PDZ scaffold regulation of astrocytic glutamate receptors and transporters

描述(申请人提供)：谷氨酸是哺乳动物中枢神经系统中的主要兴奋性神经递质。神经元之间的谷氨酸能信号是由邻近的星形胶质细胞关键调控的，星形胶质细胞包围着大多数突触连接，并表达特定的谷氨酸转运体，通过从突触间隙中移除谷氨酸来控制谷氨酸浓度。星形胶质细胞也表达谷氨酸受体，特别是代谢型谷氨酸受体亚型mGluR3和mGluR5，它们被谷氨酸激活，已知调节谷氨酸转运体的活性。虽然人们对神经元中谷氨酸受体的调节和分类知之甚少，但对星形胶质细胞中谷氨酸受体和转运体的特殊调节潜力知之甚少。MGluR亚型3和5以及星形胶质细胞谷氨酸转运体EAAT1都具有大的胞内羧基末端(CT)，在其活性控制中发挥关键作用。由于这些CTs终止于共识基序，与一类保守的蛋白质相互作用结构域(称为PDZ结构域)潜在地结合，我们筛选了一个定制的PDZ结构域蛋白质组阵列，发现mGluR3、mGluR5和EAAT1的CTs都表现出与多功能支架蛋白NHERF-2的PDZ结构域强烈而特异的相互作用。这些相互作用在细胞环境中得到证实，免疫组织化学研究显示NHERF-2在大脑中的星形胶质细胞中大量表达。我们推测NHERF-2是mGluRs和EAAT1在星形胶质细胞中活性和定位的中心调节因子，并可能促进mGluRs和谷氨酸转运体之间的相互调节。我们将通过检测NHERF-2对mGluR3、mGluR5和EAAT1功能活性和串扰的调节，以及通过对野生型和NHERF-2基因敲除小鼠的脑组织进行免疫组织化学分析，探索NHERF-2可能控制体内mGluR和EAAT1定位的可能性，来检验这一想法。这些研究具有重要的临床意义，因为代谢性谷氨酸受体和谷氨酸转运体被认为是治疗中风、阿尔茨海默病、帕金森病、肌萎缩侧索硬化症、精神分裂症和其他大脑疾病的优秀潜在治疗靶点。与公共健康相关的星形胶质细胞和神经元在整个大脑中混合在一起，星形胶质细胞拥有可以感知神经元活动的受体以及可以调节突触神经递质水平的转运体。星形细胞受体和转运蛋白通常聚集在突触附近，并被认为是高度调控的，但这种靶向和调控的机制在很大程度上是未知的。本申请中描述的工作将探索星形细胞受体和转运体，特别是那些对神经递质谷氨酸做出反应的受体和转运体被靶向和调控的机制。这些研究具有重要的临床意义，因为星形细胞谷氨酸受体和转运体被认为是治疗中风、阿尔茨海默病、帕金森病、肌萎缩侧索硬化症、精神分裂症和其他大脑疾病的优秀潜在治疗靶点。

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.