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

转位蛋白TSPO的配体是敏感和特异的生物标志物，功能强大 神经损伤的显像剂。但尽管经过了30多年的研究，该功能 这种在所有生命王国中普遍表达的古老保守蛋白质的遗迹 不清楚。许多不同生物体的一个共同主题是TSPO的上调 压力。在此背景下，我们建议TSPO作为神经保护的促进者发挥新的作用 通过激活M2小胶质细胞，这些细胞在神经炎症性疾病中发挥主要作用 如阿尔茨海默氏症和帕金森氏症。我们的目标是表征激活和应激反应 TSPO及其配体在人和小鼠小胶质细胞中的作用 回应。我们将使用RNAseq来确定常见的表达式签名和CRISPER Cas9以测试TSPO缺失和突变对该签名的影响(Aim1)。一个重要的 指导和支持这一努力的工具是高分辨率晶体结构的可用性 来自球形红杆菌的人TSPO的细菌同源物，其结构是 在脂类环境中以天然和突变的形式溶解。这种蛋白质保存得很好 并可用于诱变、纯化、结晶和生化分析。此外，一个 基于红杆菌对TSPO依赖的抗药性已开发出功能分析方法 金属毒性，这将使我们能够筛选突变形式以及各种配体来指导我们的 干预小胶质细胞系统(目标2)。尽管最初在线粒体中被认为是一种 结合苯二氮类药物的外膜蛋白，越来越多的证据表明TSPO 参与了许多复杂的细胞过程，包括胆固醇的运输， 卟啉转运、肿瘤进展、神经炎症和阿尔茨海默病。这个 该提案的优点是对以下几个系统进行了结构指导的比较分析 与炎症和应激相关的TSPO是高度诱导的。这个共同的主题 在不同的生物体之间具有揭示机制的独特见解的潜力。一个 明确TSPO结构和功能之间的机理关系将为 开发用于检测和治疗神经系统疾病的新型结构配体。

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.