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: 9789810
• 负责人: Ta Yuan CHANG
• 金额: $ 41万
• 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/9789810
• 关键词: Adult; Affect; Aging; Alleles; Alzheimer' s Disease; Alzheimer' s disease related dementia; Alzheimer' s disease risk; Amyloid beta-Protein; Apolipoprotein E; Atherosclerosis; Back; Binding; Biogenesis; Brain; Brain region; Breeding; Carrier Proteins; Cathepsins; Cell membrane; Cells; Cerebellum; Cessation of life; Childhood; Cholesterol; Cholesterol Esters; Clinical; Cytoplasmic Granules; Defect; Dementia; Disease; Elements; Endoplasmic Reticulum; Endosomes; Environmental Risk Factor; Enzymes; Esterification; Foam Cells; Genes; Genetic; Glycosphingolipids; Hepatomegaly; Homeostasis; Human; Knockout Mice; Late Onset Alzheimer Disease; Link; Lipids; Lipomucopolysaccharidoses; Liver; Longevity; Lysosomal Storage Diseases; Lysosomes; Mediating; Membrane; Membrane Microdomains; Messenger RNA; Mitochondria; Modeling; Monitor; Mus; Mutant Strains Mice; Mutation; NPC1 gene; Neuraminidase; Neurofibrillary Tangles; Neurons; Nuclear Pore Complex; Organelles; Outcome; Pathology; Pathway interactions; Peptide Hydrolases; Permeability; Play; Proteins; Public Health; Risk Factors; Sphingolipids; Sphingomyelins; Spleen; Splenomegaly; Sterol O-Acyltransferase; Subcellular structure; Supraoptic Vertical Ophthalmoplegia; Synapses; Testing; Ursidae Family; Work; acid sphingomyelinase; activator 1 protein; apolipoprotein E-4; brain cell; cell motility; density; drug candidate; efficacy testing; endosome membrane; enzyme activity; experimental study; glucosylceramidase; high risk; improved; in vivo; inhibitor/antagonist; late endosome; lipid transport; loss of function; loss of function mutation; mouse model; mutant; mutant mouse model; nervous system disorder; neuron loss; overexpression; prevent; progressive neurodegeneration; protein transport; restoration; small molecule; sterol O-acyltransferase 1; syntaxin 6; therapeutic candidate; therapeutic target; trafficking

Alzheimer’s Disease (AD) is the most prevalent dementia in the adults. It affects 35 million worldwide. Late onset AD (LOAD) involves multiple genetic and environmental factors. AD pathology includes accumulation of tangles, plaques, and lipid granules in the brain. To cite three key evidences that link lipid dys-homeostasis, endosomal abnormality with LOAD: (1). Two lipid species were elevated in vulnerable brain region of LOAD: cholesteryl esters, and the glycosphingolipid GM3. Cholesteryl esters are produced by the cholesterol storage enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1). GM3 is enriched at the plasma membranes (PM) of neurons and other cells. Degradation of GM3 occurs by the lysosomal enzyme neuraminidase 1 (NEU 1). In the lysosomal storage disease sialidosis, neuraminidase is defective causing GM3 to accumulate. (2). The soluble, oligomeric form of amyloid beta causes synapse loss and interferes with the trafficking and transport of subcellular organelles, including endosomes and mitochondria, presumably by interacting with the cholesterol rich, sphingolipid rich membrane microdomains present in these organelles. (3). The protein ATP binding cassette protein A1 (ABCA1) plays a key role in removing excess cholesterol and other lipids from brain cells, and controls the lipidation of ApoE, the major lipid transport protein in the CNS. The ApoE4 allele is the major risk factor for LOAD besides aging. In mouse models, lacking ABCA1 worsens