynamically phosphorylated by intimately associated AMPK. 2. To test the hypothesis that direct phosphorylation of GABAB receptor by AMPK regulates receptor cell surface stability and effector coupling. 3. To test the hypothesis that AMPK activity modifies GABABreceptor membrane trafficking and assembly. Together, our approaches will provide a more thorough understanding of the cell surface stability and function of GABAB receptors. The results of these studies will have the potential to make significant contributions to the development of novel therapeutic strategies for such debilitating disorders as epilepsy, nociception, depression, addiction, and mental retardation.

DESCRIPTION (provided by applicant): GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the mammalian nervous system. The fast synaptic inhibitory action of GABA is due largely to the activation of GABAA receptors, which are Cl- permeable ligand-gated ion channels. These receptors are also targets for several clinically important drug classes, including benzodiazepines, barbiturates, neurosteroids and general anesthetic. Moreover, modifications of GABAA receptor function are critical in a number of CNS pathologies including: epilepsy, anxiety, addiction, autism, and mental retardation. While the pharmacological manipulation of GABAA receptor function has been widely exploited clinically the endogenous mechanisms used by neurons to control the function and cell surface stability of these proteins remain unknown. However this issue is of central importance given the roles GABAA receptors play as mediators of synaptic inhibition, drug targets and in human disease. We hypothesize that the cell surface stability of GABAA receptors, is regulated via direct interactions with the AP2 complex, which plays an essential role in the recruitment of cargo into clathrin-coated pits to facilitate endocytosis. These interactions are dependent in turn on the phosphorylation status of GABAA receptors, which is subject to dynamic regulation by defined cell signaling pathways. Thus we will use a combination of cell biological, biochemical and electrophysiological approaches to carry out three independent but related specific aims: Specific Aim 1. To test the hypothesis that specific motifs mediate the interaction of GABAA receptors with the AP2 complex, and that these interactions are regulated by receptor phosphorylation. Specific Aim 2. To test the hypothesis that PKC activity regulates the phosphorylation, cell surface stability and activity of GABAA receptors. Specific Aim 3. To examine the significance of AP2 mediated endocytosis in the control of GABAA receptor cell surface stability. Together, our approaches will provide a more thorough understanding of the cell surface stability and function of GABAA receptors. The results of these studies will have the potential to make significant contributions to the development of novel therapeutic strategies for such debilitating disorders as epilepsy, anxiety, addiction, autism, and mental retardation.

GABA ([unreadable]y-aminobutyric acid) is the major inhibitory neurotransmitter in the mammalian nervous system. The fast synaptic inhibitory action of GABA is due largely to the activation of GABAA receptors, which are Cl~ permeable ligand-gated ion channels. These receptors are also targets for several clinically important drug classes, including benzodiazepines, barbiturates, neurosteroids and general anesthetics. Modifications of GABAA receptor function have been implicated in a range of CNS pathologies. It is essential for efficient synaptic transmission that GABAA receptors are clustered and stabilized at postsynaptic sites opposed to presynaptic GABAergic terminals. However, the mechanisms that underlie the selective accumulation and stabilization of these receptors at synaptic localizations remain unknown. We hypothesize that synaptic GABAA receptors, as opposed to extrasynaptic receptor populations, exhibit reduced lateral mobilities and enhance surface stabilities and that this difference in dynamic behavior is regulated by gephyrin, a protein implicated in the formation of inhibitory synapses. Thus we will use a combination of cell biology and biochemical approaches to carry out three independent but related specific aims: Specific Aim 1: Wewill test the hypothesis that synaptic and extrasynaptic GABAA receptors have different rates of lateral mobility. Specific Aim 2: Wewill test the hypothesis that synaptic and extrasynaptic GABAA receptors have different cell surface stabilities. Specific Aim 3: We will test the hypothesis that gephyrin regulates GABAA receptor mobility and cell surface stability at inhibitory synapses. Together, our approacheswill provide a more thorough understanding of the primary determinants that regulate accumulation of GABAA receptors at synaptic sites. The results of these studies will have the potential to make significant contributions to the development of novel therapeutic strategies for such debilitating disorders as epilepsy, anxiety,addiction, autism, and mental retardation.

