Role of MAMs in stabilization and BACE1-mediated processing of palAPP.

MAM 在 palAPP 稳定和 BACE1 介导的加工中的作用。

• 批准号: 10210441
• 负责人: RUDOLPH Emile TANZI
• 金额: $ 40.89万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10210441
• 关键词: 3-Dimensional; Acyl Coenzyme A; Address; Alzheimer like pathology; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease patient; Alzheimer' s disease therapeutic; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Axon; Biological Assay; Biotinylation; Cell physiology; Cell surface; Cells; Cellular Structures; Cholesterol; Cholesterol Esters; Cleaved cell; Clinical; Clinical Trials; Communication; Data; Dementia; Detergents; Development; Drug Targeting; Elderly; Endoplasmic Reticulum; Fatty Acids; Fibroblasts; Generations; Goals; Golgi Apparatus; Half-Life; In Vitro; Kinetics; Location; Mediating; Membrane; Membrane Microdomains; Mitochondria; Modeling; Molecular; Neurodegenerative Disorders; Neuroglia; Neurons; Organelles; Oxidative Stress; Palmitic Acids; Pathogenesis; Pathway interactions; Peptides; Play; Process; Reporting; Research; Resistance; Role; Senile Plaques; Sterol O-Acyltransferase; Testing; Time; Transgenic Mice; Vesicle; amyloid precursor protein processing; beta secretase; beta-site APP cleaving enzyme 1; cell type; dimer; experimental study; gamma secretase; human stem cells; improved; in vivo; inhibitor/antagonist; live cell imaging; novel; novel therapeutic intervention; prevent; prodromal Alzheimer' s disease; relating to nervous system; sequential proteolysis; sterol O-acyltransferase 1; therapeutic development; trafficking

The amyloid precursor protein (APP) undergoes sequential proteolysis by β- and γ- secretases to produce amyloid β (Aβ) in Alzheimer's disease (AD). Currently, clinical trials are underway targeting Aβ with β- or γ-secretase inhibitors in mild or prodromal AD patients. Alternative Aβ-lowering agents are also being actively pursued. Along these lines, we previously demonstrated that Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, e.g. CP-113,818 and CI-1011, which prevent conversion of cholesterol into cholesteryl-esters, reduce secreted Aβ levels by up to 92%, and improve AD-like pathology in hAPP transgenic mice. ACAT-inhibitors have not been clinically developed for AD largely because the molecular mechanism regarding effects on Aβ generation remains unclear. We have demonstrated, for the first time, that approximately 10% of APP is post-translationally modified by palmitic acid in vitro and in vivo. Palmitoylated APP (palAPP) is enriched in cholesterol-rich lipid rafts where it appears to serve as a preferred substrate for β-secretase (BACE1) versus total APP. ACAT-inhibition decreased lipid raft palAPP levels by up to 76%. We have also reported that ~90% of palAPP forms cis- dimers undergoing preferred BACE1 cleavage in detergent resistant membranes (DRMs). Thus, palAPP and/or cis-dimerized palAPP in lipid rafts are potentially useful drug targets for AD. Recently, we reported that palAPP is also enriched in raft-like Mitochondria- associated ER Membranes (MAMs) in vitro and in vivo, together with BACE1, γ-secretase, and ACAT. Interestingly, MAM function and ER–mitochondrial communication are increased significantly in fibroblasts from both familial and sporadic AD patients. Overall, our preliminary studies indicate that palAPP is synthesized in neuronal cells including neuronal processes and is stabilized in MAMs. Thus, we propose the following hypothesis: palAPP that is stabilized in MAM's undergoes BACE1 cleavage in neuronal cells and processes. We will explore the effects of novel ACAT inhibitors on palAPP trafficking and processing in MAM-associated/stabilized palAPP, including the use of live-cell imaging and cell surface biotinylation assays in primary neurons and our 3D human stem cell-derived neural culture models. The overarching goal of this proposal is to generate the necessary mechanistic and in vivo data to further the development of novel therapeutic strategies for AD by targeting ACAT and MAM-associated palAPP.

淀粉样蛋白前体蛋白（APP）经历了β-和γ-的顺序蛋白水解 在阿尔茨海默氏病（AD）中产生淀粉样β（Aβ）的分泌酶。目前，临床试验 在轻度或前驱AD中使用β-或γ-分泌酶抑制剂靶向Aβ 患者。还积极追求替代的降低Aβ剂。沿着这些 线，我们以前证明了酰基辅酶A：胆固醇酰基转移酶 （ACAT）抑制剂，例如CP-113,818和CI-1011，可防止胆固醇转化 进入胆汁胆固件，将分泌的Aβ水平降低多达92％，并改善类似AD的Aβ水平 HAPP转基因小鼠的病理学。 ACAT抑制剂尚未在临床上开发 AD主要是因为有关Aβ产生影响的分子机制 仍然不清楚。我们首次证明了大约10％的应用程序 在体外和体内通过棕榈酸后翻译。棕榈酰化应用 （palapp）在富含胆固醇的脂质筏中富含它似乎是首选的 β-分泌酶（BACE1）与总应用程序的底物。 ACAT抑制减少脂质筏 Palapp水平高达76％。我们还报告说，约有90％的帕拉普形成了顺式 在清洁膜（DRMS）中接受首选BACE1清洁的二聚体。 那是脂质筏中的palapp和/或顺式二聚体的帕拉普是潜在有用的药物靶标 对于广告。最近，我们报道了帕拉普（Palapp）也富含木筏样线粒体 - 相关的ER膜（MAM）体外和体内与Bace1，γ-分泌酶一起 和ACAT。有趣的是，MAM功能和ER-明骨软骨交流是 来自家族性和零星AD患者的成纤维细胞的显着增加。全面的， 我们的初步研究表明，帕拉普是在神经元细胞中合成的 神经元过程并在MAM中稳定。那，我们提出以下内容 假设：在MAM的PALAPP中稳定在Mam的Bace1裂解中 神经元细胞和过程。我们将探讨新型ACAT抑制剂对 在MAM相关/稳定的PALAPP中，Palapp贩运和处理，包括使用 原代神经元中的活细胞成像和细胞表面生物素化测定法 人类干细胞衍生的神经培养模型。该提议的总体目标是 生成必要的机械和体内数据，以进一步发展新型 通过针对ACAT和MAM相关的PALAPP来实现AD的治疗策略。