Defining the role of the Peripheral Benzodiazepine Receptor/translocator protein (TSPO) in inflammatory and stress responses in microglial cellsby comparative analysis

转运蛋白TSPO的配体是敏感和特异的生物标志物， 神经损伤的显像剂。但尽管经过30多年的研究， 这种古老的保守蛋白质在所有生命王国中都普遍表达， 不清楚在许多不同的生物体中的共同主题是TSPO的上调，以响应 应力在这种情况下，我们提出了一个新的作用，TSPO作为促进神经保护 通过激活M2小胶质细胞，其在神经炎性疾病中起主要作用， 老年痴呆症和帕金森症我们的目标是表征激活和应激反应 的人和小鼠的小胶质细胞，并探讨TSPO及其配体在这一过程中的作用。 反应我们将使用RNAseq来确定共同的表达签名和CRISPER Cas9以测试TSPO缺失和突变对该标签（Aim1）的影响。一个重要 指导和授权这项工作的工具是高分辨率晶体结构的可用性 来自球形红细菌的人TSPO的细菌同系物，其结构 在自然环境和突变体中都能溶解。这种蛋白质很保守 并且可用于诱变、纯化、结晶和生化分析。此外，A 基于红细菌的TSPO依赖性抗性， 金属毒性，这将使我们能够筛选突变形式以及各种配体，以指导我们的研究。 干预小胶质细胞系统（目标2）。虽然首先在线粒体中被认为是一种 外膜蛋白结合苯二氮卓类药物，越来越多的证据表明TSPO 参与许多复杂的细胞过程，包括胆固醇转运， 卟啉转运、肿瘤进展、神经炎症和阿尔茨海默病。的 该建议的优势是对几个系统进行结构导向的比较分析， 其TSPO是与炎症和应激相关的高度诱导的。这一共同主题 不同生物体之间的相互作用有可能揭示机制的独特见解。一 TSPO结构和功能之间的明确机制关系将为以下方面提供基础： 开发用于检测和治疗神经疾病的新型结构基配体。

Ligands of the translocator protein, TSPO, are sensitive and specific biomarkers and powerful imaging agents of neurological damage. But in spite of more than 30 years of study, the function of this ancient conserved protein that is ubiquitously expressed in all kingdoms of life, remains unclear. A common theme in many different organisms is upregulation of TSPO in response to stress. In this context, we are proposing a new role for TSPO as a promoter of neuroprotection via activation of M2 microglia cells, which play a major role in neuroinflammatory diseases such as Alzheimer’s and Parkinson’s. Our goal is to characterize the activation and stress responses of microglia cells from human and mouse and to explore the role of TSPO and its ligands in this response. We will use RNAseq to determine a common expression signature and CRISPER Cas9 to test the effects of TSPO deletion and mutation on that signature (Aim1). An important tool for guiding and empowering this effort is the availability of high resolution crystal structures of the bacterial homolog of human TSPO from Rhodobacter sphaeroides, whose structure we have solved in a lipidic environment in native and mutant forms. This protein is well conserved and accessible for mutagenesis, purification, crystallization and biochemical analysis. Further, a functional assay has been developed based on Rhodobacter’s TSPO-dependent resistance to metal toxicity, which will allow us to screen mutant forms as well as various ligands to guide our intervention in the microglial system (Aim 2). Although first recognized in mitochondria as an outer membrane protein that bound benzodiazepine drugs, mounting evidence suggests TSPO involvement in a number of complex cellular processes, including cholesterol transport, porphyrin transport, tumor progression, neuroinflammation and Alzheimer’s disease. The strength of the proposal is the structure-guided comparative analysis of several systems in which TSPO is highly induced in association with inflammation and stress. This common theme among different organisms holds the potential for revealing unique insights into mechanism. A clear mechanistic relationship between TSPO structure and function will provide the basis for developing novel structure-based ligands for detection and treatment of neurological disease.