amyloidopathy while overexpressing ABCA1 reduces amyloidopathy. In humans, a loss-of-function mutation in ABCA1 is associated with high risk of AD. Unexpectedly, expression of ABCA1 depends on the lysosomal protease cathepsin D. Thus, LOAD may be considered as a special lipid disease that involves abnormal endosomal lipid trafficking. Niemann-Pick Type C Disease (NPCD) is a rare, pediatric, genetically recessive neurological disease. This disease causes progressive neurodegeneration, hepatomegaly, splenomegaly, and ultimately early death. Currently, this disease has no cure. The disease is caused by mutations in either Npc1 or Npc2. NPC1 and NPC2 work in concert to transport cholesterol out of the late endosomes/lysosomes to various cellular compartments, including PM, endosomes, and endoplasmic reticulum (ER). Loss of function in NPC1 or NPC2 results in lysosomal accumulation of cholesterol, sphingomyelin, GM3 and GM2, sluggish endosomal motility, lower lysosomal enzymes, and lower expression of ABCA1. In these aspects, NPCD bear striking resemblances with AD, and many experts consider NPC disease as “childhood Alzheimer’s disease”. 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, significant amount of cholesterol can translocate from the PM to the ER as the substrate for ACAT1 for esterification, in an 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 subcellular organelles. 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 show that in mutant NPC1 cells, A1B, either by using Acat1 KO or by using a potent, small molecule ACAT inhibitor, dissimilates the cholesterol laden late endo/lysosomes into several subcellular structures with heavier densities. A1B also restored the lower cathepsin D enzyme activity and the lower ABCA1 protein; it also increases biogenesis of many other lysosomal degradation enzymes, through activation of the CLEAR pathway. To account for the actions of A1B, we formulate the following model: A1B restores the membrane cholesterol contents of various membrane organelles, including the limiting membrane of the endosomes. These effects restore endosomal motility and causes a decrease in luminal lysosomal contents of cholesterol and other lipids, and restores the expressions of various lysosomal enzymes and ABCA1. We propose three specific aims to test this model and to further investigate A1B actions in vivo. Aim 1. Elucidate the mechanism of A1B on endosomal motility in mutant mouse NPC cells. a. Monitor cholesterol content in the limiting membrane of NPC1-associated endosomes. b. Monitor the endosomal motility. Aim 2. Monitor the mRNA, protein, and enzyme activity of various lysosomal enzymes in mutant NPC cells, and in various brain regions of the mutant NPC mouse. a. Monitor lysosomal sphingomyelin, and the degrading enzyme acid sphingomyelinase. b. Monitor lysosomal GM2 and GM3, and the degrading enzymes glucocerebrosidase and NEU1. c. Monitor the lysosomal enzyme cathepsin D (that controls ABCA1 expression). Aim 3. Test efficacy of a brain permeable small molecule ACAT inhibitor F12511, a clinically tested candidate drug originally intended to treat atherosclerosis, in ameliorating NPC disease. 2 Relevance to Public Health, and to AD/ADRD. In several aspects, NPCD bears striking resemblances with AD. Our lab now has strong genetic evidence that in mouse models, inactivating the Acat1 gene can benefit both diseases. The outcome of this proposal can provide a fresh spark, that is needed to treat both NPC disease and AD, as well as other ADRDs.

