The Role of KCa3.1 in Microglial function and in Parkinsons disease pathogenesis

摘要 最近的研究已经开始揭示小胶质细胞介导的神经炎症在帕金森病中的中心作用 疾病(PD)发病机制。越来越多的证据表明，小胶质细胞驱动的先天性免疫可能会进一步 增强有害的α-突触核蛋白(α-Syn)聚集和进行性神经变性。然而，我们缺乏一个 深入了解αSYN诱导先天免疫的细胞调控机制。因此， 识别帕金森病病理反应中调节小胶质细胞功能的信号机制可能 导致帕金森病新的免疫调节疗法的发展。我们最近发现文字记录 和钙激活钾通道KCa3.1的蛋白表达水平，最为人所知的是它在 免疫细胞钙信号在帕金森病死后脑内和临床前激活的小胶质细胞中升高 帕金森病的模型。我们进一步发现，FYN或STAT1的干扰抑制了反应性小胶质细胞的激活 聚集型α突触蛋白(αSynagg)刺激的原代小胶质细胞通过调节炎症介质的反应。 重要的是，高选择性和口服活性的KCa3.1抑制剂Senicapoc减少了神经炎症和 黑质多巴胺(DA)能神经毒性在帕金森病临床前小鼠模型中的作用，提示KCa3.1在 主治疾病病理学的多方面作用。尽管有这些令人鼓舞的发现，但确切的细胞 KCa3.1在突触核病背景下调节小胶质细胞功能的机制仍不清楚 特色化的。在此，我们提出了三个综合目标来检验KCa3.1促进的中心假设 α突触素通过激活小胶质细胞Fyn-1介导的黑质能神经退行性变过程 STAT1信号轴，体内抑制KCa3.1可恢复小胶质细胞内稳态，并为 突触核病背景下的DAR能神经保护。在AIM-1中，我们将测试上调的假设 KCa3.1致炎小胶质细胞活化表型及黑质DA能神经元丢失 联核症的背景。在AIM-2中，我们将测试Fyn-STAT1信号轴驱动 小胶质细胞对帕金森病样病理的反应依赖于KCa3.1。在AIM-3中，我们将检验这一假设 抑制KCa3.1活性可有效减少反应性小胶质细胞活化和进行性PD样变 疾病病理学。拟议的研究是创新的，利用了转录图谱、RNA 原位杂交、图像分析、αSynagg种子RT-QuIC法、CRISPR/Cas9KCNN4 基因敲除(KO)小鼠、转基因条件性KO小鼠模型和电生理记录，以测试 小胶质细胞KCa3.1影响帕金森病的进行性神经退变过程。这些研究解决了关键 关于KCa3.1在帕金森病发病机制中的作用的机制，可能有助于识别 可靶向减缓或阻止帕金森病进展和/或重新调整用途的新分子决定因素 Senicapoc用于帕金森病治疗。 1

Abstract Recent studies have begun to uncover the central role of microglia-mediated neuroinflammation in Parkinson’s disease (PD) pathogenesis. Increasing evidence suggests that microglia-driven innate immunity could further potentiate deleterious α-synuclein (αSyn) aggregation and progressive neurodegeneration. However, we lack an in-depth understanding of the cellular mechanisms regulating αSyn-induced innate immunity. Therefore, identifying signaling mechanisms that regulate microglial function in response to Parkinsonian pathology may lead to the development of novel immunomodulatory therapies for PD. We recently discovered that the transcript and protein expression levels of the calcium-activated potassium channel KCa3.1, best known for its role in immune cell calcium signaling, are elevated in activated microglia in both postmortem PD brains and in preclinical models of PD. We further identified that disruption of either FYN or STAT1 dampens reactive microglia activation responses via modulation of inflammatory mediators in aggregated αSyn (αSynagg)-stimulated primary microglia. Importantly, the highly selective and orally active KCa3.1 inhibitor Senicapoc reduced neuroinflammation and nigral dopamin(DA)ergic neurotoxicity in a preclinical mouse model of PD, suggesting that KCa3.1 plays a multifaceted role by governing disease pathology. Despite these encouraging findings, the exact cellular mechanisms by which KCa3.1 regulates microglial function in the context of synucleinopathy remain poorly characterized. Herein, we propose three integrated aims to test the central hypothesis that KCa3.1 promotes αSynagg-mediated progressive nigral DAergic neurodegenerative processes via activation of the microglial Fyn- STAT1 signaling axis and that the in vivo inhibition of KCa3.1 restores microglial homeostasis and affords DAergic neuroprotection in the context of synucleinopathy. In Aim-1, we will test the hypothesis that upregulation of KCa3.1 induces the proinflammatory microglial activation phenotype and nigral DAergic neuronal loss in the context of synucleinopathy. In Aim-2, we will test the hypothesis that the Fyn-STAT1 signaling axis drives microglial responses to PD-like pathology in a KCa3.1-dependent manner. In Aim-3, we will test the hypothesis that inhibiting KCa3.1 activation is efficacious in reducing reactive microglial activation and progressive PD-like disease pathology. The proposed studies are innovative, utilizing a combination of transcriptomic profiling, RNA in situ hybridization (ISH), imaging analysis, the RT QuIC assay for αSynagg seeding, CRISPR/Cas9 KCNN4 knockout (KO) mice, transgenic conditional KO mouse models, and electrophysiological recordings to test how microglial KCa3.1 influences progressive neurodegenerative processes in PD. These studies address key mechanistic aspects regarding the functional roles of KCa3.1 in PD pathogenesis and may aid in the identification of new molecular determinants that can be targeted for slowing or halting PD progression and/or repurposing Senicapoc for PD therapy. 1