The Role of KCa3.1 in Neuroinflammation in Alzheimer Disease

描述(申请人提供)：阿尔茨海默病(AD)是导致老年人痴呆症的最常见原因。为了应对AD带来的公共健康挑战，国家阿尔茨海默氏症项目法设定的目标是到2025年预防或治疗AD。因此，目前迫切需要发现新的治疗靶点并开发相应的化合物。一种沉积在AD大脑中的蛋白质称为淀粉样β蛋白(Abeta)，已被假设在AD的发病机制中发挥关键作用。Abeta可以激活小胶质细胞清除Abeta，但同时也刺激小胶质细胞释放细胞毒物质，导致神经元损伤。利用本课题组合成的小分子TRAM-34作为药理工具，我们最近证实了钙激活钾通道KCa3.1在小胶质细胞激活和小胶质细胞神经毒性中起重要作用。体内证据表明，TRAM-34阻断KCa3.1可以抑制小胶质细胞介导的神经元杀伤，而不影响其迁移和吞噬活性。与AD相关，我们的 结果表明，TRAM-34可阻断Abeta激活的小胶质细胞所致的神经毒性，但不抑制其吞噬Abeta的有益功能。因此，KCa3.1阻断是一种潜在的治疗AD的新方法。在这笔赠款的帮助下，我们希望进行原则验证研究，通过以下三个具体目标验证KCa3.1作为减少阿尔茨海默病(AD)小胶质细胞介导的神经毒性和小胶质细胞功能障碍的新治疗靶点：目的-1：确定KCa3.1阻断对抗体诱导的小胶质细胞激活的影响。我们将用Abeta聚集体(寡聚体和原纤维)处理培养的小胶质细胞，并评估TRAM-34处理(Aim-1a)或基因敲除(Aim-1b)阻断KCa3.1对趋化和吞噬活性、信号通路以及趋化因子、细胞因子、活性氧和一氧化氮产生的影响。目的-2：使用基因敲除方法评估KCa3.1在APPswe/PS1dE9(APP-PS1)模型中的AD样病理和认知缺陷中的作用。我们将使APP-PS1小鼠与KCa3.1-/-小鼠杂交，并评估KCa3.1的降低是否会影响APP-PS1小鼠的神经病理和行为异常。我们将进一步确定KCa3.1的减少是否可以缓解APP-PS1小鼠的小胶质细胞功能障碍。目的-3：通过使用TRAM-34进行临床前研究，验证KCa3.1作为AD的治疗靶点。我们将确定APP-PS1小鼠服用KCa3.1的选择性抑制剂TRAM-34 2个月的疗程是否会减少神经炎症，缓解小胶质细胞功能障碍，并改善APP-PS1小鼠的神经病理和行为结果。由于KCa3.1阻断是相对安全的，我们的临床前研究将对未来通过抑制有害的小胶质细胞功能来治疗轻度认知障碍或AD的药物的开发具有翻译意义。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of dementia in the elderly. To meet the public health challenge posed by AD, a goal set by the National Alzheimer's Project Act is to prevent or treat AD by 2025. Therefore currently there is an urgent need for new therapeutic target discovery and corresponding compound development. A protein deposited in AD brains called amyloid-beta (Abeta) has been hypothesized to play a critical role in AD pathogenesis. Abeta can activate microglia to clear Abeta but at the same time also stimulate microglia to release cytotoxic substances that cause neuronal damage. Using the small molecule TRAM-34, which was synthesized by our group, as a pharmacological tool we recently demonstrated that the calcium-activated potassium channel KCa3.1 plays an important role in microglia activation and microglial neurotoxicity. In vivo evidence indicates that blockade of KCa3.1 by TRAM-34 can inhibit microglia-mediated neuronal killing without affecting their migration and phagocytotic activities. Relevant to AD, our results suggest that TRAM-34 blocks the neurotoxicity induced by Abeta-activated microglia, but does not inhibit their beneficial function of phagocytosing Abeta. KCa3.1 blockade, therefore, is a potential new approach for the treatment of AD. With the help of this grant we wish to perform proof-of-principle studies to validate KCa3.1 as a novel therapeutic target for reducing microglia-mediated neurotoxicity and microglial dysfunction in Alzheimer's disease (AD), through the following three Specific Aims: Aim-1: Determine the effect of KCa3.1 blockade on Ab-induced microglial activation. We will treat cultured microglia with Abeta aggregates (oligomer and fibril) and evaluate the effect of KCa3.1 blockade by TRAM-34 treatment (Aim-1a) or genetic knockout (Aim-1b) on chemotactic and phagocytotic activities, signaling pathways, and the production of chemokines, cytokines, reactive oxygen species, and nitric oxide. Aim-2: Assess the contribution of KCa3.1 to AD-like pathology and cognitive deficits seen in the APPswe/PS1dE9 (APP-PS1) model using the genetic knockout approach. We will cross-breed APP-PS1 mice with KCa3.1-/- mice and evaluate whether KCa3.1 reduction affects neuropathological and behavioral abnormalities in APP-PS1 mice. We will further determine if KCa3.1 reduction can alleviate microglial dysfunction seen in APP-PS1 mice. Aim-3: Validate KCa3.1 as a therapeutic target for AD by performing preclinical studies with TRAM-34. We will determine if a 2-month course of TRAM-34, a selective inhibitor of KCa3.1, administered to APP-PS1 mice will reduce neuroinflammation, alleviate microglial dysfunction, and improve the neuropathological and behavioral outcomes of APP-PS1 mice. Because KCa3.1 blockade is relatively safe, our preclinical studies will have translational significance for future developmen of drugs for treating individuals with mild cognitive impairment or AD through inhibition of detrimental microglia functions.