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

描述（由申请人提供）：阿尔茨海默病（AD）是美国第六大死亡原因，其特征是神经纤维缠结的形成和β -淀粉样蛋白（A¿）在大脑中的沉积。A¿在大脑中的积累是阿尔茨海默病发病的关键因素，是认知障碍的预测因素。减少阿尔茨海默病的策略主要针对阿尔茨海默病的产生机制，到目前为止，这些机制在临床试验中基本上是不成功的。现在的证据表明，阿尔茨海默病患者大脑中A -的过度积累不是由于异常的A -产生，而是由于大脑中A -的清除受损。因此，靶向清除相关通路可能被证明在减少AD大脑中A¿积累方面是最有效的。低密度脂蛋白受体相关蛋白1 （LRP1）是主要负责脑内A¿消除的血脑屏障（BBB）转运蛋白。与LRP1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最强的遗传风险因素。然而，apoE在血脑屏障中A¿交换中的作用尚未明确定义。几项细胞培养研究表明，apoE存在时，a¿内化受到刺激，而其他研究则报道apoE破坏血脑屏障中a¿的清除。在我们的初步研究中，我们试图阐明apoE在血脑屏障清除A¿中的作用。我们的研究和其他人的工作表明，当apoE与A¿结合时，A¿在血脑屏障上的运输显著减弱。然而，我们首次证明了大脑中未结合的apoE可以以一种亚型特异性的方式促进A¿BBB的清除（apoE3 >> apoE4）。在BBB水平，很少有研究调查apoE亚型对LRP1功能的影响，特别是LRP1脱落（即形成可溶性LRP1，无功能，不转糖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基因型相比，大脑中LRP1的脱落增加。结合我们在体内的A¿血脑屏障转运数据，我们的初步研究表明，LRP1的脱落与A¿在血脑屏障中的转运之间存在反比关系，这是apoE基因型特异性的。我们假设apoE4在防止LRP脱落方面的效率低于其他apoE亚型，LRP脱落导致血脑屏障A¿清除率降低。我们的初步研究结果还表明，在体外血脑屏障模型中，LRP1内化的调节导致LRP1脱落增强，A¿转运减少。由于LRP1内化和胞吞作用是从大脑中消除A¿的必要步骤，我们将研究apoE亚型对LRP1介导的内化和血脑屏障中A¿的亚细胞运输的影响。总的来说，目前的建议将研究几种潜在的机制来阐明我们在LRP1脱落和A¿穿越血脑屏障中观察到的apoE依赖性差异。这些研究将提高我们对apoE和LRP1在AD中的关系的理解，并可能为这种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.