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

项目摘要 阿尔茨海默病（AD）是一种破坏性的神经退行性疾病，其特征在于 淀粉样蛋白-β（Aβ）和tau蛋白、神经炎症、神经元损失和痴呆。目前没有 改变疾病的治疗方法小胶质细胞是大脑中的免疫细胞，代表了有希望的细胞靶点 用于治疗开发以改变AD进展的过程。小胶质细胞过渡到一个独特的状态 在AD中称为疾病相关小胶质细胞（DAM），其通过保护和 有害的反应。DAM的一个子集，称为促炎性DAM，促进神经元损伤， 有效吞噬Aβ。初步研究表明，促炎性DAM经历了生物能量转变 糖酵解状态来维持有害反应。因此，确定 糖酵解转变在确定抑制AD中促炎反应的新途径中至关重要。我们实验室 发现促炎性DAM高度表达一种称为Kv1.3的钾通道，该通道调节钾离子 外排和细胞功能。我们实验室的工作表明，在AD小鼠模型中， 病理学减少神经炎症和Aβ病理学，但Kv1.3如何调节促炎性 DAM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通道是一个关键的调节器， AD病理学中小胶质细胞的生物能量转换和促炎反应。我将在体外和体内使用 研究Kv1.3如何调节DAM的生物能量学和炎症反应的方法。的 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 目的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功能。 小胶质细胞的功能将被Aβ激活。目标1将使用海马分析，活细胞成像，免疫 分析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体内小胶质细胞中的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的影响， 通过免疫分析、蛋白质印迹、关键代谢物测定和 免疫组化这两个平行的目标将允许我阐明Kv1.3 调节AD中的促炎性DAM反应。通过了解Kv1.3小胶质细胞在AD中的影响，我将 有助于理解AD背后的生物学，并建立一个潜在的治疗标志物。通过 我将从这次建议中获得培训，我将为我未来的职业目标做好充分的准备，成为一名独立的 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.