Influence of apoE on LRP1 function and Beta-Amyloid Transport Across the BBB

描述(申请人提供)：阿尔茨海默病(AD)是美国第六大死亡原因，其特征是神经原纤维缠结的形成和大脑中β-淀粉样蛋白(A？)的沉积。大脑中的A积聚是AD发病的关键因素，也是认知障碍的预测指标。AD的减少策略主要针对的是A的产生机制，到目前为止，这些机制在临床试验中基本上没有成功。现在的证据表明，阿尔茨海默病患者大脑中A？的过度积累并不是由于A？的异常产生，而是A？从大脑中清除受损的结果。因此，靶向清除相关通路可能被证明是最有效的减少A？在AD大脑中的积聚。血脑屏障(BBB)转运体是低密度脂蛋白受体相关蛋白1(LRP1)。LRP1与载脂蛋白E(ApoE)相互作用的配体之一是载脂蛋白E(ApoE)，拥有E4等位基因是AD最大的遗传危险因素。然而，载脂蛋白E在血脑屏障的A？交换中的作用还没有明确的定义。在细胞培养中的几项研究表明，在存在apoE的情况下，A？内化被刺激，而另一些研究报告apoE对跨血脑屏障的A？清除有干扰作用。在我们的初步研究中，我们试图澄清载脂蛋白E在清除血脑屏障中的作用。我们的研究和其他人的工作表明，当载脂蛋白E与A°结合时，A°通过血脑屏障的运输显著减弱。然而，我们首次证明了大脑中未结合的apoE可以促进A？BBB以异构体特异性的方式清除(apoE3和gt；&gt；apoE4)。在血脑屏障水平上，很少有研究研究apoE亚型对LRP1功能的影响，特别是LRP1的脱落(即形成可溶性LRP1，它是无功能的，不跨细胞糖A)。在apoE4转基因小鼠和携带E4等位基因的AD患者中，我们观察到与apoE3基因相比，LRP1在大脑中的脱落增加。当结合我们体内的A？BBB转运数据时，我们的初步研究表明，LRP1的脱落和A？跨BBB的转运之间存在反向关系，这种关系是载脂蛋白E基因特有的。我们假设apoE4在阻止LRP脱落方面不如其他apoE亚型有效，这会导致血脑屏障上的A？清除减少。我们的初步发现还表明，LRP1内化的调节导致LRP1的脱落增强，并减少了A？通过体外血脑屏障模型的转运。由于LRP1的内化和跨细胞作用是从脑中清除Aβ的重要步骤，我们将研究apoE亚型对LRP1介导的Aβ在血脑屏障中的内化和亚细胞转运的影响。总体而言，目前的提案将调查几种可能的机制，以阐明我们在LRP1脱落和A？跨血脑屏障转运中观察到的载脂蛋白E依赖的差异。这些研究将加深我们对载脂蛋白E和LRP1在AD中的关系的理解，并可能为该疾病提供新的治疗方式。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the sixth leading cause of death in the United States and is characterized by the formation of neurofibrillary tangles and the deposition of beta-amyloid (A¿) proteins in the brain. A¿ accumulation in the brain is a key factor in AD pathogenesis and is a predictor of cognitive impairment. A¿ reducing strategies in AD have primarily targeted A¿ production mechanisms which, to this point, have been largely unsuccessful in clinical trials. Evidence now suggests that the excessive accumulation of A¿ in the AD brain is not due to aberrant A¿ production, but the result of impaired A¿ removal from the brain. Thus, targeting clearance-related pathways may prove most effective in attenuating A¿ accumulation in the AD brain. The blood-brain barrier (BBB) transporter primarily responsible for the brain elimination of A¿ is the low density lipoprotein receptor-related protein 1 (LRP1). One of the ligands with which LRP1 interacts is apolipoprotein E (apoE) and possession of the E4 allele represents the strongest genetic risk factor for AD. However, the role of apoE in the exchange of A¿ across the BBB is not clearly defined. Several studies in cell culture have shown a stimulation of A¿ internalization in the presence apoE, while others have reported apoE is disruptive to A¿ clearance across the BBB. In our preliminary studies we sought to clarify the role of apoE in the BBB clearance of A¿. Our studies and the work of others indicate that when apoE is bound to A¿, the transport of A¿ across the BBB is dramatically attenuated. However, for the first time, we demonstrate that unbound apoE in the brain can facilitate A¿ BBB clearance in an isoform-specific manner (apoE3 >> apoE4). At the BBB level, few studies have investigated the influence of apoE isoforms on LRP1 function, in particular LRP1 shedding (i.e., formation soluble LRP1, which is nonfunctional and does not transcytose A¿). In apoE4 transgenic mice and AD patients carrying the E4 allele, we observe elevated LRP1 shedding in the brain compared to apoE3 genotypes. When combined with our in vivo A¿ BBB transit data, our preliminary studies demonstrate an inverse relationship between LRP1 shedding and A¿ transport across the BBB, one that is apoE genotype-specific. We hypothesize that apoE4 is less efficient than other apoE isoforms in preventing LRP shedding, which leads to reduced A¿ clearance across the BBB. Our preliminary findings also show that modulation of LRP1 internalization leads to enhanced LRP1 shedding and reduced A¿ transport across an in vitro model of the BBB. Since LRP1 internalization and transcytosis are essential steps in eliminating A¿ from the brain, we will examine the effect of apoE isoforms on LRP1-mediated internalization and subcellular trafficking of A¿ in the BBB. Overall, the current proposal will investigate severa potential mechanisms to elucidate the apoE- dependant differences we observe in LRP1 shedding and A¿ transit across the BBB. These studies will improve our understanding of the relationship between apoE and LRP1 in AD and may provide new treatment modalities for this disease.