Alleviating lysosomal lipid defects in ADRD by blocking cholesterol storage

阿尔茨海默病(AD)是成人中最常见的痴呆。它影响着全球3500万人。晚发性阿尔茨海默病(LOAD)涉及多种遗传和环境因素。AD病理包括脑内缠结、斑块和脂肪颗粒的堆积。引用三个将脂代谢紊乱--内稳态、内体异常与负荷联系起来的关键证据：(1)。两种脂类物质在易受负荷的脑区升高：胆固醇酯和神经鞘糖脂GM3。胆固醇酯是由胆固醇储存酶酰基辅酶A：胆固醇酰基转移酶1(ACAT1)产生的。GM3在神经元和其他细胞的质膜(PM)中富含。GM3的降解是通过溶酶体酶神经氨酸酶1(Neu1)进行的。在溶酶体储存疾病唾液化症中，神经氨酸酶缺陷导致GM3积聚。(2)。可溶的低聚形式的淀粉样β蛋白会导致突触丢失，并干扰亚细胞细胞器的运输和运输，包括内小体和线粒体，可能是通过与这些细胞器中存在的富含胆固醇、鞘磷脂的膜微域相互作用而实现的。(3)。蛋白三磷酸腺苷结合盒蛋白A1(ABCA1)在清除脑细胞中多余的胆固醇和其他脂质方面起着关键作用，并控制着中枢主要脂质运输蛋白ApoE的脂化。除年龄因素外，载脂蛋白E4等位基因也是负荷的主要危险因素。在小鼠模型中，缺乏ABCA1会加重淀粉样变性，而过表达ABCA1则会减少淀粉样变性。在人类中，ABCA1功能丧失突变与AD的高风险相关。出乎意料的是，ABCA1的表达依赖于溶酶体组织蛋白酶D。因此，LOAD可能被认为是一种特殊的脂类疾病，涉及到内体脂质的异常转运。尼曼-皮克C型疾病(NPCD)是一种罕见的儿科、遗传隐性神经疾病。这种疾病会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，这种疾病还没有治愈的方法。这种疾病是由NPC1或NPC2的突变引起的。NPC1和NPC2协同工作将胆固醇从晚期的内小体/溶酶体输送到不同的细胞室，包括PM、内小体和内质网(ER)。NPC1或NPC2功能丧失导致溶酶体胆固醇、鞘磷脂、GM3和GM2积聚，内体运动迟缓，溶酶体酶活性降低，ABCA1表达降低。在这些方面，NPCD与AD有着惊人的相似之处，许多专家将鼻咽癌疾病称为儿童阿尔茨海默病。ACAT1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。缺乏功能的NPC1或NPC2会显著减缓胆固醇从晚期内切/溶酶体到内质网的转运速度。然而，大量的胆固醇可以不依赖于NPC的方式从PM转移到作为ACAT1酯化底物的ER。我们假设ACAT1阻断(A1B)导致胆固醇在内质网积聚；这个胆固醇池移动到其他亚细胞膜。在突变的鼻咽癌细胞中，A1B作用导致亚细胞细胞器部分满足胆固醇需求。为了验证这一假设，我们进行了一项小鼠遗传学实验，通过培育一种新的鼻咽癌疾病突变小鼠模型和Acat1基因KO小鼠。结果表明，Acat1基因KO显著延缓了NPC1突变小鼠的临床发病，延长了NPC1突变小鼠的寿命34%，部分阻止了小脑浦肯野神经元的丢失，并显著改善了肝和脾中泡沫细胞的病理变化。我们还表明，在突变的NPC1细胞中，A1B无论是通过使用Acat1KO还是通过使用有效的小分子ACAT抑制剂，都会将富含胆固醇的晚期内切/溶酶体异化为几个密度更高的亚细胞结构。A1B还恢复了较低的组织蛋白酶D酶活性和较低的ABCA1蛋白；它还通过激活透明途径增加了许多其他溶酶体降解酶的生物合成。为了解释A1B的作用，我们建立了以下模型：A1B恢复各种膜细胞器的膜胆固醇含量，包括内体的限制膜。这些作用恢复了内膜体的运动，导致管腔内溶酶体中胆固醇和其他脂质含量的下降，并恢复了各种溶酶体酶和ABCA1的表达。我们提出了三个具体目标来测试这一模型，并进一步研究A1B在体内的作用。目的1.阐明A1B对突变型小鼠鼻咽癌细胞内膜运动的影响机制。A.监测NPC1相关内体限制膜中的胆固醇含量。B.监测内质体动能。目的2.检测突变的鼻咽癌细胞和突变的鼻咽癌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GM2和GM3，以及降解酶葡萄糖脑苷酶和NEU1。C.监测溶酶体酶组织蛋白酶D(控制ABCA1表达)。目的3.脑透性小分子ACAT抑制剂F12511是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，它在改善鼻咽癌疾病方面的疗效。2与公共卫生和AD/ADRD的相关性。在几个方面，NPCD与AD有着惊人的相似之处。我们的实验室现在有强有力的遗传学证据表明，在小鼠模型中，灭活Acat1基因对这两种疾病都有好处。这项提议的结果可以提供新的火花，这是治疗鼻咽癌疾病和AD以及其他ADRDS所需的。

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.