Development of Site-Directed Caspase-Cleavage Antibodies

阿尔茨海默病(AD)的一个显著特征是细胞凋亡导致的神经元丢失。细胞凋亡的特征是质膜出血、核固缩和DNA片段化，并由天冬氨酸蛋白酶家族Capspase的激活启动。细胞凋亡的启动涉及蛋白水解酶将原天冬氨酸酶顺序激活为活性形式。这个家族的两个关键成员是caspase-8和caspase-3，caspase-8是caspase最顶端的成员，caspase-3通常被称为该家族的刽子手成员。目前，研究细胞凋亡在神经退行性疾病中的作用的主要技术包括能够检测细胞中DNA片段化的TUNEL或ISEL方法。然而，这些方法有几个“陷阱”，即它们可能会检测到凋亡和坏死细胞中的DNA链断裂。此外，DNA链断裂可随着死亡时间的延长而增加，是一种晚期核事件，可在各种情况下发生，而不会发生细胞凋亡。此外，观察到的关键形态特征，如细胞体圆整和表面出血，即使在没有细胞核的情况下也会发生，并被归因于caspase对细胞骨架蛋白的蛋白水解性切割。由于这些局限性，需要更新、更特异的细胞凋亡探针来证实TUNEL实验提供的证据。因为caspase是特定的，在天冬氨酸残基之后裂解，这将产生抗原性不同的caspase裂解产物(CCP)，因此是切割定点定向抗体的理想靶点。利用这种方法，我们设计了一种针对神经元细胞骨架蛋白Fodrin CCPS的抗体，并显示这些产物在AD中广泛积累。在目前的建议中，我们建议1)进一步表征这种抗体，以确定是否有多条途径的凋亡导致共同的效应器caspase的激活；2)开发针对caspase-8活性片段的切割位点定向抗体，并使用凋亡模型系统来表征这些抗体；3)将该抗体与forbin CCP抗体结合使用，以确定caspase激活和ccp积累与其他与AD相关的事件的关系，包括β-淀粉样蛋白沉积和神经纤维缠结形成。

A prominent feature of Alzheimer's disease (AD) is loss of neurons by apoptotic cell death. Apoptosis is characterized by plasma membrane bleeding, nuclear condensation, and DNA fragmentation and is initiated by the activation of capspases, a family of aspartate proteases. The initiation of apoptosis involves the sequential activation of procaspases to their active form by proteolysis. Two key members of this family are caspase-8, the most apical member of the caspase, and caspase-3 which is commonly referred to as the executioner member of this family. Presently, the major technique to examine the role of apoptosis in neurodegenerative diseases consist of TUNEL or ISEL methods that are able to detect DNA fragmentation in cells. However, there are several 'pitfalls' associated with these methods, namely that they may detect DNA strand breaks in both apoptotic and necrotic cells. In addition, DNA strand breaks can be increase with postmortem interval and are a late stage nuclear event that can occur in a variety of situations without apoptosis. Furthermore, key morphological features observed such as rounding up of the cell body and surface bleeding occur even in the absence of nuclei and have been attributed to proteolytic cleavage of cytoskeletal proteins by caspases. Due to these limitations, newer and more specific probes for apoptosis are necessary to confirm evidence provided by TUNEL experiments. Because caspases are specific, cleaving after aspartic residues, this will generate caspase cleavage products (CCPs) that are antigenically distinct and therefore, represent desirable targets for cleavage site-directed antibodies. Using this approach, we designed an antibody to CCPs of fodrin, a neuronal cytoskeleton protein, and showed widespread accumulation of these products in AD. In the present proposal we propose to 1) further characterize this antibody to determine whether multiple pathways of apoptosis lead to the activation of a common effector caspase; 2) develop cleavage site-directed antibodies against the active fragments of caspase-8 and characterize these antibodies using model systems of apoptosis; 3) use this antibody together with the forbin CCP antibody to determine the relationship between caspase activation and accumulation of CCPs with other events associated with AD including beta-amyloid deposition and neurofibrillary tangle formation.