DESCRIPTION (provided by applicant): ?-aminobutyric acid (GABAA) receptors are key sites of synaptic inhibition in the brain and are critical drug targets for therapeutic agents including benzodiazepines, barbiturates, general anesthetics and neurosteroids. Moreover compromised GABAA receptor function is central to a number of CMS pathologies including epilepsy, anxiety, sleep disorders, addiction, autism, mental retardation, depression and schizophrenia. Ubiquitination of lysine residues is a commonly used cellular mechanism to regulate both protein half-life and the endocytic fate. This accepted paradigm together with our preliminary studies has led us to formulate a central hypothesis driving the experiments described in this proposal: GABAA receptors are subject to direct ubiquitination of lysine residues with the intracellular domains of individual receptor subunits, a process that is subject to dynamic modulation by neuronal activity. Depending on the subunit ubiquitinated this covalent modification acts to decrease receptor half-life within the secretory pathway or enhances lysozomal targeting, thereby modulating GABAA receptor cell surface stability and the efficacy of synaptic inhibition. Our efforts will center on four complementary but distinct experimental goals: 1. To test the hypothesis that GABAA receptor [unreadable]3 subunit ubiquitination regulates receptor cell surface stability by modulating insertion at the plasma membrane 2. To test the hypothesis that GABAA receptor ?2 subunit ubiquitination regulates receptor cell surface stability by modulating lysozomal targeting 3. To test the hypothesis that ubiquitination modulates the efficacy of synaptic inhibition mediated by GABAA receptors 4. To test the hypothesis that neuronal activity regulates GABAA receptor cell surface stability by modulating receptor ubiquitination Together, our approaches will provide a more thorough understanding of the primary determinants that regulate accumulation of GABAA receptors at synaptic sites. The results of these studies will have the potential to make significant contributions to the development of novel therapeutic strategies for such debilitating disorders as epilepsy, anxiety, sleep disorders, addiction, autism, mental retardation, depression and schizophrenia.

DESCRIPTION (provided by applicant): Research will be carried out primarily in Chile at the University of Chile in collaboration with Dr. Andris Couve, as an extension of NIH grant R01 NS048045. GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian nervous system. The slow action of GABA is mediate by GABAB receptors (GABABRs), which belong the group C G protein- coupled receptors. They function as heterodimers composed of GABABR1 and GABABR2 and couple to adenylyl cyclase, presynaptic voltage-gated Ca2+ channels and postsynaptic inwardly rectifying K+ channels. They have been implicated in epilepsy, nociception, depression and cognition. They also represent attractive targets for the treatment of withdrawal symptoms from addictive drugs such as cocaine. We suggest that GABABRs mediate their plethora of functions as part of multi-protein complexes that are subject to exquisite modulation. Thus we have proposed our general hypothesis: "GABABRs modulate the efficacy of inhibitory synaptic transmission as part of multi-protein complexes." Previous attempts at identifying these complexes have been only partially successful. We have now employed a unique approach to identify GABABR associated proteins in neurons using immunopurification and mass spectrometry. As a result we have obtained several candidates involved in protein turnover. To maximize the impact of our screen and open a new line of research we have prioritized the study of one candidate, namely dynamin-1, a protein with an established role in protein trafficking. Here we propose to investigate its role in GABABR turnover in the context of the following hypothesis: "Dynamin-1 defines the trafficking properties and membrane availability of GABABRs." We will develop the following specific aims: Specific Aim 1: Characterize the endocytosis of GABABRs in neurons. Specific Aim 2: Evaluate the specificity of the interaction to dynamin-1. Specific Aim 3: Test the functional consequences of the interaction to dynamin-1. Together with the parent grant, our proposed studies are likely to contribute to uncover novel mechanisms of trafficking and assembly of GABABRs into inhibitory sites and the identification of signaling events with potential therapeutic value. PUBLIC HEALTH RELEVANCE: Together with the parent grant, our proposed studies are likely to contribute to uncover novel mechanisms of trafficking and assembly of GABABRs into inhibitory sites and the identification of signaling events with potential therapeutic value.

