Regulation of Microglial Bioenergetics and Neuroinflammation by Kv1.3 Channels in Alzheimer's Disease

项目总结 阿尔茨海默病(AD)是一种毁灭性的神经退行性疾病，其特征是积聚 淀粉样蛋白-β(A-β)和tau蛋白，神经炎症，神经元丢失，和痴呆。目前，没有 阿尔茨海默病的疾病改变疗法。小胶质细胞是大脑的免疫细胞，代表着很有希望的细胞靶点。 用于治疗开发，以改变AD的进展过程。小胶质细胞向独特状态的转变 在AD中被称为疾病相关小胶质细胞(DAM)，它通过保护性和 有害的反应。DAM的一个子集，称为促炎性DAM，促进神经元损伤，并未能 有效地吞噬Aβ。初步研究表明，促炎大坝经历了生物能量的转变 进入糖酵解状态以维持它们的有害反应。因此，确定 糖酵解转变是确定抑制AD促炎反应的新途径的关键。我们的实验室 发现促炎因子高度表达一种名为Kv1.3的钾通道，该通道调节钾 外排和细胞功能。我们实验室的工作显示在AD小鼠模型中Kv1.3通道被阻断 病理减少神经炎症和β病理，但Kv1.3如何调节促炎 大坝方面的反应仍不清楚。我认为Kv1.3通过控制炎症反应来调节炎症反应 小胶质细胞的生物能量学。我的中心假设是Kv1.3通道是 阿尔茨海默病病理中小胶质细胞的生物能量转换和促炎反应。我会在体外和体内使用 研究Kv1.3如何调节DAM的生物能量学和炎症反应的方法。这个 该项目的长期目标是研究Kv1.3在小胶质细胞生物能量学和炎症中的作用。在……里面 目标1，我将使用体外系统来研究Kv1.3如何改变小胶质细胞的生物能量学和免疫 小胶质细胞的功能，它将被β激活。AIM 1将使用海马体分析、活细胞成像、免疫 和流式细胞术来描述这些生物能量学和炎症反应的变化。在目标2中， 我将利用一种新的遗传方法在AD小鼠模型中选择性地删除小胶质细胞中的Kv1.3。 这将使我能够测量小胶质细胞中Kv1.3缺失对小胶质细胞生物能量学和 通过免疫分析、免疫印迹、关键代谢物分析、以及 免疫组织化学。这两个平行的目标将使我能够阐明Kv1.3 调节阿尔茨海默病的炎症反应。通过了解Kv1.3对小胶质细胞在AD中的影响，我将 有助于理解阿尔茨海默病背后的生物学，并建立一个潜在的治疗标记。通过 我将在这个建议中获得培训，我将为我未来成为一名独立人士的职业目标做好准备 调查员。

PROJECT SUMMARY Alzheimer’s disease (AD) is a devastating neurodegenerative disease is characterized by accumulation of amyloid-β (Aβ) and tau proteins, neuroinflammation, neuronal loss, and dementia. Currently, there are no disease-altering therapies for AD. Microglia, the immune cells of the brain, represent promising cellular targets for therapeutic development to modify the course of AD progression. Microglia transition to a unique state called disease associated microglia (DAM) in AD which contribute to AD pathogenesis via both protective and detrimental responses. A subset of DAM, known as proinflammatory DAM, promote neuronal injury and fail to phagocytize Aβ effectively. Preliminary studies indicate proinflammatory DAM undergo a bioenergetic shift toward a glycolytic state to sustain their detrimental responses. Therefore, identifying the regulators of the glycolytic shift is critical in determining novel avenues to inhibit proinflammatory responses in AD. Our lab found proinflammatory DAM highly express a potassium channel called Kv1.3, which regulates potassium efflux and cellular functions. Our lab’s work showed blockade of Kv1.3 channels in mouse models of AD pathology reduces neuroinflammation and Aβ pathology, but how Kv1.3 regulates the proinflammatory response in DAM remains unclear. I propose that Kv1.3 regulates inflammatory responses by controlling the bioenergetics of microglia. My central hypothesis is the Kv1.3 channel is a critical regulator of the bioenergetic switch and proinflammatory responses of microglia in AD pathology. I will use in vitro and in vivo approaches to examine how Kv1.3 modulates the bioenergetics and inflammatory responses of DAM. The long-term goal of this project is to examine the role of Kv1.3 in microglial bioenergetics and inflammation. In Aim 1, I will use an in vitro system to examine how Kv1.3 alters the microglial bioenergetics and immune function of microglia which will be activated by Aβ. Aim 1 will use seahorse assays, live cell imaging, immune profiling, and flow cytometry to describe these changes in bioenergetics and inflammatory response. In Aim 2, I will utilize a novel genetic approach to delete Kv1.3 selectively in microglia in vivo in an AD mouse model. This will allow me to measure the effects of Kv1.3 deletion in microglia on microglial bioenergetics and inflammatory responses via immune profiling, western blots, assays for key metabolites, and immunohistochemistry. These two parallel aims will allow me to elucidate a mechanism by which Kv1.3 regulates proinflammatory DAM responses in AD. By understanding Kv1.3 influence of microglia in AD, I will contribute to the understanding of biology behind AD and establish a potential therapeutic marker. Through the training I will gain in this proposal, I will be well prepared for my future career goal of becoming an independent investigator.