Function of Astrocytic GPCR Signaling Cascades in Physiology and Mental Illness

描述（由申请人提供）：本拨款提案的目标是开发遗传工具，可用于激活或灭活g蛋白偶联受体（GPCR）信号级联，在严格鉴定的星形胶质细胞群体中，使用可被研究界易于使用的方法；我们相信这些工具的可用性将在理解星形胶质细胞在生理、行为和精神疾病中的作用方面具有变革性。虽然星形胶质细胞占中枢神经系统的很大一部分，并表达多种在神经元活动期间被激活的gpcr，但对星形胶质细胞gpcr的作用知之甚少。这方面信息的缺乏源于我们在测量生理和行为参数时无法选择性地激活或灭活星形细胞信号级联。重要的是，临床上用于治疗精神疾病的大多数治疗药物影响GPCR信号级联反应。最近，遗传工具已经开发出来，可用于选择性地激活或灭活特定细胞类型中的GPCR信号级联。一种这样的药物遗传学方法利用受体家族统称为DREADD受体（由设计药物独家激活的设计受体）来激活特定细胞类型中的GPCR信号级联。DREADD受体是通过M3-ACh受体的定向分子进化制备的，不响应任何已知的内源性GPCR配体，由氯氮平- n -氧化物（CNO）激活。重要的是，CNO穿过血脑屏障，首次通过腹腔注射CNO选择性激活特定星形胶质细胞群中的GPRC信号。我们计划使用交叉基因激活，这需要两个细胞类型特异性启动子来激活基因表达，从而大大提高基因表达的细胞特异性。交叉基因激活将用于在严格定义的星形胶质细胞群体中表达Gq-、Gs-和Gi-DREADD受体。任何药理学方法都需要关注的是所产生的表型（如LTP、行为等）是否反映了所研究系统的过度刺激。我们计划开发遗传工具，可用于在严格定义的星形胶质细胞群体中灭活GPCR信号级联；这些遗传工具也将利用交叉基因激活。在严格定义的星形胶质细胞群体中激活或灭活GPCR信号级联的结合将为理清星形胶质细胞在生理、行为和精神疾病中的作用提供有力的方法；将这些小鼠模型分发给研究界可能会带来变革。本提案中开发的遗传工具将用于测试星形细胞GPCR信号级联的激活调节突触传递的假设；这一假设是我们理解星形胶质细胞在脑功能中的作用的基础，目前在研究界极具争议。

DESCRIPTION (provided by applicant): The goal of this grant proposal is to develop genetic tools that can be used to activate or inactivate G-protein coupled receptor (GPCR) signaling cascades in rigorously identified populations of astrocytes using approaches that can be readily used by the research community; we believe the availability of such tools will be transformational with respect to understanding the role of astrocytes in physiology, behavior, and mental illness. While astrocytes make up a large portion of the CNS and express a wide variety of GPCRs that are activated during neuronal activity, the role of astrocytic GPCRs is poorly understood. The lack of information in this area stems from our inability to selectively activate or inactivate astrocytic signaling cascades while measuring physiological and behavioral parameters. Importantly, the majority of therapeutic agents used clinically to treat mental illness effect GPCR signaling cascades. Recently, genetic tools have been developed that can be used to selectively activate or inactivate GPCR signaling cascades in specific cell types. One such pharmacogenetic approach utilizes a receptor family referred to collectively as DREADD receptors (for Designer Receptor Exclusively Activated by Designer Drug) to activate GPCR signaling cascades in specific cell types. DREADD receptors were prepared by directed molecular evolution of M3-ACh receptor DREADD receptors, fail to respond to any known endogenous GPCR ligand, and are activated by clozepine-N-oxide (CNO). Importantly, CNO crosses the blood-brain-barrier enabling, for the first time, the selective activation of GPRC signaling in specific populations of astrocytes via an intraperitoneal (ip) injection of CNO. We plan to use intersectional gene activation which requires two cell- type specific promoters to activate gene expression and greatly increases the cell specificity of gene expression. Intersectional gene activation will be used to express Gq-, Gs-, and Gi-DREADD receptors in rigorously defined populations of astrocytes. A concern with any pharmacological approach is whether the resulting phenotype (e.g., LTP, behavior, etc.) reflects the over stimulation of the system under study. We plan to develop genetic tools that can be used to inactivate GPCR signaling cascades in rigorously defined populations of astrocytes; these genetic tools will also take advantage of intersectional gene activation. The combination of being able to activate or inactivate GPCR signaling cascades in rigorously defined populations of astrocytes will provide a powerful approach to sorting out the role of astrocytes in physiology, behavior, and mental illness; distribution of these mouse models to the research community is likely to be transformational. The genetic tools developed in this proposal will be used to test the hypothesis that the activation of astrocytic GPCR signaling cascades modulates synaptic transmission; this hypothesis is fundamental to our understanding of the role of astrocytes in brain function and is currently extremely controversial within the research community.