In Vivo Neuronal Modulation via Engineered G Protein-Coupled Receptors

描述（由申请人提供）：已知G蛋白偶联受体（GPCR）信号通路是多种人类疾病的贡献者。在中枢神经系统内，诸如抑郁症、焦虑症、精神分裂症和帕金森病的疾病只是GPCR信号传导发挥作用的许多病理中的几种。重要的是，GPCR代表了这些和其他疾病的最大一类药物靶点，开发新的治疗方法需要了解这些受体在特定神经元群体内信号转导中的作用。研究这些作用的传统药理学方法受到脱靶药物作用和组成性受体活性的限制。为了克服这一障碍，Roth实验室设计了一个GPCR家族，来自人类毒蕈碱受体，其失去了对其天然配体乙酰胆碱的亲和力，缺乏任何可检测的内在活性，并被生物可利用的惰性合成配体氯氮平-N-氧化物（CNO）选择性激活。我们将这些受体称为设计师受体，仅由设计师药物激活，或DREADD。这项研究计划调查的假设，DREADD的使用将允许前所未有的时空控制神经元GPCR信号，并提供了一种新的系统来探测GPCR信号的神经元病理的贡献。具体目的是（1）概念验证研究，以表征Gq偶联DREADD在转基因小鼠前脑中选择性表达时的表达和激活效应;以及（2）确定通过DREADD选择性激活和沉默多巴胺能神经元的体内生化和信号传导效应。为了实现这些目标，将使用各种技术，包括免疫组织化学、体内微透析、测量磷酸肌醇积累、免疫印迹和立体定位病毒输注。后续研究将采用精神疾病模型进行小鼠行为测试。这个新的系统有可能彻底改变理解和理解的方法。在体内操纵神经元GPCR信号传导。此外，这项提案将调查的特定信号通路可能揭示有关精神疾病病因和治疗的关键信息。

DESCRIPTION (provided by applicant): G protein-coupled receptor (GPCR) signaling pathways are known contributors to a variety of human diseases. Within the central nervous system, diseases such as depression, anxiety, schizophrenia, and Parkinson's disease are but a few of the many pathologies in which GPCR signaling plays a role. Importantly, GPCRs represent the largest class of drug targets for these and other disorders, and developing new therapeutic approaches requires an understanding of the role these receptors play in signal transduction within specific neuronal populations. Traditional pharmacological approaches to investigating these roles are limited by off-target drug actions and constitutive receptor activity. To overcome this barrier, the Roth lab has engineered a family of GPCRs, derived from the human muscarinic receptors, which have lost affinity for their native ligand, acetylcholine, lack any detectable intrinsic activity, and are selectively activated by the bioavailable inert synthetic ligand, clozapine-N-oxide (CNO). We refer to these receptors as Designer Receptors Exclusively Activated by Designer Drug, or DREADDs. This research proposal investigates the hypothesis that the use of DREADDs will allow for unprecedented spatiotemporal control of neuronal GPCR signaling and provide a novel system to probe the contributions of GPCR signaling to neuronal pathologies. The specific aims are (1) a proof-of-concept study to characterize the effects of expression and activation of a Gq-coupled DREADD when selectively expressed in the forebrain of transgenic mice; and (2) to determine the in vivo biochemical and signaling effects of selectively activating and silencing serotonergic neurons via DREADDs. To achieve these aims, a variety of techniques will be used, including immunohistochemistry, in vivo microdialysis, measurement of inositol phosphate accumulation, immunoblotting, and stereotaxic viral infusion. Follow-up studies will incorporate mouse behavioral testing using models of psychiatric disease. This novel system has the potential to revolutionize the approach to understanding and. manipulating neuronal GPCR signaling in vivo. Additionally, the particular signaling pathways this proposal will investigate could reveal critical information regarding the etiology and treatment of mental illness.