Alleviating lysosomal lipid defects in ADRD by blocking cholesterol storage

鼻咽癌是一种罕见的儿科遗传隐性神经系统疾病。这种疾病是由基因突变引起的 NPC1或NPC2。NPC1或NPC2蛋白功能的丧失会导致胆固醇的积累 晚期内体/溶酶体中的鞘磷脂。这种疾病会导致进行性神经变性， 肝和脾肿大，最终过早死亡。目前，这种疾病还没有治愈的方法。许多 专家认为鼻咽癌是“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，将胆固醇运出 晚期内小体/溶酶体到各种细胞室，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(ACAT1)是一种常驻酶 位于急诊室。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NPC1或NPC2显著减缓晚期胆固醇的转运速度 内质体/溶酶体到内质网。然而，其他人和我们已经表明，大量的胆固醇 在鼻咽癌中，可以从PM转运到ER作为ACAT1酯化的底物 以独立的方式。我们假设ACAT1阻断(A1B)导致胆固醇在 呃，这个胆固醇池转移到了其他亚细胞膜上。在突变的鼻咽癌细胞中，A1B作用导致 膜中胆固醇的部分满足需求。为了验证这一假设，我们进行了一种小鼠基因 实验，通过培育新的鼻咽癌基因突变小鼠模型和Acat1基因KO小鼠。这个 结果表明，Acat1基因KO显著延迟了突变体的临床发病，延长了患者的生存期 NPC1小鼠减少34%，部分阻止了小脑浦肯野神经元的丢失，并明显改善 肝和脾的泡沫细胞病理。我们还在细胞培养水平上进行了初步研究，并显示 在突变的NPC1细胞中，通过使用Acat1 KO或使用有效的小分子ACAT1，A1B 抑制剂，使胆固醇结合的t-陷阱--Synaxin 6的错误定位恢复到TGN。此外, A1B使胆固醇含量高、密度高的晚期内切/溶酶体分化为几种亚细胞结构 密度更大。A1B还恢复了在突变体NPC1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，我们提出了进一步调查A1B行动的三个具体目标。 目的1.确定A1B介导的Synaxin 6恢复为TGN所涉及的元件。 目的2.确定A1B介导的组织蛋白酶D活性恢复的元件。 目的3.检测脑渗透性小分子ACAT抑制剂改善鼻咽癌疾病的疗效。

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.