ABCA1 knock-in mouse model to study molecular pathology of Alzheimer's Disease

描述（申请人提供）：研究表明，阿尔茨海默病（AD）患者的动脉粥样硬化比年龄匹配的非AD对照组更严重，并建立了AD患者脑动脉粥样硬化疾病程度与AD典型神经病理学特征之间的正相关性。然而，胆固醇（CL）代谢影响AD发病机制仍不确定。CL流出和HDL的产生由ABCA 1膜转运蛋白介导，ABCA 1膜转运蛋白由肝X核受体（LXR）转录控制。ABCA 1突变导致严重的HDL缺乏，如低脂蛋白血症和丹吉尔病（TD）。本研究的一个主要目标是揭示ABCA 1在AD分子发病机制中的作用，从而探索和测试新的治疗策略。核心假设是ABCA 1影响ABeta形成/沉积和清除。支持我们的研究（目前正在出版）由NIA R21和R 03奖资助，表明APP 23小鼠中ABCA 1缺乏导致可溶性apoE急剧减少和ABeta在大脑中沉积增加。我们还证明，体内应用合成的LXR配体T0901317（TO）增加了CNS中ABCA 1的表达，并降低了脑中ABeta种类的量。我们发现TD患者的原代细胞产生并分泌更多的ABeta，并且它们对LXR合成配体的反应是不同的，并且取决于ABCA 1突变的类型。重要的是，具有引起N935 S氨基酸取代的点突变的主要ABCA 1突变细胞系之一是从患有痴呆和脑中大量淀粉样蛋白沉积但没有心血管病理学的TD患者中建立的。因此，ABCA 1中的特定突变差异干扰其对β-淀粉样蛋白沉积和动脉粥样硬化的调节作用，因此我们假设ABCA 1突变通过至少部分不同的机制确定在患者中观察到的临床表型。这项NIA试点资助的目标是开发和表征表达突变ABCA 1 N935 S的小鼠品系。目标1.创造基因工程小鼠表达突变ABCA 1 N935 S。该小鼠系将通过利用“敲入”方法在小鼠ES细胞中的ABCA 1基因座中引入N935 S突变来创建。这些ABCA 1 S/S小鼠将普遍表达ABCA 1 N935 S，突变基因将保持在内源性ABCA 1转录控制下，突变蛋白将取代野生型ABCA 1。目标二。表征ABCA 1 N935 S基因敲入小鼠。我们将通过测量血浆CL和脂蛋白来分析ABCA 1 N935 s的体内表达及其作为胆固醇转运蛋白的功能。将确定突变的ABCA 1 N93 ss对脑脂蛋白和内源性APR加工以及体内ABeta生成的影响，作为初步步骤，以证实未来详细的生化研究。

DESCRIPTION (provided by applicant): Studies have now revealed that Alzheimer's disease (AD) patients have more severe atherosclerosis than age-matched controls without AD, and established a positive correlation between the degree of atherosclerotic disease of cerebral arteries in AD patients and neuropathological features typical for AD. However, mechanisms by which cholesterol (CL) metabolism influences AD pathogenesis remain uncertain. CL efflux and generation of HDL are mediated by ABCA1 membrane transporter which is transcriptionally controlled by Liver X nuclear Receptors (LXR). ABCA1 mutations cause severe HDL deficiencies, like hypoalphalipoproteinemia and Tangier Disease (TD). A major goal of our research is to reveal the role of ABCA1 in the molecular pathogenesis of AD and thus to explore and to test new therapeutic strategies. The central hypothesis is that ABCA1 affects ABeta formation/deposition and clearance. In support are our studies (currently in press) funded by NIA R21 and R03 awards, showing that ABCA1 deficiency in APP23 mice leads to a dramatic decrease of soluble apoE and an increased deposition of ABeta in the brain. We have also demonstrated that application of the synthetic LXR ligand T0901317 (TO) in vivo increases the expression of ABCA1 in CNS and decreases the amounts of ABeta species in the brain. We found that primary cells from TD patients generate and secrete more ABeta and that their response to LXR synthetic ligands is different and depend on the type of ABCA1 mutation. Importantly, one of the primary ABCA1 mutant cell lines with a point mutation causing N935S amino acid substitution was established from a TD patient with dementia and abundant amyloid deposits in the brain, but without cardiovascular pathology. Thus, specific mutations in ABCA1 differentially disturb its regulatory role on beta-amyloid deposition and atherosclerosis, and therefore we hypothesize that ABCA1 mutations determine the clinical phenotype observed in patients by at least partly different mechanisms. The goal of this NIA pilot grant is to develop and characterize a mouse line that expresses mutated ABCA1N935S. Aim 1. To create genetically engineered mice that express mutated ABCA1N935S. This mouse line will be created by utilizing a "Knock-in" approach to introduce an N935S mutation in the ABCA1 locus in mouse ES cells. These ABCA1S/S mice will ubiquitously express ABCA1N935S, the mutated gene will remain under the endogenous ABCA1 transcriptional control and the mutant protein will replace wild type ABCA1. Aim 2. To characterize ABCA1N935S knock-in mice. We will analyze in vivo the expression of ABCA1N935s and its function as a cholesterol transporter by measuring plasma CL and lipoproteins. The effect of the mutated ABCA1N93ss on brain lipoproteins and endogenous APR processing and ABeta generation in vivo will be determined as a preliminary step to substantiate future detailed biochemical studies.