DESCRIPTION (provided by applicant): Fast neuronal inhibition in the adult brain is critically dependent on the ability of neurons to synthesize the inhibitory neurotransmitter ?-aminobutyric acid (GABA) that mediates its actions via ionotropic GABAA and metabotropic GABAB receptors. Deficits in GABAergic inhibition are central to epilepsy and a plethora of other neuropsychiatric disorders. The major metabolic precursor for GABA synthesis by neurons is glutamine, which in turn is supplied by astrocytes. The ability of astrocytes to export glutamine i dependent upon the activity of the astrocyte-specific enzyme glutamine synthetase (GS). The significance of GS for brain function has been revealed by the use of specific inhibitors and gene deletion. These manipulations lead to seizures and death that result from decreased synaptic inhibition. Consistent with this, deficits in GS expression are found in the brains of epileptics and animal models of epilepsy. To date, however, there have been no systematic experiments to evaluate how the activity of GS is regulated to meet the demands of neurons for glutamine, and if deficits in these processes contribute to epileptogenesis. These issues will be addressed here. Preliminary results suggest that GS expression is subject to powerful regulation by astrocytic GABABRs. 2+ GABABRs are heterodimeric G-protein coupled receptors which couple to Gi/o, to modulate Ca transients, and inhibit the activity of adenylate cyclase. Preliminary studies have revealed that astrocytic GABABRs act to stabilize GS by reducing its ubiquitination and subsequent degradation. To understand the significance of this finding, we have created a mouse in which the expression of astrocytic GABABRs can be specifically ablated. These mice have decreased steady state expression levels of GS, spontaneous seizures, and premature death. Based on these observations we hypothesize that: Astrocytic GABABRs prevent the ubiquitin-dependent degradation of GS and thereby ensure the continued availability of glutamine for neuronal GABA synthesis. This proposal will center on three aims that are detailed below: Aim 1. To test the hypothesis that astrocytic GABABRs regulate the stability of GS. Aim 2. To test the hypothesis that ablating the expression of astrocytic GABABRs results in GS degradation, spontaneous seizures and death. Aim 3. To test the hypothesis that reducing astrocytic GABABR expression compromises synaptic inhibition and neuronal viability. Together these experiments will provide unique insights into the role that astrocytic GABABRs play in regulating GS expression, fast synaptic inhibition and epileptogenesis. Collectively these studies may lead to the development of novel therapies to increase the activity of GS to alleviate the burdens of epilepsy

DESCRIPTION (provided by applicant): Epilepsy is a curable disease, but nearly one out of four patients develops seizures that are resistant to anti- epileptic medications. Seizures are neurological conditions characterized by abnormal brain waves and decreased inhibition. The fundamental determinant of inhibition in the brain is the protein KCC2, which dictates how key anti-epileptic drug targets work. Without KCC2 these targets would no longer function properly, and consequently, neither would the first- and second-line anti-epileptic drug therapies. KCC2 was only recently found to be severely diminished in the brains of people suffering from drug-resistant epilepsy. This proposal represents the first attempt to unequivocally identify the loss o this protein as the common feature of drug- resistant seizures. Given the fundamental role of this protein, it is vital that the proposed experiments are completed for the benefit of people suffering from this as yet incurable disorder. The project will consist of an array of molecular, electrophysiological, and genetic experiments to confirm the hypothesized role of KCC2 in animal models of drug-resistant seizures. The first project aim utilizes a new strategy to examine impaired KCC2 function on the molecular scale. This will demonstrate that a deficit in KCC2 function impedes inhibitory signaling between brain cells. The second project aim utilizes a model of drug-resistant seizures in animal brain tissue to directly demonstrate that the deficit in KCC2 function causes this disorder. This will be the first demonstration of this process in any model of drug-resistant seizures. The final project aim will directly address a longstanding unanswered question in medicine. Physicians have known for years that drug-resistant seizures develop with time, slowly and sometimes rapidly evolving from a state that can be treated into one that cannot. These experiments will be the first demonstration in a living animal brain that the progressive loss of KCC2 function underlies the development of these drug-resistant seizures. Such a finding would suggest that rescuing KCC2 function could restore the therapeutic effectiveness of current anti-epileptic drugs. The overarching research objective is to lay the foundation for immediate testing of a novel therapeutic strategy that targets KCC2 function to improve the quality of life of those suffering from drug-resistant seizures.

