Alleviating lysosomal lipid defects in ADRD by blocking cholesterol storage

通过阻断胆固醇储存来缓解 ADRD 中的溶酶体脂质缺陷

• 批准号: 9933635
• 负责人: Ta Yuan CHANG
• 金额: $ 24.76万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9933635
• 关键词: Acyl Coenzyme A; Alzheimer' s Disease; Alzheimer' s disease related dementia; Back; Binding; Brain; Breeding; Cathepsins; Cell Culture Techniques; Cell membrane; Cells; Cerebellum; Cessation of life; Childhood; Cholesterol; Cholesterol Esters; Clinical; Defect; Disease; Elements; Endoplasmic Reticulum; Enzymes; Esterification; Foam Cells; Genes; Genetic; Golgi Apparatus; Hepatomegaly; Knockout Mice; Lipids; Liver; Longevity; Lysosomes; Mediating; Membrane; Mus; Mutation; NPC1 gene; Nuclear Pore Complex; Outcome; Pathology; Permeability; Pilot Projects; Proteins; Sphingolipids; Spleen; Splenomegaly; Sterol O-Acyltransferase; Subcellular structure; Supraoptic Vertical Ophthalmoplegia; Testing; Work; density; efficacy testing; enzyme activity; experimental study; improved; inhibitor/antagonist; late endosome; loss of function; mutant; mutant mouse model; nervous system disorder; neuron loss; prevent; progressive neurodegeneration; restoration; small molecule; sterol O-acyltransferase 1; syntaxin 6; target SNARE proteins; therapeutic candidate; therapeutic target

NPC is a rare, pediatric, genetically recessive neurological disease. The disease is caused by mutations in either Npc1 or Npc2. Loss of function in either the NPC1 or NPC2 protein results in accumulation of cholesterol and sphingolipids within the late endosomes/lysosomes. This disease causes progressive neurodegeneration, hepatomegaly and splenomegaly, and ultimately early death. Currently, this disease has no cure. Many experts consider NPC disease as “childhood Alzheimer’s disease”. Both NPC1 and NPC2 bind to cholesterol. These 2 proteins work in concert to transport cholesterol out of the late endosomes/lysosomes to various cellular compartments, including plasma membrane (PM), Golgi, and endoplasmic reticulum (ER). Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme located at the ER. It utilizes cholesterol arriving at the ER as substrate to produce cholesteryl esters. Lacking functional NPC1 or NPC2 considerably slows the transport rate of cholesterol from the late endosomes/lysosomes to the ER. However, others and we have shown that, significant amount of cholesterol can translocate from the PM to the ER as serve as the substrate for ACAT1 for esterification, in NPC independent manner. We hypothesize that ACAT1 blockage (A1B) causes cholesterol to accumulate at the ER; this cholesterol pool moves to other subcellular membranes. In mutant NPC cells, the A1B action leads to partial fulfillment of cholesterol needs in membranes. To test this hypothesis, we conducted a mouse genetic experiment, by breeding a new mutant mouse model for NPC disease and the Acat1 gene KO mouse. The results show that Acat1 gene KO significantly delayed the clinical onset, prolonged the lifespan of the mutant Npc1 mouse by 34%, partially prevented Purkinje neuron loss in the cerebellum, and significantly improved foam cell pathology in the liver and spleen. We also performed pilot studies at the cell culture level and showed that, in mutant NPC1 cells, A1B, either by using Acat1 KO or by using a potent, small molecule ACAT1 inhibitor, restored the mislocalization of syntaxin 6, a cholesterol binding t-SNARE, back to TGN. In addition, A1B dissimilates the cholesterol laden, buoyant density late endo/lysosomes into several subcellular structures with heavier densities. A1B also restored the lower cathepsin D enzyme activity observed in the mutant NPC1 cells. In the current proposal, we propose three specific aims to further investigate the A1B actions. Aim 1. Identify elements involved in the A1B mediated restoration of syntaxin 6 back to TGN. Aim 2. Identify elements involved in the A1B mediated restoration in cathepsin D activity. Aim 3. Test efficacy of a brain permeable small molecule ACAT inhibitor in ameliorating NPC disease.

NPC是一种罕见的，儿科，遗传性隐性神经系统疾病。该疾病是由突变引起的 NPC1或NPC2。 NPC1或NPC2蛋白的功能损失会导致胆固醇的积累 以及晚期内体/溶酶体内的鞘脂。这种疾病会导致进行性神经退行性， 肝肿大和脾肿大，最终是早期死亡。目前，这种疾病无法治愈。许多 专家认为NPC疾病是“童年时期的阿尔茨海默氏病”。 NPC1和NPC2都与胆固醇结合。这两种蛋白质共同运输于 晚期内体/溶酶体到各种细胞室，包括质膜（PM），高尔基体和 内质网（ER）。酰基辅酶A：胆固醇酰基转移酶1（ACAT1）是一种居民酶 位于急诊室。它利用到达ER的胆固醇作为底物产生胆固醇酯。缺乏 功能性NPC1或NPC2大大减慢了晚期胆固醇的运输速率 内体/溶酶体向ER。但是，其他人也表明，大量胆固醇 可以从PM转移到ER，作为ACAT1进行酯化的底物，在NPC中 独立的方式。我们假设ACAT1阻塞（A1b）导致胆固醇在 er;该胆固醇池移至其他亚细胞膜。在突变的NPC细胞中，A1b的作用导致 膜中胆固醇需求的部分满足。为了检验该假设，我们进行了小鼠通用 实验，通过繁殖用于NPC疾病和ACAT1基因KO小鼠的新型突变小鼠模型。这 结果表明，ACAT1基因KO显着延迟了临床发作，延长了突变体的寿命 NPC1小鼠占34％，部分阻止了小脑的Purkinje神经元丧失，并显着改善 肝脏和脾脏中的泡沫细胞病理学。我们还在细胞培养水平进行了试点研究，并显示 在突变的NPC1细胞中，A1b，通过使用ACAT1 KO或使用电势小分子ACAT1 抑制剂，恢复了语法6（胆固醇结合的T-nare）的错误定位，回到TGN。此外， A1b将胆固醇女士降低，浮力密度晚期/溶酶体成几个亚细胞结构 较重的密度。 A1b还恢复了在突变NPC1中观察到的下部组织蛋白酶D酶活性 细胞。在当前的提案中，我们提出了三个特定的目的，以进一步研究A1B的行动。 AIM 1。确定与A1b介导的语法恢复6回到TGN有关的元素。 AIM 2。确定在组织蛋白酶D活动中涉及的A1b介导的恢复的元素。 AIM 3。可渗透的小分子ACAT抑制剂在改善NPC疾病中的测试效率。