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

淀粉样前体蛋白(APP)经历β-和γ-的连续蛋白降解 阿尔茨海默病(AD)中产生淀粉样蛋白(Aβ，Aβ)的分泌酶。目前，临床试验 正在用β-或β-分泌酶抑制剂靶向Aγ治疗轻度或前驱AD 病人。另一种A类β降低剂也在积极寻找。沿着这些 株，我们先前证明了酰基辅酶A：胆固醇酰基转移酶 (ACAT)抑制剂，如CP-113,818和CI-1011，可防止胆固醇转化 转化为胆固醇酯，使分泌的Aβ水平降低高达92%，并改善类AD HAPP转基因小鼠的病理学。ACAT抑制剂尚未在临床上开发出来 对于AD，很大程度上是因为影响β生成的分子机制 目前仍不清楚。我们首次展示了大约10%的APP 在体外和体内被棕榈酸翻译后修饰。Palmitoylated应用程序 (PalAPP)富含高胆固醇的脂筏，似乎是首选的 β-分泌酶底物(BACE1)与总APP。ACAT抑制减少脂筏 PalAPP水平最高可达76%。我们还报告了~90%的PalAPP形成顺式- 在耐洗涤剂膜(DRM)中发生BACE1优先裂解的二聚体。 因此，脂筏中的PalAPP和/或顺式二聚化的PalAPP是潜在的有用的药物靶点 对于AD。最近，我们报道了PalAPP也富含筏状线粒体- 体内外相关ER膜(MAM)与BACE1、γ分泌酶、 和ACAT。有趣的是，MAM功能和内质网-线粒体通讯 家族性和散发性AD患者成纤维细胞显著增加。总的来说， 我们的初步研究表明，PalAPP是在神经细胞中合成的，包括 神经元突起，并在巨噬细胞瘤中稳定。因此，我们提出如下建议 假设：稳定在MAM中的PalAPP经历BACE1裂解 神经细胞和突起。我们将探索新型ACAT抑制剂对ACAT的影响 MAM相关/稳定的PalAPP的贩运和加工，包括使用 在原代神经元和我们的3D中的活细胞成像和细胞表面生物素化分析的 人类干细胞来源的神经培养模型。这项提议的首要目标是 产生必要的机械性和活体数据，以进一步开发新的 通过靶向ACAT和MAM相关的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.