Neuroactive steroids (NASs) such as allopregnanolone (ALLO) play a central role in regulating behavior via their potent anxiolytic, anticonvulsant, sedative, and hypnotic actions. Accordingly, modifications in the levels of NASs contribute to anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome. Classically, NASs are thought to act by rapidly boosting neuronal inhibition by positive allosteric modulation of the activity of ?-aminobutyric acid type A receptors (GABAARs). In addition to their allosteric actions, we have recently shown that NASs act via a protein kinase C-dependent mechanism to enhance the phosphorylation of residues including Serine?s 408 and 409 in the ?3 subunit (S408/9), a process that increases GABAAR number on the plasma membrane leading to a sustained increase in the efficacy of GABAergic inhibition. Although we have shown that NASs do not directly activate PKC, the mechanism by which NASs lead to changes in phosphorylation of GABAAR subunits are unknown. It is emerging that in addition to their positive allosteric modulation of GABAARs, NASs can directly activate membrane progesterone receptors (mPRs); G-protein coupled receptors that regulate PKC signaling. However, no information is available on the role that mPRs play in regulating GABAAR activity. Likewise, the behavioral significance of the sustained mPR-mediated metabotropic actions of NASs remains unexplored. To address these issues we have created mice in which S408/9 in the ?3 subunit have been mutated to alanines, mutations that are predicted to reduce the metabotropic actions of NASs on GABAAR function. Preliminary studies using these tools have allowed us to formulate a central hypothesis that will be tested here; NASs activate mPRs to enhance the phosphorylation of GABAARs on residues including S408/9 in the ?3 subunit, a mechanism that underlies their anticonvulsant efficacy. In contrast, their anxiolytic efficacy is mediated via allosteric potentiation of GABAAR activity, a process dependent upon Q241 in the ?2 subunit. Our experiments will focus on the following aims. Aim 1. To test the hypothesis that the ability of NAS to induce sustained effects on GABAergic inhibition is dependent upon S408/9A in the ?3 subunit. Aim 2. To test the hypothesis that the anxiolytic, and anticonvulsant efficacy of NASs is dependent upon S408/9 in the ?3 subunit. Aim 3. To test the hypothesis that NASs mediate their metabotropic effects on GABAARs via the activation of mPRs. Collectively, our proposal will identify the molecular mechanisms by which NAS exert their therapeutic actions. This information may aid the development of new therapeutic strategies to alleviate the burdens of anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome.

The electroneutral K+/Cl- co-transporter 2 (KCC2) allows neurons to maintain low intracellular Clconcentrations, an essential prerequisite for fast synaptic inhibition mediated by type A ?-aminobutyric acid (GABAAR). Consistent with this, deficits in KCC2 activity lead to seizures and are believed to be central to the pathology of Status Epilepticus (SE). SE is the most devastating form of epilepsy, and accounts for 42,000 deaths per year in the US, and hundreds of thousand more cases of severe brain damage. SE becomes less tractable with time, leading to the development of drug resistant seizures, resulting in increased mortality and morbidity. SE is associated with a cost of $4.8 billion per year in the US. Consistent with its essential role in regulating neuronal Cl- homeostasis, deficits in KCC2 activity are seen in patients with intractable epilepsy, and in animal models of SE. Therefore, understanding the mechanisms by which SE leads to inactivation of KCC2 is of clear clinical significance. KCC2 function is subject to both positive and negative modulation via phosphorylation of key regulatory residues within the C-terminal intracellular domain of this protein. Specifically, phosphorylation of serine 940 (S940) by protein kinase C enhances KCC2 activity, while phosphorylation of the adjacent threonine residues 906 and 1007 by with-no-lysine kinases (WNKs) decreases transporter activity (Lee et al., 2007; 2011; Riehart, 2009). Thus, one mechanism that may contribute to KCC2 inactivation during SE is modifications in the phosphorylation of these key regulatory residues. To address this issue, we have utilized phospho-specific antibodies against S940 and T906. In addition, we have created mice in which the phosphorylation of these key regulatory residues has been prevented via mutation to alanines. Finally, we have made use of mice deficient in WNK3, the principle WNK isoform expressed in the adult brain. Preliminary studies using these novel reagents have allowed us to formulate an overarching hypothesis that will be tested here; ?Persistent elevations in neuronal activity during SE lead to dephosphorylation of S940, but enhanced phosphorylation of T906/1007, events that lead to rapid inhibition of KCC2, reductions in the efficacy of GABAergic inhibition that directly contribute to the pathophysiology of SE?. Our studies will focus on the following specific aims. Specific Aim 1. To test the hypothesis that deficits in KCC2 phosphorylation contribute to the development and lethality of SE Specific Aim 2. To test the hypothesis that S940A mice exhibit enhanced T906 phosphorylation and a selective deficit in KCC2 activity during SE Specific Aim 3. To test the hypothesis that reducing WNK dependent phosphorylation of KCC2 prevents the development of SE. Collectively these experiments will provide key mechanistic insights into the pathophysiology of SE, and may aid the development of novel therapeutics to limit the impact of this devastating disorder.

