Mechanisms that determine Glutamine synthetase activity in the brain

抽象的。神经回路功能需要γ-氨基丁酸（GABA）介导的突触抑制。 谷氨酰胺是神经元GABA合成的主要代谢前体，并通过神经元供应给神经元。 星形胶质细胞特异性酶谷氨酰胺合成酶（GS）的活性。抑制或脑特异性基因消融 导致GABA能抑制受损、难治性癫痫和死亡。与此相一致， GS表达导致癫痫并与神经变性有关。然而，迄今为止， 目前还没有系统的研究来评估GS活性如何调节以满足神经元对谷氨酰胺的需求。 在这里，我们将讨论cAMP依赖性磷酸化在调节GS活性中的作用，如果这一点是正确的， 这一过程导致GABA能抑制的缺陷，从而导致癫痫。为此，我们将确定 在对照条件下和癫痫持续状态（SE）期间， 癫痫和紧急医疗情况。磷酸化对GS活性的影响将是 使用高分辨率酶动力学测定。调节GS磷酸化的细胞机制 将使用设计师药物独家激活的设计师受体（DREADD）进行探索， 调节星形胶质细胞中PKA信号传导。最后，我们将评估GS磷酸化对疗效的意义。 使用病毒表达的GABA能抑制，用突变体取代内源性GS分子， 关键调节残基的磷酸化被阻止。初步研究使我们能够 提出了一个中心假设，将在这里测试：GS活性是负调控PKA- 依赖性磷酸化，这一过程导致GABA能抑制的缺陷， 是癫痫病理生理学的基础我们的建议将围绕以下具体目标： 目标1.为了验证PKA介导的GS磷酸化导致酶活性降低的假设， 活动 目标2.验证癫痫发作增强GS磷酸依赖性失活的假设 活动 目标3.为了验证GS磷酸化导致GABA能缺乏的假设， 在SE期间抑制。 总的来说，这些研究将提供第一个证据表明，GS是受磷酸依赖性调制 并且该过程导致SE中所见的GABA能抑制的缺陷。这种见解可能会导致 改善治疗方法以减少癫痫的影响。 !

Abstract. Neural circuit function requires synaptic inhibition mediated by γ-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. !