Alzheimer's Disease: simultaneous modulation of NMDAR, BACE1, APP and Fyn activity by palmitoylation-targeting CIRC825

项目摘要/摘要 阿尔茨海默病(AD)的主要特征是淀粉样斑块和神经原纤维tau 缠结和这些肽的可溶性寡聚体被认为是有毒的；因此，针对这些组织病理学 HARMARKS导致了一系列的临床试验，但到目前为止，这些试验都失败了。耐人寻味的，反常的 已有多个蛋白质的棕榈酰化在AD中被报道。因此，我们在这里寻求同时 调节多种AD相关蛋白的位置和活性(神经递质受体NMDAR， 淀粉样斑块相关的BACE1和APP，以及神经原纤维缠结相关的Fyn激酶) 共享翻译后修饰，棕榈酰化。为了这项努力，我们将把一种药物的学习 从蛋白质中去除棕榈酸酯的小分子N-(叔丁基)-羟胺的开发计划 (NtBuHA，称为CIRC825)在这种定义明确的神经退行性疾病中，与过度- 棕榈酰化，CLN1巴顿(CLN1)。对这项AD相关的赠款非常重要的是，突触外(E)NMDAR 以GluN2B亚基为主的亚基被认为在AD的发病机制中被过度激活。 在一项证明我们的方法将在人类阿尔茨海默病的背景下工作的可行性研究中，我们的初步发现 最近的研究表明，CIRC825治疗几乎消除了脑梗塞患者异常的超兴奋表型。 人AD组与WT组HIPSC脑皮质神经元比较。此外，作为可行性数据，我们调查了 CIRC825在过表达APP的CHOAPP细胞中，发现它降低了APP的棕榈酰化，并降低了APP的棕榈酰化 β42代(致病形式)，但不降低可溶性Aβ40。这一阶段工作的目标是 重点扩大了我们对APP、BACE1、 Fyn和NMDAR在Cln1/-小鼠神经元和人(H)iPSC来源的2D皮质神经元培养中的表达 以及具有家族性AD突变的3D脑器官。将使用的人类IPSCs包括wPSEN1 M146V/WT和APPSWE/WT HiPSC系以及PSEN1ΔE9/WT HiPSC系(带有WT等基因对照)。 我们将通过用CIRC825处理这些模型来调节棕榈酰化。最后，在Cln1-/-鼠标中 模型中，我们将比较CIRC825和美金刚的生理益处。具体地说，在目标1中，我们将 评估CIRC825在培养的CLN1-/-神经元和(H)IPSC来源的AD神经元中的作用 APP、BACE1、Fyn和NMDAR的棕榈酰化；2)抑制突触外树突状细胞钙内流 钙成像，以及3)在兴奋性毒性试验中减轻谷氨酸诱导的细胞死亡。在《目标2》中我们将 CIRC825对(H)iPSC衍生的3D脑器官的测试浓度依赖的生理效应 家族性AD突变和WT对照)，使用包括膜片钳电生理、钙 成像、多电极阵列实验和免疫组织化学。在目标3中，我们将调查剂量- CIRC825和美金刚对CLN1-/-小鼠的依赖反应，用旋转棒评估运动缺陷 通过水迷宫和Y迷宫评估认知功能障碍，通过脑电波控制癫痫发作。在……上面 完成这项工作后，我们的目标是更有信心地证明棕榈酰化是一个可用药的靶点。 在AD中，据我们所知，这是一种治疗AD的新的药理学方法。此外，由于CIRC825正在被 在支持IND的研究中进行了调查，我们的目标是在CIRC825上开发一个数据包，证明更严格的 研究其在AD模型中的疗效，最终目标是临床翻译CIRC825(或其他 羟胺类化合物)。这项工作的结果将指导第二阶段计划，重点是更多 Cln1/-绵羊的靶向剂量范围评估和更广泛的AD模型研究，包括在 Happ-J20和5XfAD转基因小鼠。

PROJECT SUMMARY/ABSTRACT Alzheimer’s Disease (AD) has predominantly been characterized by amyloid plaques and neurofibrillary tau tangles, and soluble oligomers of these peptides are thought to be toxic; hence, targeting these histopathological hallmarks has led to a long series of clinical trials, but to date they have all failed. Intriguingly, aberrant palmitoylation of multiple proteins has been reported in AD. Accordingly, we seek here to simultaneously modulate the location and activity of multiple AD-associated proteins (neurotransmitter receptor NMDAR, amyloid plaque-associated BACE1 and APP, and neurofibrillary tangle-associated Fyn kinase) by altering their shared post-translational modification, palmitoylation. To this effort, we will transfer learnings from a drug development program of a small molecule that removes palmitate from proteins, N-(tert-butyl)-hydroxylamine (NtBuHA, called CIRC825) in the well-defined neurodegenerative disease that is associated with over- palmitoylation, CLN1 Batten (CLN1). Of great importance to this AD-related grant, extrasynaptic (e)NMDARs containing predominantly GluN2B subunits have been implicated to be overactivated in the pathogenesis of AD. In a feasibility study demonstrating our approach will work in the context of human AD, our preliminary findings have recently shown that CIRC825 treatment virtually eliminates the aberrant hyperexcitable phenotype in human AD hiPSC cerebrocortical neurons compared to WT. Additionally as feasibility data, we investigated CIRC825 in CHOAPP cells (which overexpress APP), and found it lowered palmitoylation of APP, and it decreased Aβ42 generation (the pathogenic form) without decreasing soluble Aβ40. The aims of this Phase 1 work are focused on expanding our understanding the pathomolecular importance of the palmitoylation of APP, BACE1, Fyn, and NMDAR in both Cln1-/- mouse neurons and in human (h)iPSC-derived 2D cortical neuronal cultures and 3D cerebral organoids that have familial AD mutations. Human iPSCs to be used include wPSEN1 M146V/WT and APPswe/WT hiPSC lines as well as the PSEN1 ΔE9/WT hiPSC line (with WT isogenic controls). We will modulate palmitoylation in these models by treating them with CIRC825. Finally, in a Cln1-/- mouse model, we will compare physiologic benefits of CIRC825 to those of memantine. Specifically, in aim 1 we will evaluate CIRC825 in cultured CLN1-/- neurons and (h)iPSC-derived AD neurons for 1) effects on decreasing palmitoylation of APP, BACE1, Fyn, and NMDAR; 2) suppression of extra-synaptic dendritic Ca2+ influxes by calcium imaging, and 3) mitigation of glutamate-induced cell death in an excitotoxicity assay. In aim 2 we will test concentration-dependent physiologic effects of CIRC825 on (h)iPSC-derived 3D cerebral organoids (with familial AD mutations and WT controls), using multiple assays including patch-clamp electrophysiology, calcium imaging, multi-electrode array experiments, and immunohistochemistry. In aim 3 we will investigate the dose- dependent response of CIRC825 and memantine in CLN1-/- mice, assessing for motor deficits by a rotarod analysis, cognitive deficits by a water-maze and Y-maze assessment, and control of seizures by EEG. On completion of this work, we aim to demonstrate with greater confidence that palmitoylation is a druggable target in AD, which, to our knowledge, is a novel pharmacologic approach in AD. Further, as CIRC825 is being investigated in IND-enabling studies, we aim to develop a data package on CIRC825 that justifies more rigorous investigations of its efficacy in AD models, with the ultimate goal of clinical translation of CIRC825 (or other hydroxylamine derivatives) in AD. Results of this work will guide a Phase II program that is focused on a more targeted dose range assesment in Cln1-/- sheep and investigations in a broader set of AD models, including in hAPP-J20 and 5XfAD transgenic mice.