? DESCRIPTION (provided by applicant): Autism spectrum disorders (ASD) have common core symptoms which include increases in anxiety, and repetitive behaviors, with reduced sociability. It is widely believed that ASDs arise from subtle differences in the equilibrium between excitatory and inhibitory GABAergic neurotransmission. The electroneutral K+/Cl- co- transporter 2 (KCC2, or SLC12A5) is selectively expressed in the CNS after birth and allows neurons to maintain low intracellular Cl- concentrations an essential pre-requisite for the postnatal development of inhibitory neurotransmission. Consistent with its role in facilitating neuronal inhibition, deficits in KCC2 expression are evident in patients with ASDs and multiple autism animal models. KCC2 activity is subject to both positive and negative modulation via phosphorylation of serine's 940 and threonine residues 906 and 1007 respectively. Here, we will directly test if these regulatory processes influence the postnatal development of GABAergic inhibition, and the pathophysiology of ASDs. To do so we have created mice in which positive modulation of KCC2 activity by phosphorylation has been ablated via mutation of S940 to an alanine (S940A). T906/1007 are phosphorylated by with with-no-lysine kinases (WNKs), and we have also obtained line mouse lines with deficits in WNK activity. Preliminary studies using these new reagents have allowed us to formulate a novel hypothesis that will be tested here: The postnatal activation of KCC2 is facilitated by the reciprocal phosphorylation of S940 and dephosphorylation of T906/1007. Compromising these processes decreases the efficacy of GABAergic and directly contributes to the pathophysiology of ASDs. The experiments we will perform to test our hypothesis are detailed in the following aims. Specific Aim 1. To test the hypothesis that phosphorylation of S940 in KCC2 is a critical determinant for the postnatal development of GABAergic inhibition. Specific Aim 2. To test the hypothesis that WNK dependent phosphorylation of KCC2 slows the postnatal development of hyperpolarizing GABAergic inhibition. Specific Aim 3. To test the hypothesis that slowing the postnatal development of GABAergic inhibition reproduces the core behavioral deficits of ASDs. Collectively, these experiments will provide key mechanistic insights into how deficits in KCC2 phospho-dependent modulation contribute to the pathophysiology of ASD. This information may lead to the development of more effective therapies targeting KCC2 activity to ultimately improve patient outcomes for ASDs. Such strategies may also be relevant for other neuropsychiatric disorders, such as epilepsy, neuropathic pain, and schizophrenia in which deficits of KCC2 activity are believed to be of significance.

GABAA receptors are Cl? permeable ion channels that mediate hyperpolarizing fast synaptic inhibition in the adult brain, while in immature developing neurons GABAA receptors depolarize and excite neurons. This shift in the signaling of GABAA receptors is due to the postnatal increase in the activity of KCC2, the neuron-specific K+/Cl- co-transporter. KCC2 is the major protein mechanism that allows neurons to pump Cl? out of the cell. In rodents KCC2 expression is evident at birth and increases substantially during the critical periods of early brain development between postnatal days 7 and 14. Deficits in KCC2 activity in humans lead to epilepsy, and are strongly implicated in chronic pain and developmental disorders such as Fragile X and Rett syndromes. Therefore, understanding the mechanisms by which neurons determine the proper postnatal increase of KCC2 activity and its maintenance in adults is clinically significant. KCC2 function is dynamically controlled by signals within neurons that can rapidly and reversibly modify its structure. Modification of KCC2's structure in one region increases its activity, while modification of KCC2's structure in another region decreases its activity. Our overarching hypothesis is that the correct balance between these opposing modifications contributes to the proper early postnatal development and adult maintenance of synaptic inhibition in the brain. To address this issue we have created two new genetic tools that can prevent the modification of KCC2's structure. Importantly, one of the genetic tools is the first of its kind to allow scientists to increase the function of KCC2, and so our proposal constitutes the first test of the theory which states that increasing KCC2 function can be utilized as a therapy. To date, no medications exist that can directly and rapidly increase the function of KCC2. The aims of our proposal are threefold: 1) demonstrate that preventing the modification of KCC2's structure is critical during early brain development; 2) examine the mechanisms by which disease causing factors influence the structure of KCC2 both in immature and more mature neurons; and 3) demonstrate that increasing the function of KCC2 can reduce the likelihood and severity of epileptic seizures. Our study will provide new insights on how KCC2 structure and function is controlled under normal conditions and during disease states. This information may aid in the development of new and improved treatments to alleviate the burdens of a range of neurological disorders.

