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是一种罕见的儿童隐性遗传神经系统疾病。这种疾病是由基因突变引起的， Npc1或Npc2。NPC1或NPC2蛋白功能的丧失导致胆固醇的积累 和晚期内体/溶酶体内的鞘脂。这种疾病会导致进行性神经退化， 肝肿大和脾肿大，最终导致早期死亡。目前，这种疾病没有治愈方法。许多 专家认为NPC病是“儿童阿尔茨海默病”。 NPC1和NPC2都与胆固醇结合。这两种蛋白质协同工作，将胆固醇从胃中运输出来。 晚期内体/溶酶体到各种细胞区室，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（ACAT 1）是一种常驻酶， 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显著减慢了胆固醇从晚期的转运速率， 内体/溶酶体到ER。然而，其他人和我们已经表明，大量的胆固醇 可从PM转移到ER，作为ACAT 1酯化的底物，在NPC中 独立的方式。我们假设ACAT1阻断（A1B）导致胆固醇在 胆固醇池转移到其他亚细胞膜上。在突变的NPC细胞中，A1B作用导致 部分满足细胞膜对胆固醇的需求。为了验证这一假设，我们进行了一项小鼠遗传学研究。 实验中，通过培育一种新的NPC病突变小鼠模型和Acat 1基因敲除小鼠。的 结果表明Acat1基因KO能显著延迟突变体的临床发病，延长突变体的寿命 Npc1小鼠34%，部分防止小脑浦肯野神经元丢失， 肝脏和脾脏中的泡沫细胞病理。我们还在细胞培养水平上进行了初步研究， 在突变的NPC1细胞中，A1B，无论是通过使用Acat1 KO还是通过使用有效的小分子ACAT1， 抑制剂，恢复了突触融合蛋白6的错误定位，胆固醇结合t-SNARE，回到TGN。此外，本发明还提供了一种方法， A1B将胆固醇负载的、浮力密度的晚期内体/溶酶体异化成几种亚细胞结构 密度更大。A1 B还恢复了突变NPC 1中观察到的较低的组织蛋白酶D酶活性 细胞在目前的提案中，我们提出了三个具体目标，以进一步调查A1B行动。 目标1。鉴定参与A1B介导的突触融合蛋白6恢复到TGN的元件。 目标二。鉴定参与A1B介导的组织蛋白酶D活性恢复的元件。 目标3.测试脑渗透性小分子ACAT抑制剂在改善NPC疾病中的功效。