阿尔茨海默氏病（AD）是成年人中最普遍的痴呆症。它影响了全球3500万。晚期AD（负载）涉及多种遗传和环境因素。 AD病理包括大脑中缠结，斑块和脂质颗粒的积累。为了引用三个关键证据，可以将脂质dys-homeostasis联系起来，内体异常与负载：（1）。在脆弱的脑部脑区域中，两种脂质物种升高：胆固醇酯和糖磷脂GM3。胆固醇储存酶酰基-COA：胆固醇酰基转移酶1（ACAT1）产生胆固醇酯。 GM3富含神经元和其他细胞的质膜（PM）。 GM3的降解是由溶酶体酶神经酶1（NEU 1）发生的。在溶酶体储存疾病唾液酸病中，神经瘤酶有缺陷，导致GM3积累。 （2）。淀粉样蛋白β的可溶性，寡聚形式会导致突触丧失，并干扰包括内体和线粒体在内的细胞细胞器的贩运和运输，这可能是通过与富含胆固醇的，辛辛糖脂富含浓度的膜膜微粒子相互作用。 （3）。蛋白ATP结合盒蛋白A1（ABCA1）在去除多余的胆固醇和其他脂质中起着关键作用，并控制了CNS中主要的脂质转运蛋白APOE的脂质化。除衰老外，APOE4等位基因是负载的主要危险因素。在小鼠模型中，缺乏ABCA1会使淀粉样病变恶化，而过表达ABCA1会降低淀粉样病。在人类中，ABCA1的功能丧失突变与AD的高风险有关。出乎意料的是，ABCA1的表达取决于溶酶体蛋白组织蛋白酶D.，该负载可能被认为是一种涉及异常内体脂质运输的特殊脂质疾病。 Niemann-Pick型C疾病（NPCD）是一种罕见的，儿科，遗传性神经系统疾病。这种疾病会导致进行性神经变性，肝肿大，脾肿大，并最终早期死亡。目前，这种疾病无法治愈。该疾病是由NPC1或NPC2突变引起的。 NPC1和NPC2协同工作，将胆固醇从晚期的内体/溶酶体传输到各种细胞室，包括PM，内体和内质网（ER）。 NPC1或NPC2中功能的损失导致胆固醇，鞘磷脂，GM3和GM2的溶酶体积累，内体运动缓慢，较低的溶酶体酶以及ABCA1的较低表达。在这些方面，NPCD与AD相似，许多专家将NPC疾病视为“阿尔茨海默氏病”。 ACAT1是位于急诊室的常驻酶。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认为降低了从晚期内体/溶酶体到ER的胆固醇的转运率。但是，大量胆固醇可以从PM转换为ER作为ACAT1的底物进行酯化，以NPC独立的方式。我们假设ACAT1阻塞（A1b）会导致胆固醇在ER处积聚。该胆固醇池移至其他亚细胞膜。在突变的NPC细胞中，A1b的作用导致亚细胞细胞器中胆固醇需求的部分满足。为了检验这一假设，我们通过育种新的NPC疾病和ACAT1基因KO小鼠的新突变小鼠模型进行了小鼠基因实验。结果表明，ACAT1基因KO显着延迟了临床发作，将突变体NPC1小鼠的寿命延长了34％，部分阻止了小脑Purkinje神经元的丧失，并显着改善了肝脏和脾脏中的泡沫细胞病理学。我们还表明，在突变的NPC1细胞A1B中，无论是使用ACAT1 KO还是使用潜在的小分子ACAT抑制剂，将载有胆固醇的胆固醇降低到具有较重密度的几个亚细胞结构中。 A1b还恢复了下部组织蛋白酶D酶活性和下ABCA1蛋白。它还通过激活透明途径来增加许多其他溶酶体降解酶的生物发生。为了说明A1b的作用，我们制定了以下模型：A1B恢复各种膜细胞器的膜胆固醇含量，包括内体的限制膜。这些作用恢复了内体运动，并导致胆固醇和其他脂质的腔溶酶体含量减少，并恢复各种溶酶体酶和ABCA1的表达。我们提出了三个特定的目的，以测试该模型并进一步研究体内A1b的作用。 AIM 1。阐明A1b在突变小鼠NPC细胞中内体运动的机制。一个。在NPC1相关的内体运动的极限膜中监测胆固醇含量。 b。监视内体运动。 AIM 2。监测突变NPC细胞中各种溶酶体酶的mRNA，蛋白质和酶活性，以及​​在突变NPC小鼠的各个大脑区域中。一个。监测溶酶体鞘磷脂和降解酶酸鞘磷脂酶。 b。监测溶酶体GM2和GM3，以及降解酶葡萄糖辣椒酶和NEU1。 c。监测溶酶体酶组织蛋白酶D（控制ABCA1表达）。 AIM 3。大脑可渗透小分子ACAT抑制剂F12511的测试效率，这是一种临床测试的候选药物，最初旨​​在治疗动脉粥样硬化，以改善NPC疾病。 2与公共卫生以及广告/ADRD相关。在一些方面，NPCD与AD相似。现在，我们的实验室有强有力的遗传证据，即在小鼠模型中灭活ACAT1基因可以使两种疾病受益。该提案的结果可以提供新的火花，这是治疗NPC疾病和AD以及其他ADRD所需的。