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

淀粉样前体蛋白（APP）在β-和γ- 在阿尔茨海默病（AD）中，分泌酶产生淀粉样蛋白β（Aβ）。目前，临床试验 正在进行中，在轻度或前驱AD中使用β或γ分泌酶抑制剂靶向Aβ 患者也正在积极寻求替代的Aβ降低剂。沿着这些 线，我们以前证明酰基辅酶A：胆固醇酰基转移酶， （ACAT）抑制剂，例如CP-113，818和CI-1011，其防止胆固醇转化 转化为胆固醇酯，降低分泌的Aβ水平高达92%，并改善AD样 hAPP转基因小鼠的病理学。ACAT抑制剂尚未在临床上开发 对于AD，很大程度上是因为关于对Aβ生成的影响的分子机制 仍不清楚我们首次证明，大约10%的APP 在体外和体内被棕榈酸修饰。棕榈酰化APP （palAPP）富含富含胆固醇的脂筏，在那里它似乎是作为优选的脂筏。 β-分泌酶（BACE 1）底物与总APP。ACAT抑制降低脂筏 palAPP水平高达76%。我们还报道了~90%的palAPP形成顺式- 在耐洗涤剂膜（DRM）中经历优选BACE 1裂解的二聚体。 因此，脂筏中的palAPP和/或顺式二聚palAPP是潜在有用的药物靶标 对于AD。最近，我们报道palAPP也富含筏状线粒体- 结合ER膜（MAMs）在体外和体内，连同BACE 1，γ-分泌酶， 和ACAT。有趣的是，MAM功能和ER-线粒体通讯是 在家族性和散发性AD患者的成纤维细胞中显著增加。总的来说， 我们的初步研究表明palAPP在神经元细胞中合成， 神经元过程，并在MAMs中稳定。因此，我们提出以下建议 假设：在MAM’s中稳定的palAPP在MAM’s中经历BACE 1切割， 神经元细胞和过程。我们将探讨新的ACAT抑制剂对 MAM相关/稳定的palAPP的palAPP贩运和加工，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 人干细胞衍生的神经培养模型。本提案的总体目标是 产生必要的机制和体内数据，以进一步开发新的 通过靶向ACAT和MAM相关palAPP治疗AD的策略。

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.