GABAergic regulation of glutamine synthetase and its role in preventing epilepsy

描述(申请人提供)：成人大脑中的快速神经元抑制严重依赖于神经元合成抑制性神经递质--氨基丁酸(GABA)的能力，GABA通过亲离子GABAA和亲代谢性GABAB受体介导其作用。GABA能抑制缺陷是癫痫和其他过多神经精神障碍的核心。神经元合成GABA的主要代谢前体是谷氨酰胺，而谷氨酰胺又由星形胶质细胞提供。星形胶质细胞输出谷氨酰胺I的能力取决于星形胶质细胞特异性酶谷氨酰胺合成酶(GS)的活性。GS对脑功能的意义已经通过使用特定的抑制剂和基因缺失来揭示。这些操作导致癫痫发作和死亡，这是由于突触抑制减少所致。与此一致的是，在癫痫患者和癫痫动物模型的大脑中发现了GS表达的缺陷。然而，到目前为止，还没有系统的实验来评估GS的活性如何被调节来满足神经元对谷氨酰胺的需求，以及这些过程中的缺陷是否有助于癫痫的发生。这些问题将在这里得到解决。初步结果表明，GS的表达受星形细胞GABRs的强大调控。2+GABRs是一种异二聚体G蛋白偶联受体，与Gi/o偶联，调节钙瞬变，抑制腺苷环化酶的活性。初步研究表明，星形细胞GABRs通过减少GS的泛素化和随后的降解来稳定GS。为了理解这一发现的意义，我们创造了一只小鼠，在其中星形细胞GABA受体的表达可以被特异性地消融。这些小鼠降低了GS的稳定表达水平，自发性癫痫发作和过早死亡。基于这些观察，我们假设：星形细胞的GABRs阻止了依赖泛素的GS的降解，从而确保了谷氨酰胺持续用于神经元GABA的合成。这项建议将围绕以下三个目标展开：目标1.验证星形细胞GABRs调节GS稳定性的假说。目的2.验证阻断星形细胞GABRs表达导致GS降解、自发性癫痫发作和死亡的假说。目的3.验证减少星形细胞GABABR表达会影响突触抑制和神经元活性的假说。总之，这些实验将为星形细胞GABRs在调节GS表达、快速突触抑制和癫痫发生中所起的作用提供独特的见解。总的来说，这些研究可能导致开发新的治疗方法，以增加GS的活性，以减轻癫痫的负担

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