GABAergic regulation of glutamine synthetase and its role in preventing epilepsy

描述（由申请人提供）：成人大脑中的快速神经元抑制关键取决于神经元合成抑制性神经递质γ-氨基丁酸（GABA）的能力，GABA通过离子型GABAA和代谢型GABAB受体介导其作用。 GABA 能抑制的缺陷是癫痫和许多其他神经精神疾病的核心。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的能力取决于星形胶质细胞特异性谷氨酰胺合成酶（GS）的活性。通过使用特定抑制剂和基因删除揭示了 GS 对大脑功能的重要性。这些操作会因突触抑制减弱而导致癫痫发作和死亡。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 表达缺陷。然而，迄今为止，还没有系统的实验来评估如何调节 GS 的活性以满足神经元对谷氨酰胺的需求，以及这些过程的缺陷是否会导致癫痫发生。这些问题将在这里得到解决。初步结果表明，GS 表达受到星形胶质细胞 GABABR 的强力调节。 2 GABABR 是异二聚体 G 蛋白偶联受体，可与 Gi/o 偶联，调节 Ca 瞬变，并抑制腺苷酸环化酶的活性。初步研究表明，星形胶质细胞 GABABR 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，我们创造了一种星形细胞 GABABR 表达可以被特异性消除的小鼠。这些小鼠的 GS 稳态表达水平降低，出现自发性癫痫发作和过早死亡。基于这些观察，我们假设： 星形胶质细胞 GABABR 可以防止 GS 泛素依赖性降解，从而确保谷氨酰胺持续用于神经元 GABA 合成。该提案将围绕以下三个目标展开： 目标 1. 检验星形胶质细胞 GABABR 调节 GS 稳定性的假设。目标 2. 检验以下假设：消除星形细胞 GABABR 的表达会导致 GS 降解、自发性癫痫发作和死亡。目标 3. 检验减少星形胶质细胞 GABABR 表达会损害突触抑制和神经元活力的假设。这些实验将为星形细胞 GABABR 在调节 GS 表达、快速突触抑制和癫痫发生中的作用提供独特的见解。总的来说，这些研究可能会导致新疗法的开发，以增加 GS 的活性，从而减轻癫痫的负担

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