Mechanisms of Dantrolene Neuroprotection in Alzheimer's Disease

摘要 针对淀粉样蛋白的阿尔茨海默病(AD)治疗的临床试验 在很大程度上失败了。钙离子调节失调可能是另一种机制。在家族性AD(FAD)中， N-甲基-D-天冬氨酸(NMDAR)和兰尼定(RyR)的过度激活破坏了钙稳态 受体导致胞浆钙升高和随后的认知功能障碍 神经病理学。载脂蛋白E4是散发性阿尔茨海默病(SAD)的主要危险因素，也会导致钙离子 与NMDAR/RyR过度激活有关的调节失调。丹曲林，RyR拮抗剂和 临床上可用的药物，已被证明可以减轻淀粉样蛋白病理，神经变性， 时髦动物模型中的突触和记忆丧失。我们的初步研究表明 丹曲林鼻腔给药可消除5XFAD小鼠的记忆丧失。此外， 丹曲林促进体外诱导的多能干细胞向神经元分化 伴有APOE4的SAD或RyR/NMDAR过度抑制的家族性AD(FAD)患者 激活。我们的长期目标是检查丹曲林治疗AD的疗效。整体而言 本研究的目的是探讨丹曲林对血管紧张素转换酶的影响及其机制。 人和啮齿动物SAD和FAD细胞的成体神经发生以及鼻腔给药的影响 丹曲林对AD转基因小鼠成年神经发生和认知功能的影响。 我们的中心假设是丹曲林鼻腔内抑制细胞过度激活 RYRs和NMDAR在AD中的作用和促进成人神经发生，改善认知功能 减少AD的神经病理改变。我们将通过以下具体目标来检验这一假设。 具体目的1.确定丹曲林对RyR和NMDAR表达的影响 AD患者胞浆、线粒体和内质网钙离子浓度及AD生物标志物的研究 SAD患者成纤维细胞诱导多能干细胞(IPSC)的干细胞应用 (ApoE4危险因子)或FAD(PSEN1突变)，以及分离的神经前体细胞(NPC) 来自E4FAD(APOE4+5XFAD)和5XFAD转基因的特异靶2.检测效果 丹曲林对RyR和NMDAR活性、神经发生、增殖和细胞的影响 AD细胞来源的神经元和胶质细胞的功能。使用与SA1相同的单元。 具体目的3.确定丹曲林对成人神经发生、认知的影响 阿尔茨海默病动物模型的功能和神经病理学。我们预计丹曲林将 促进阿尔茨海默病小鼠成体神经发生和恢复认知功能，减轻AD病理改变 通过缓解RYR和/或NMDAR的过度激活来建立模型，特别是通过 鼻腔入路。

Abstract Clinical trials for the treatment of Alzheimer's disease (AD) targeting amyloid have largely failed. Ca2+ dysregulation may be an alternative mechanism. In familial AD (FAD), calcium homeostasis is disrupted by over activation of NMDA (NMDAR) and ryanodine (RYR) receptors leading to increased cytosolic calcium and subsequent cognitive dysfunction and neuropathology. APOE4, a major risk factor for sporadic AD (SAD), also causes Ca2+ dysregulation related to NMDAR/RYR over activation. Dantrolene, a RYR antagonist and clinically available drug, has been shown to mitigate amyloid pathology, neurodegeneration, synaptic and memory loss in a FAD animal model. Our preliminary studies suggested that intranasal dantrolene administration abolished memory loss in 5XFAD mice. Furthermore, dantrolene promoted neuronal differentiation in induced pluripotent stem cells (iPSC) from patients with either SAD with APOE4 or familial AD (FAD) by inhibition of RYR/NMDAR over- activation. Our long term goal is to examine the efficacy of dantrolene to treat AD. The overall objective of this study is to investigate the effects and underlying mechanisms of dantrolene on adult neurogenesis in human and rodent SAD and FAD cells, as well as the effects of intranasal dantrolene administration on adult neurogenesis and cognitive function in AD transgenic mice. Our central hypothesis is that intranasal dantrolene inhibits the excessive activation of RYRs and NMDARs in AD and promotes adult neurogenesis, improved cognitive function and reduced AD neuropathology. We will test the hypothesis by the following specific aims. Specific Aim 1. To determine the effects of dantrolene on RYR and NMDAR expression and on cytosolic, mitochondrial, and ER Ca2+ concentrations and AD biomarkers in AD stem cells using induced pluripotent stem cells (iPSC) from fibroblasts patients with either SAD (APOE4 risk factor) or FAD (PSEN1 mutations), as well as neuroprogenitor cells (NPC) isolated from E4FAD (APOE4+5XFAD) and 5XFAD transgenic Specific Aim 2. To examine the effects of dantrolene on RYR and NMDAR activity, neurogenesis, proliferation, and the cellular function of derived neurons and glia from AD cells. using the same cells as in SA1. Specific Aim 3. To determine the effects of dantrolene on adult neurogenesis, cognitive function, and neuropathology in animal models of AD. We expect that dantrolene will promote adult neurogenesis and restore cognition and reduce AD pathology in AD mouse models by alleviating the excessive activation of RYRs and/or NMDARs, especially with the intranasal approach.