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

阿尔茨海默病：通过棕榈酰化靶向 CIRC825 同时调节 NMDAR、BACE1、APP 和 Fyn 活性

• 批准号: 10257994
• 负责人: Devin Thomas Wiley
• 金额: $ 50.73万
• 依托单位: CIRCUMVENT PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10257994
• 关键词: 3-Dimensional; Action Potentials; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease therapeutic; Amyloid beta-42; Biochemical Pathway; Biological Assay; Biology; Brain Diseases; CLN1 gene; Calcium; Cell Death; Cell Survival; Cells; Cerebrum; Clinical; Clinical Trials; Cognition; Cognitive deficits; DLG4 gene; Data; Diffusion; Disease; Disease Progression; Doctor of Philosophy; Dose; Drug Targeting; Electroencephalography; Electrophysiology (science); Epilepsy; Event; Fatty acid glycerol esters; Feasibility Studies; Frequencies; Generations; Glutamates; Goals; Grant; Human; Hydroxylamine; Image; Immunohistochemistry; Investigation; J20 mouse; Location; Marshal; Memantine; Modeling; Motor; Mus; Mutation; Namenda; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Neurotransmitter Receptor; Organoids; Palmitates; Pathogenesis; Pathogenicity; Peptides; Performance; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Phosphotransferases; Physiological; Post-Translational Protein Processing; Program Development; Proteins; Psychological Transfer; Reporting; Research Personnel; Seizures; Senile Plaques; Series; Sheep; Synapses; Testing; Therapeutic; Transgenic Mice; Work; beta-site APP cleaving enzyme 1; brain cell; clinical translation; cognitive function; cytotoxicity; drug development; druggable target; excitotoxicity; experimental study; familial Alzheimer disease; improved; induced pluripotent stem cell; motor deficit; mouse model; multi-electrode arrays; new therapeutic target; novel; novel therapeutics; overexpression; palmitoylation; patch clamp; presenilin-1; programs; response; small molecule; tau aggregation; virtual; water maze

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.

项目摘要/摘要 阿尔茨海默氏病（AD）的主要特征是淀粉样蛋白斑块和神经纤维tau 这些宠物的缠结和可溶性低聚物被认为是有毒的。因此，针对这些组织病理学 标志已导致了一系列漫长的临床试验，但迄今为止，它们都失败了。有趣的是，异常 AD中已经报道了多种蛋白质的棕榈酰化。根据，我们在这里寻求简单 调节多种AD相关蛋白的位置和活性（神经递质受体NMDAR， 淀粉样菌斑相关的BACE1和APP，以及神经纤维缠结相关的Fyn激酶），通过改变其 共享的翻译后修饰，棕榈酰化。为了这项努力，我们将从药物中转移学习 从蛋白质中去除棕榈酸盐的小分子的开发程序，n-（tert丁基） - 羟胺 （NTBUHA，称为Circ825）在明确定义的神经退行性疾病中，与过度有关 Palmitoylation，Cln1 Batten（CLN1）。对于这个与广告相关的赠款非常重要 在AD的发病机理中，含有主要含有Glun2b亚基的主要含量。 在一项可行性研究中，表明我们的方法将在人类广告的背景下起作用，我们的初步发现 最近表明，Circ825治疗实际上消除了异常过度表型 与WT相比，人AD HIPSC脑皮层神经元。此外，作为可行性数据，我们研究了 Chip825在Choapp单元格（过表达应用）中，发现其降低了APP的棕榈酰化，并改进了 Aβ42生成（致病形式）而不降低固体Aβ40。这一阶段工作的目的是 专注于扩大我们理解App，Bace1， Fyn和cln1 - / - 小鼠神经元和人（H）IPSC衍生的2D皮质神经元培养的NMDAR 和具有家庭广告突变的3D大脑器官。使用的人IPSC包括WPSEN1 M146V/WT和APPSWE/WT HIPSC线以及PSEN1ΔE9/WT HIPSC系（带有WT同源控制）。 我们将通过使用Circ825处理这些模型来调节棕榈酰化。最后，在Cln1 - / - 鼠标中 模型，我们将将Circ825的生理益处与纪念元素的生理益处进行比较。具体来说，在AIM 1中，我们将 评估培养的CLN1 - / - 神经元和（H）IPSC衍生的AD神经元中的Circ825，以减少降低 App，Bace1，Fyn和NMDAR的棕榈酰化； 2）抑制外突触的树突状Ca2+影响 钙成像和3）在兴奋性测定中缓解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大脑器官的测试浓度依赖性生理效应（带有 家族性广告突变和WT对照），使用多种测定法，包括斑块钳电生理学，钙 成像，多电极阵列实验和免疫组织化学。在AIM 3中，我们将调查剂量 - Cln1 - / - 小鼠中Circ825和美容的依赖性响应，评估电动机的定义 分析，通过水迷宫和Y迷宫评估的认知缺陷以及脑电图控制癫痫发作。在 完成这项工作，我们旨在更加自信地证明棕榈酰化是可吸毒的目标 据我们所知，AD是AD中的一种新型药理方法。此外，随着Circ825的存在 在进行的研究中，我们旨在在Circ825上开发一个数据包，以证明更严格 对其在AD模型中的效率进行了调查，其最终目标是Circ825的临床翻译（或其他 AD中的羟胺衍生物）。这项工作的结果将指导II阶段计划，该计划的重点更多 Cln1 - / - 绵羊中的有针对性剂量范围评估和更广泛的广告模型中的调查，包括 Happ-J20和5XFAD转基因小鼠。