Mechanisms of Dantrolene Neuroprotection in Alzheimer's Disease

摘要 冠状病毒病2019年(新冠肺炎)，一种影响周围数以百万计患者的大流行 世界上，由严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起，没有 有效的药物治疗。病毒在宿主细胞中的感染和转染都需要 胞浆和内体中钙离子浓度异常升高通过过度激活 Ryanodine受体(RyRs)钙通道位于内质膜 网状结构(ER)。丹曲林是RyRs的拮抗剂，已被证明可以抑制SARS- 冠状病毒-2介导的宿主细胞毒性和损伤。在RyRs过度激活和 胞浆和内体钙离子浓度过高和异常升高，峰值 病毒的蛋白(S1、S2)在结合、融合和病毒复制中发挥重要作用 宿主细胞，最终导致细胞损伤或死亡。我们的长期目标是研究 丹曲林治疗阿尔茨海默病的疗效及机制。这项研究的总体目标是 丹曲林对宿主细胞的保护作用及其机制研究 SARS-CoV-2刺突蛋白介导的RyRs和RyRs过度激活诱导的损伤或死亡 相关的钙离子失调。我们的中心假设得到初步数据的支持，即 丹曲林通过抑制SARS-CoV-2刺突蛋白介导的细胞损伤 通过过度激活RyRs和恢复AD细胞内钙稳态。我们 我将通过以下具体目标来检验这一假设。特定目标1(SA1)。要确定 丹曲林对S SARS冠状病毒S蛋白介导的钙离子异常调节的影响 以及使用诱导多能干细胞(IPSC)在AD细胞中通过凋亡造成的细胞损伤，来自 散发性(SAD)或家族性(FAD)患者和人类皮肤成纤维细胞 神经母细胞瘤细胞(SH-SY5Y)，敲入AD早老素1突变(M146L)。特定的 目标2(SA2)。丹曲林对SARS-CoV-2的作用及机制研究 SPEKE(S)蛋白介导的导致宿主细胞损伤的病理途径。我们会 研究S蛋白对线粒体耗氧量、三磷酸腺苷生成、 活性氧(ROS)的产生、内质网应激和细胞因子的释放。我们将相互关联 SA1和SA2之间的结果。我们预计丹曲林将抑制S蛋白介导的 RyRs的过度激活及其相关的细胞内钙稳态和细胞内平衡的破坏 由细胞凋亡引起的死亡。

Abstract Coronavirus disease 2019 (COVID-19), a pandemic affecting millions of patients around the world, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with no effective drug treatments. Both infection and transfection of virus in host cells require abnormally elevated Ca2+ concentrations in cytosol and endosome via overactivation of ryanodine receptors (RyRs) Ca2+ channel located on the membrane of the endoplasmic reticulum (ER). Dantrolene, an antagonist of RyRs, has been demonstrated to inhibit SARS- CoV-2-mediated host cell toxicity and damage. In the presence of RyRs overactivation and excessive and abnormal elevation of cytosolic and endosome Ca2+ concentrations, the spike proteins (S1, S2) of the virus play important roles in binding, fusion, and virus replication in the host cell, eventually leading to cell damage or death. Our long-term goal is to examine the efficacy and mechanisms of dantrolene to treat AD. The overall objective of this study is to investigate the effects and underlying mechanisms of dantrolene to protect against host cell damage or death induced by SARS-CoV-2 spike proteins-mediated overactivation of RyRs and associated Ca2+ dysregulation. Our central hypothesis supported by preliminary data is that dantrolene inhibits SARS-CoV-2 spike proteins-mediated cell damage by inhibiting the overactivation of RyRs and by restoring intracellular Ca2+ homeostasis in AD cells. We will test this hypothesis with the following specific aims. Specific Aim 1 (SA1). To determine the effects of dantrolene on SARS-CoV-2 spike (S) proteins-mediated Ca2+ dysregulation and cell damage by apoptosis in AD cells using induced pluripotent stem cells (iPSC), from skin fibroblasts of patients with either sporadic (SAD) or familial (FAD), and human neuroblastoma cells (SH-SY5Y), with knocked-in AD presenilin 1 mutation (M146L). Specific Aim 2 (SA2). To determine the effects and mechanisms of dantrolene on SARS-CoV-2 spike (S) proteins-mediated pathological pathways leading to host cell damage. We will examine the effects of S proteins on mitochondria oxygen consumption, ATP production, reactive oxygen species (ROS) production, ER stress, and cytokine release. We will correlate the results between SA1 and SA2. We expect that dantrolene will inhibit S proteins-mediated overactivation of RyRs and associated disruption of intracellular Ca2+ homeostasis and cell death by apoptosis.