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: 9920896
• 负责人: RUDOLPH Emile TANZI
• 金额: $ 5.11万
• 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/9920896
• 关键词: 3-Dimensional; Acyl Coenzyme A; Address; Alzheimer' s Disease; Alzheimer' s disease model; 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; Pathology; Pathway interactions; Patients; Peptides; Play; Process; Reporting; Research; Resistance; Role; Senile Plaques; Sterol O-Acyltransferase; Testing; Therapeutic; 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 therapeutics; prevent; 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.

淀粉样前体蛋白 (APP) 通过 β- 和 γ- 进行连续蛋白水解 阿尔茨海默病 (AD) 中分泌酶产生 β 淀粉样蛋白 (Aβ)。目前，临床试验 正在使用 β- 或 γ-分泌酶抑制剂靶向 Aβ，治疗轻度或前驱 AD 患者。人们也在积极寻找替代性 Aβ 降低药物。沿着这些 线，我们之前证明了酰基辅酶 A：胆固醇酰基转移酶 (ACAT) 抑制剂，例如CP-113,818 和 CI-1011，防止胆固醇转化 转化为胆固醇酯，将分泌的 Aβ 水平降低高达 92%，并改善 AD 样症状 hAPP 转基因小鼠的病理学。 ACAT抑制剂尚未临床开发 对于 AD 来说，很大程度上是因为影响 Aβ 生成的分子机制 仍不清楚。我们首次证明，大约 10% 的 APP 在体外和体内均被棕榈酸翻译后修饰。棕榈酰化APP (palAPP) 富含富含胆固醇的脂筏，它似乎是首选 β-分泌酶 (BACE1) 底物与总 APP 的比较。 ACAT 抑制减少脂筏 palAPP 水平高达 76%。我们还报道约 90% 的 palAPP 形成顺式 二聚体在耐去垢剂膜 (DRM) 中经历优先的 BACE1 裂解。 因此，脂筏中的 palAPP 和/或顺式二聚化 palAPP 是潜在有用的药物靶点 对于AD。最近，我们报道了 palAPP 也富含筏状线粒体 - 体外和体内相关 ER 膜 (MAM) 以及 BACE1、γ-分泌酶、 和 ACAT。有趣的是，MAM 功能和 ER-线粒体通讯 家族性和散发性 AD 患者的成纤维细胞显着增加。全面的， 我们的初步研究表明 palAPP 在神经元细胞中合成，包括 神经元过程并在 MAM 中稳定。因此，我们提出以下建议 假设：在 MAM 中稳定的 palAPP 在 神经元细胞和过程。我们将探讨新型 ACAT 抑制剂对 MAM 相关/稳定的 palAPP 中的 palAPP 贩运和处理，包括使用 原代神经元的活细胞成像和细胞表面生物素化测定以及我们的 3D 人类干细胞衍生的神经培养模型。该提案的总体目标是 生成必要的机械和体内数据，以进一步开发新型药物 通过针对 ACAT 和 MAM 相关 palAPP 的 AD 治疗策略。