Abstract. Neural circuit function requires synaptic inhibition mediated by ?-aminobutyric acid (GABA). Glutamine is the major metabolic precursor for neuronal GABA synthesis and is supplied to neurons via the activity of the astrocyte-specific enzyme glutamine synthetase (GS). Inhibition or brain-specific genetic ablation of GS leads to impaired GABAergic inhibition, intractable epilepsy, and death. Consistent with this, deficits in GS expression lead to epilepsy and are implicated in neurodegeneration. However, to date, there have been no systematic studies to evaluate how GS activity is regulated to meet the demand of neurons for glutamine. Here we will address the role that cAMP-dependent phosphorylation plays in regulating GS activity, and if this process contributes to the deficits in GABAergic inhibition that result in epilepsy. To do so, we will identify sites of phosphorylation within GS, under control conditions and during Status Epilepticus (SE), the most severe form of epilepsy and a medical emergency. The effects that phosphorylation have on GS activity will then be determined using high-resolution enzyme kinetics. The cellular mechanisms that regulate GS phosphorylation will be explored using Designer Receptors Exclusively Activated by Designer Drugs (DREADD) to selectively modulate PKA signaling in astrocytes. Finally, we will assess the significance of GS phosphorylation for the efficacy of GABAergic inhibition using viral expression to replace endogenous GS molecules with mutants in which phosphorylation of critical regulatory residues has been prevented. Preliminary studies have allowed us to formulate a central hypothesis that will be tested here: GS activity is negatively regulated by PKA- dependent phosphorylation, and this process contributes to the deficits in GABAergic inhibition that are fundamental to the pathophysiology of epilepsy. Our proposal will center on the following specific aims: Aim 1. To test the hypothesis that PKA-mediated phosphorylation of GS leads to decreased enzyme activity. Aim 2. To test the hypothesis that phospho-dependent inactivation of GS is enhanced by seizure activity. Aim 3. To test the hypothesis that GS phosphorylation contributes to the deficits in GABAergic inhibition during SE. Collectively, these studies will provide the first evidence that GS is subject to phospho-dependent modulation and that this process contributes to the deficits in GABAergic inhibition seen in SE. Such insights may lead to improved therapies to reduce the impact of epilepsy. !

