Function of Astrocytic GPCR Signaling Cascades in Physiology and Mental Illness

描述(由申请者提供)：这项资助提案的目标是开发基因工具，利用研究界容易使用的方法，在严格识别的星形胶质细胞群体中，用来激活或灭活G蛋白偶联受体(GPCR)信号级联信号；我们相信，这种工具的可用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产生革命性的作用。虽然星形胶质细胞构成了中枢神经系统的很大一部分，并表达在神经元活动期间被激活的各种GPCRs，但对星形胶质细胞GPCRs的作用知之甚少。这方面信息的缺乏源于我们无法在测量生理和行为参数时选择性地激活或停用星形细胞信号级联。重要的是，大多数临床上用于治疗精神疾病的治疗剂都会影响GPCR信号转导通路。最近，基因工具已经被开发出来，可以用来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被统称为DREADD受体的受体家族(针对由Designer Drug独家激活的Designer Receptor)来激活特定细胞类型中的GPCR信号级联。DREADD受体是由M3-ACh受体DREADD受体定向分子进化而来的，不能与任何已知的内源性GPCR配体反应，可被氯西平-N-氧化物(CNO)激活。重要的是，CNO跨越血脑屏障，首次能够通过腹腔注射CNO选择性地激活特定星形胶质细胞群体中的GPRC信号。我们计划使用交叉性基因激活，这需要两个细胞型的特异性启动子来激活基因表达，大大提高了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考虑的一个问题是，由此产生的表型(例如，LTP、行为等)反映了正在研究的系统的过度刺激。我们计划开发基因工具，可以用来在严格定义的星形胶质细胞群体中灭活GPCR信号级联；这些基因工具也将利用交叉基因激活。能够激活或灭活严格定义的星形胶质细胞群体中的GPCR信号级联，将为区分星形胶质细胞在生理、行为和精神疾病中的作用提供一种强有力的方法；向研究界分发这些小鼠模型可能是变革性的。在这项建议中开发的遗传工具将被用来检验星形胶质细胞GPCR信号级联激活调节突触传递的假说；这一假说是我们理解星形胶质细胞在脑功能中作用的基础，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.