Regulation of Matrix Metallopeptidase 9 by Apolipoprotein E

 描述（由申请人提供）： 阿尔茨海默病（Alzheimer's disease，AD）是一种以β-淀粉样蛋白（beta- amyloid protein，Aβ）在脑和血管系统中沉积为特征的神经退行性疾病。虽然AD一直是我们老龄化退伍军人的一个问题，但在战斗中头部受伤的倾向使我们的退伍军人比一般人群面临更大的发展AD的风险。越来越多的证据表明，AD中Aβ的过度蓄积是Aβ从大脑清除受损的结果。血脑屏障内的脂蛋白受体有助于Aβ的脑-血消除。然而，这些受体易受细胞表面蛋白水解的影响（即，胞外域脱落），这降低了它们转运配体的能力， Aβ。与脂蛋白受体密切相关的配体之一是载脂蛋白E（apoE），其在人类中以三种同种型（apoE 2、apoE 3和apoE 4）存在。重要的是，拥有apoE 4等位基因是晚发性AD最强的遗传风险因素。最近的报道，包括我们自己的报道，表明Aβ从脑中的清除受apoE亚型表达的差异调节。我们的初步研究表明，apoE 4在保护脂蛋白受体免于脱落过程中不如其他亚型熟练，这导致Aβ从脑中清除减少。然而，在这个阶段，载脂蛋白E影响脂蛋白受体蛋白水解的机制尚未阐明。 MMP 9是一种内肽酶，可以结合和蛋白水解（即，脱落）脂蛋白受体。在AD中，与对照受试者相比，大脑和外周中的MMP 9水平显著升高。到目前为止，对apoE和MMP 9之间的关系几乎没有研究，但我们的初步数据表明apoE在介导MMP 9对脂蛋白受体脱落的作用中起关键作用。在我们的初步研究中，从apoE 2转基因小鼠分离的脑血管暴露于活化的MMP 9后，与apoE 4血管相比，脂蛋白受体脱落减少。此外，我们发现，给apoE 4转基因小鼠施用MMP 9抑制剂可减少脑中脂蛋白受体脱落，并将Aβ穿过BBB的清除率提高至接近未治疗的apoE 3动物中观察到的水平。这些研究表明apoE和MMP 9之间存在亚型特异性关系。该建议的假设是apoE 4在调节MMP 9功能方面比其他apoE亚型效率低，这导致脂蛋白受体脱落增加和Aβ从脑中消除减少。该提案的目的是阐明apoE调节MMP 9功能并影响脂蛋白受体脱落和Aβ从脑中清除的机制。目前的建议将检查几种机制，以确定apoE和MMP 9之间的关系。Aim 1将通过检查AD小鼠和人脑（各自具有不同的apoE基因型）的分离血管系统中的MMP 9水平来评估apoE基因型对MMP 9表达的影响。Aim 2将检查每种apoE亚型对MMP 9分布的影响。这些研究将研究apoE对MMP 9细胞分泌、pro-MMP 9转化为活性MMP 9和MMP 9活性的影响。Aim 3将测试apoE和MMP 9之间的结合亲和力，并确定apoE对MMP 9与脂蛋白受体结合的影响。此外，目前的建议将检查MMP 9定向治疗对转基因小鼠（Aim 4）中AD表型的影响。据我们所知，还没有研究在AD动物模型中研究MMP 9抑制的效果。预计这些研究将为在apoE 4转基因动物和携带apoE 4等位基因的AD患者中观察到的Aβ脑负荷升高和治疗反应有限提供依据，并为AD的新治疗策略铺平道路。

 DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative process characterized by the deposition of beta- amyloid proteins (Aβ) in the brain and cerebrovasculature. While AD has always been a concern for our aging Veterans, the propensity for head injuries sustained in combat has placed our Veterans at even greater risk for developing AD than the general population. Mounting evidence now suggests the excessive accumulation of Aβ in AD is the result of impaired Aβ clearance from the brain. Lipoprotein receptors within the blood-brain barrier contribute to the brain-to-blood elimination of Aβ. However, these receptors are vulnerable to proteolysis at the cell surface (i.e., ectodomain shedding), which diminishes their ability to transport ligands, such as Aβ. One of the ligands closely associated with the lipoprotein receptors is apolipoprotein E (apoE), which exists as three isoforms in humans (apoE2, apoE3, and apoE4). Importantly, possession of the apoE4 allele represents the strongest genetic risk factor for late-onset AD. Recent reports, including our own, indicate Aβ clearance from the brain is differentially regulate by the type of apoE isoform expressed. Our preliminary studies demonstrate that apoE4 is less adept than the other isoforms in protecting lipoprotein receptors from the shedding process, which results in reduced Aβ clearance from the brain. At this stage, however, the mechanism by which apoE influences lipoprotein receptor proteolysis has yet to be elucidated. MMP9 is an endopeptidase that can bind and proteolyze (i.e., shedding) lipoprotein receptors. In AD, MMP9 levels are significantly elevated in the brain and periphery compared to control subjects. To this stage there has been little investigation into the relationship between apoE and MMP9, but our preliminary data suggest a key role for apoE in mediating the effect of MMP9 on lipoprotein receptor shedding. In our preliminary studies, cerebral vessels isolated from apoE2 transgenic mice showed reduced lipoprotein receptor shedding compared to apoE4 cerebrovessels following exposure to activated MMP9. In addition, we found that administration of an MMP9 inhibitor to apoE4 transgenic mice reduced lipoprotein receptor shedding in the brain and improved Aβ clearance across the BBB to levels near that observed in untreated apoE3 animals. These studies suggest an isoform-specific relationship between apoE and MMP9. The hypothesis of this proposal is that apoE4 is less efficient than other apoE isoforms in regulating MMP9 function, which leads to increased lipoprotein receptor shedding and reduced Aβ elimination from the brain. The goal of this proposal is to elucidate the mechanism by which apoE regulates MMP9 function and influences lipoprotein receptor shedding and Aβ removal from the brain. The current proposal will examine several mechanisms to determine the relationship between apoE and MMP9. Aim1 will evaluate the impact of apoE genotype on MMP9 expression by examining MMP9 levels in isolated cerebrovasculature from AD mouse and human brains, each with different apoE genotypes. Aim2 will examine the influence of each apoE isoform on MMP9 disposition. These studies will investigate the effect of apoE on MMP9 cellular secretion, conversion of pro-MMP9 to active MMP9, and MMP9 activity. Aim3 will test the binding affinity between apoE and MMP9 and determine the effect of apoE on MMP9 binding to the lipoprotein receptor. In addition, the current proposal will examine the effect of MMP9-directed therapy on the AD phenotype in transgenic mice (Aim4). To our knowledge, no study has investigated the effect of MMP9 inhibition in an animal model of AD. It is anticipated these studies will provide rationale for the elevated Aβ brain burden and limited response to treatment that is observed in apoE4 transgenic animals and AD patients carrying the apoE4 allele, and pave the way for new therapeutic strategies in AD.