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: 10202476
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202476
• 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（Alzheimer's Disease，AD）是最常见的老年痴呆症。它影响了全世界3500万人。晚发性AD（LOAD）涉及多种遗传和环境因素。AD病理学包括缠结、斑块和脂质颗粒在脑中的积累。列举脂质代谢紊乱、内体异常与LOAD相关的三个关键证据：（1）。两种脂质在LOAD的脆弱脑区升高：胆固醇酯和鞘糖脂GM 3。胆固醇酯由胆固醇储存酶酰基辅酶A：胆固醇酰基转移酶1（ACAT 1）产生。GM 3在神经元和其他细胞的质膜（PM）处富集。GM 3的降解通过溶酶体酶神经氨酸酶1（NEU 1）发生。在溶酶体贮积病唾液酸沉积症中，神经氨酸酶有缺陷，导致GM 3积聚。（二）、β淀粉样蛋白的可溶性低聚物形式导致突触丧失，并干扰亚细胞器（包括内体和线粒体）的运输和转运，可能是通过与这些细胞器中存在的富含胆固醇、富含鞘脂的膜微区相互作用。（三）、蛋白ATP结合盒蛋白A1（ABCA 1）在从脑细胞去除过量胆固醇和其他脂质中起关键作用，并控制CNS中主要脂质转运蛋白ApoE的脂化。ApoE 4等位基因是LOAD的主要危险因素。在小鼠模型中，缺乏ABCA 1会导致淀粉样蛋白病，而过表达ABCA 1会减少淀粉样蛋白病。在人类中，ABCA 1的功能缺失突变与AD的高风险相关。出乎意料的是，ABCA 1的表达依赖于溶酶体蛋白酶组织蛋白酶D。因此，LOAD可能被认为是一种涉及异常内体脂质运输的特殊脂质疾病。C型尼曼-皮克病（NPCD）是一种罕见的儿科隐性遗传神经系统疾病。这种疾病导致进行性神经变性、肝肿大、脾肿大，最终导致早期死亡。目前，这种疾病没有治愈方法。这种疾病是由Npc 1或Npc 2突变引起的。NPC 1和NPC 2协同工作，将胆固醇从晚期内体/溶酶体转运到各种细胞区室，包括PM、内体和内质网（ER）。NPC 1或NPC 2的功能丧失导致胆固醇、鞘磷脂、GM 3和GM 2的溶酶体蓄积、内体运动迟缓、溶酶体酶降低和ABCA 1表达降低。在这些方面，NPCD与AD有着惊人的相似性，许多专家将NPC疾病称为“儿童阿尔茨海默病”。ACAT 1是位于ER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NPC 1或NPC 2显著减慢胆固醇从晚期内体/溶酶体到ER的转运速率。然而，大量的胆固醇可以从PM转移到ER作为ACAT 1的底物进行酯化，以NPC非依赖性方式。我们假设ACAT 1阻断（A1 B）导致胆固醇在ER积聚;这种胆固醇池移动到其他亚细胞膜。在突变的NPC细胞中，A1 B作用导致亚细胞器中胆固醇需求的部分满足。为了验证这一假设，我们进行了小鼠遗传学实验，通过培育一种新的NPC疾病突变小鼠模型和Acat 1基因敲除小鼠。结果表明，Acat 1基因KO显着延迟了突变Npc 1小鼠的临床发病，使其寿命延长了34%，部分阻止了小脑浦肯野神经元的丢失，并显着改善了肝脏和脾脏的泡沫细胞病理学。我们还表明，在突变的NPC 1细胞，A1 B，无论是通过使用Acat 1 KO或通过使用一个有效的，小分子ACAT抑制剂，异化成几个亚细胞结构的胆固醇负载晚期内/溶酶体密度较重。A1 B还恢复了较低的组织蛋白酶D酶活性和较低的ABCA 1蛋白;它还通过激活CLEAR途径增加了许多其他溶酶体降解酶的生物合成。为了解释A1 B的作用，我们建立了以下模型：A1 B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，并恢复了各种溶酶体酶和ABCA 1的表达。我们提出了三个具体的目标来测试这个模型，并进一步研究A1 B在体内的作用。目标1。阐明A1 B对突变型小鼠鼻咽癌细胞内体运动的影响机制。a.监测NPC 1相关内体的界膜中的胆固醇含量。B.监测内体运动。目标二。监测突变型NPC细胞和突变型NPC小鼠不同脑区中各种溶酶体酶的mRNA、蛋白质和酶活性。a.监测溶酶体鞘磷脂和降解酶酸性鞘磷脂酶。B.监测溶酶体GM 2和GM 3以及降解酶葡萄糖脑苷脂酶和NEU 1。C.监测溶酶体酶组织蛋白酶D（控制ABCA 1表达）。目标3。测试脑渗透性小分子ACAT抑制剂F12511在改善NPC疾病中的功效，F12511是一种临床测试的候选药物，最初用于治疗动脉粥样硬化。2与公共卫生和AD/ADRD的相关性。NPCD与AD在许多方面具有惊人的相似性。我们的实验室现在有强有力的遗传证据表明，在小鼠模型中，Acat 1基因的失活可以使这两种疾病受益。这项提案的结果可以提供一个新的火花，这是需要治疗NPC疾病和AD，以及其他ADRD。

项目成果

Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

• DOI: 10.3390/ijms24065525
• 发表时间: 2023-03-14
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: Harned, Taylor C.;Stan, Radu V.;Cao, Ze;Chakrabarti, Rajarshi;Higgs, Henry N.;Chang, Catherine C. Y.;Chang, Ta Yuan
• 通讯作者: Chang, Ta Yuan