Abstract Neural circuit function requires synaptic inhibition mediated by ?-aminobutyric acid (GABA). Glutamine is the major metabolic precursor for neuronal GABA synthesis and is supplied to neurons via the activity of the astrocyte-specific enzyme glutamine synthetase (GS). Inhibition or brain-specific genetic ablation of GS leads to impaired GABAergic inhibition, intractable epilepsy, and death. Consistent with this, deficits in GS expression are found in the brains of patients and animal models of epilepsy. Deficits in GS levels have also been reported in the brains of patients with Alzheimer's disease and schizophrenia. Despite the critical role GS plays in neuronal physiology there have been no systematic studies to evaluate how GS activity is regulated in the mammalian brain to meet the demand of neurons for glutamine. GS structure is highly conserved through evolution, but all mammalian isoforms contain unique amino acids that are potential substrates for phosphorylation which are adjacent to, or within the catalytic site of this enzyme. These residues include threonine residue 301 (T301) and serine residue 343 (S343), which are consensus sites for a phosphorylation by a variety of classical 2nd messenger dependent protein kinases including cAMP-dependent (PKA) kinase. However, it remains to be determined if GS is a phospho-protein in the brain and if this covalent modification impacts on enzyme activity and/or stability. To address this issue, we will use stable isotope labeling of amino acids in culture (SILAC) coupled with liquid chromatography-tandem mass spectrometry (LC- MS/MS) to 1) identify sites of phosphorylation, and 2) quantify their stoichiometry of phosphorylation under basal conditions and after the activation of PKA in situ, in vitro and in vivo. To complement our LC-MS/MS studies we will use recently developed phospho-specific antibodies to further analyze GS phosphorylation. In parallel, we will assess the effects phosphorylation have on GS activity and stability. Finally, we will determine if seizure activity induced by chemico-convulsants modifies GS phosphorylation and activity in the mouse brain. Preliminary studies have allowed us to formulate a central hypothesis that will be tested in this proposal; GS activity is inhibited by PKA-dependent phosphorylation of T301/S343, and enhanced phosphorylation of these residues contributes to the deficits in GS activity seen during epilepsy. Our proposal will center on the following specific aims: Aim 1. To test the hypothesis that PKA mediated phosphorylation of GS leads to decreased enzyme activity. Aim 2. To test the hypothesis that ?phospho-dependent? inactivation of GS is enhanced by seizure like activity. Collectively these studies will provide the first evidence of the mechanism by which astrocytes regulate GS activity. Therefore, they may lead to the development of novel therapeutic strategies to increase GS activity to alleviate the burdens of epilepsy and neurodegenerative disorders.

Type A ?-aminobutyric receptors (GABAAR) are Cl--preferring ligand gated ion channels that mediate fast phasic synaptic inhibition in the adult brain and are are drug targets for barbiturates, benzodiazepines, intravenous anesthetics, and neurosteroids. Consistent with their essential role in regulating neuronal excitability, modification in GABAAR activity contribute to anxiety, autism, depression, epilepsy, substance abuse and schizophrenia. The receptors are heteropentamers that can be assembled from ?(1-6), ?(1-3), ?(1-3), ?, ?, ? and ? subunits. Surprisingly, to date our comprehension of the molecular structure of native GABAARs subtypes, and how these proteins are targeted to inhibitory synapses is rudimentary. However, these issues are of fundamental importance given the critical role GABAARs play in determining neuronal excitability, in neuropsychiatric diseases and as drug targets. To address these issues, we will determine the structure of the principal GABAAR subtypes assembled from ?1 and ?2 subunits in the brain. We will then assess which proteins co-purify with these receptor subtypes. The roles that the most abundant ?receptor-associated proteins? play in the synaptic targeting of distinct GABAAR subtypes will then be examined. Our studies will be facilitated by the novel mouse lines we have developed in which the N-terminus of the ?1 and ?2 subunits have been modified with distinct fluorescent reporters; pHlourin/9E10-?2 (pH?2) and mKate/FLAG-?1 (mK?1). These additions are functionally silent but allow sequential affinity purification of individual GABAAR subtypes, and analysis of their structure using blue-native polyacrylamide gel electrophoresis (BN-PAGE) followed by liquid chromatography coupled mass spectroscopy (LC-MS/MS). Preliminary experiments using these new tools have allowed us to formulate a central hypothesis that will be tested here; ?2- and ?1-subunits are assembled into two distinct GABAAR subtypes; ??2/?1? containing equimolar amounts of both ? subunits (?2?1??2), and ??1/?1? which does not contain an ?2 subunit (?1??2). These subtypes are targeted to distinct subsets of synapses by spectrins, which are intimately associated with these GABAAR subtypes. Our experiments will focus on the following specific aims: Aim 1. To test the hypothesis that neurons assemble GABAAR subtypes containing ?2?1??2 and ?1??2 subunits. Aim 2. To test the hypothesis that the ?2/?1 and ?1/?1 GABAAR subtypes are associated with distinct spectrins. Aim 3. To test the hypothesis that spectrins facilitate the synapse-specific targeting of GABAARs. Collectively, our study will provide novel insights into the structure of native GABAARs and the processes that regulate their accumulation at inhibitory synapses, information that may lead to improved understanding of, and treatments for epilepsy and other neuropsychiatric disorders.