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: 9977871
• 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/9977871
• 关键词: Adult; Affect; Aging; Alleles; Alzheimer' s Disease; Alzheimer' s disease pathology; 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; 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 genetics; mouse model; mutant; mutant mouse model; nervous system disorder; neuron loss; overexpression; prevent; progressive neurodegeneration; replication factor C; 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.

阿尔茨海默病 (AD) 是成年人中最常见的痴呆症，它影响着全球 3500 万人。 AD 病理学包括大脑中的缠结、斑块和脂质颗粒的积累。引用了三个关键证据，将脂质稳态失衡、内体异常与 LOAD 联系起来：(1) 两种脂质在 LOAD 的易受影响的大脑区域中升高：胆固醇酯和脂质。鞘糖脂 GM3 由胆固醇储存酶酰基辅酶 A：胆固醇酰基转移酶 1 (ACAT1) 产生，GM3 富集于神经元和其他细胞的质膜 (PM)，溶酶体酶神经氨酸酶 1 会降解 GM3。 NEU 1). 在溶酶体贮积病唾液酸贮积症中，神经氨酸酶存在缺陷，导致 GM3 积聚，导致突触丢失并干扰亚细胞细胞器（包括内体和线粒体）的运输和运输 (2)。这些细胞器中存在的微结构域 ATP 结合盒蛋白 A1 (ABCA1) 在去除多余胆固醇和其他脂质方面发挥着关键作用。 ApoE4 等位基因是 LOAD 的主要危险因素，在小鼠模型中，缺乏 ABCA1 会加重淀粉样变性，而过度表达 ABCA1 会减少人类淀粉样变性。 ABCA1 的功能缺失突变与 AD 的高风险相关，但意想不到的是，ABCA1 的表达依赖于溶酶体蛋白酶组织蛋白酶。 D. 因此，LOAD 可能被认为是一种特殊的脂质疾病，涉及异常的内体脂质运输（NPCD），是一种罕见的儿科遗传隐性神经系统疾病，这种疾病会导致进行性神经变性、肝肿大、脾肿大。目前，这种疾病尚无治愈方法。该疾病是由 Npc1 或 Npc2 协同作用将胆固醇排出体外引起的。核内体/溶酶体进入各种细胞区室，包括 PM、核内体和内质网 (ER)。 NPC1 或 NPC2 功能丧失导致胆固醇、鞘磷脂、GM3 和 GM2 的溶酶体积累、内体运动迟缓、溶酶体酶降低和降低。在这些方面，NPCD 与 AD 有着惊人的相似之处。专家认为NPC疾病是“儿童阿尔茨海默病”，它是一种位于内质网的常驻酶，它利用到达内质网的胆固醇作为底物来产生胆固醇酯，缺乏功能性的NPC1或NPC2会大大减慢后期胆固醇的转运速度。然而，大量的胆固醇可以从 PM 转移到 ER，作为 ACAT1 酯化的底物。我们帮助 ACAT1 阻断 (A1B) 导致胆固醇在 ER 处积聚；在突变的 NPC 细胞中，A1B 的作用导致部分满足了亚细胞器的胆固醇需求。检验这一假设，我们进行了小鼠基因实验，通过培育新的鼻咽癌突变小鼠模型和Acat1基因KO小鼠，结果显示Acat1基因KO显着推迟了临床发病，延长了病程。突变型 Npc1 小鼠的寿命延长了 34%，部分防止了小脑中的浦肯野神经元损失，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，在突变型 NPC1 细胞中，A1B 通过使用 Acat1 KO 或使用一种有效的小分子 ACAT 抑制剂，可将富含胆固醇的晚期内切酶/溶酶体异化为密度较高的几个亚细胞结构，也可恢复较低的 A1B 水平。组织蛋白酶 D 酶活性和较低的 ABCA1 蛋白；它还通过激活 CLEAR 途径增加许多其他溶酶体降解酶的生物合成。为了解释 A1B 的作用，我们制定了以下模型：A1B 恢复各种膜胆固醇含量。膜细胞器，包括内体的限制膜，这些作用恢复内体运动并导致胆固醇和其他脂质的管腔溶酶体含量减少，并恢复我们提出了三个具体目标来测试该模型并进一步研究 A1B 的体内作用。 目的 1. 阐明 A1B 对突变小鼠 NPC 细胞内体运动的机制。 NPC1 相关内体的限制膜 b. 监测内体运动。 目的 2. 监测各种溶酶体酶的 mRNA、蛋白质和酶活性。在突变的 NPC 细胞中，以及在突变的 NPC 小鼠的各个大脑区域中，监测溶酶体鞘磷脂，以及降解酶 GM2 和 GM3，以及降解酶葡萄糖脑苷脂酶和 NEU1。组织蛋白酶 D（控制 ABCA1 表达） 目标 3. 测试 a 的功效。脑渗透性小分子 ACAT 抑制剂 F12511，一种经过临床测试的候选药物，最初用于治疗动脉粥样硬化，改善 NPC 疾病。 在多个方面，NPCD 与 AD 具有惊人的相似性。有强有力的遗传证据表明，在小鼠模型中，灭活 Acat1 基因可以使这两种疾病受益。该提案的结果可以提供治疗这两种疾病所需的新火花。鼻咽癌疾病和 AD，以及其他 ADRD。