Molecular Mechanisms of ApoE4 Proteolysis in Alzheimer's Disease

描述(申请人提供)：阿尔茨海默病(AD)是一种进行性神经退行性疾病，可能由遗传和环境因素共同引起。在已确定的遗传危险因素中，34 kDa的载脂蛋白(Apo)E4是 非常重要，因为apoE4携带者占所有AD病例的65%-80%。虽然apoE4在脂蛋白运输中发挥正常作用，但它如何在AD发病中起作用目前尚不清楚。新的数据表明，apoE4对蛋白水解性切割很敏感，因此可能通过功能丧失参与了与AD相关的潜在分子病理。然而，apoE4蛋白水解性裂解的分子机制，包括所涉及的蛋白酶的特性，还不是很清楚。这项研究的目的是确定负责ApoE4裂解的蛋白酶，并确定这种裂解事件是否发生在AD脑中。利用针对apoE4裂解的定点抗体，我们最近从大肠杆菌中纯化apoE4后，确定apoE4的一个18 KDa的主要裂解片段以重组形式存在，并原位存在于AD脑切片中。我们假设，这种蛋白降解事件代表了apoE4裂解的一条新途径，而caspase是AD脑中这种裂解的原因。目标1中描述的实验将严格测试apoE4被半胱氨酸酶切割产生18 kDa氨基末端片段的假设。这些实验将通过分析纯化的apoE4或重组形式与caspase-3在无细胞系统中孵育或在凋亡模型系统中caspase激活后apoE4片段的产生来表征切割事件。这些实验将通过开发一种定点切割抗体来辅助，该抗体可以在D172处切割后特异性地检测apoE4的氨基末端18 kDa片段。目标2中概述的实验将确定蛋白水解性切割是否只对E4亚型是独有的，而不是包括APOE2或apoE3在内的其他已知亚型。我们假设，与其他异构体相比，只有apoE4对蛋白水解性切割唯一敏感，因此，这将作为一个潜在的事件，将apoE4蛋白水解性与晚发性AD风险增加联系起来。使用我们的定点切割抗体，目标3的实验将确定AD大脑中是否产生了相同的apoE4片段，如果是，则确定是什么细胞类型。由于半胱氨酸酶在裂解tau和促进 关于神经原纤维缠结的形成，我们假设apoE4的这个18 kDa片段将定位于AD脑的神经原纤维缠结中。使用固定的死后脑切片的免疫组织化学分析以及AD样本的Western印迹分析都将被进行，并与年龄匹配的对照组进行比较。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive neurodegenerative disease likely caused by a combination of both genetic and environmental factors. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (apo) E4, is of significant importance as apoeE4 carriers account for 65-80 percent of all AD cases. Although apoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. Emerging data suggests that apoE4 is sensitive to proteolytic cleavage and thus contributes to the underlying molecular pathology associated with AD possibly through a loss of function. However, the molecular mechanisms underlying the proteolytic cleavage of apoE4, including the identity of the protease involve, has not been clarified. The purpose of this study is to identify the protease responsible for ApoE4 cleavage and to determine whether or not this cleavage event occurs in the AD brain. Using a site-directed antibody to cleaved apoE4 we have recently determined a major cleavage fragment of apoE4 of 18 KDa is present in recombinant forms following purification of apoE4 from E. coli and is present in situ in AD brain sections. We hypothesize that this proteolytic event represents a novel pathway for apoE4 cleavage and that caspases are responsible for this cleavage in the AD brain. Experiments described in Aim 1 will rigorously test the hypothesis that apoE4 is cleaved by caspases to generate a 18 kDa amino-terminal fragment. These experiments will characterize the cleavage event by analyzing the production of the apoE4 fragment following incubation of purified apoE4 or recombinant forms with caspase-3 in a cell-free system or following caspase activation in a model system of apoptosis. These experiments will be aided through the development of a site-directed cleavage antibody that specifically detects the amino-terminal 18 kDa fragment of apoE4 following cleavage at D172. Experiments outlined in Aim 2 will determine whether proteolytic cleavage is unique to only the E4 isoform, and not other known isoforms including apoE2 or apoE3. We hypothesize that only apoE4 will be uniquely sensitive to proteolytic cleavage compared to other isoforms and thus, this will serve as an underlying event connecting apoE4 proteolysis to an enhanced risk of late-onset AD. Using our site-directed cleavage antibodies, experiments in Aim 3 will determine if this same apoE4 fragment is generated in the AD brain and if so, what cell type. Due to the role of caspases in cleaving tau and contributing to the formation of neurofibrillary tangles, we hypothesize this 18 kDa fragment of apoE4 will localize within neurofibrillary tangles of the AD brain. Both immunohistochemical analysis using fixed post mortem brain sections as well as Western blot analysis from AD samples will be performed and compared to age-matched controls.