Development of Site-Directed Caspase-Cleavage Antibodies

阿尔茨海默病（AD）的一个突出特征是通过凋亡性细胞死亡引起的神经元损失。细胞凋亡的特征在于质膜出血、核浓缩和DNA片段化，并且由天冬氨酸蛋白酶家族capspases的激活启动。细胞凋亡的启动涉及通过蛋白水解将半胱氨酸蛋白酶原顺序激活为它们的活性形式。该家族的两个关键成员是胱天蛋白酶-8（胱天蛋白酶的最顶端成员）和胱天蛋白酶-3（通常被称为该家族的刽子手成员）。目前，检测细胞凋亡在神经退行性疾病中的作用的主要技术包括TUNEL或ISEL方法，其能够检测细胞中的DNA片段化。然而，有几个“陷阱”与这些方法，即，他们可以检测DNA链断裂凋亡和坏死细胞。此外，DNA链断裂可以随着死后间隔而增加，并且是可以在没有细胞凋亡的各种情况下发生的晚期核事件。此外，观察到的关键形态学特征，如细胞体的圆形化和表面出血，即使在没有细胞核的情况下也会发生，并归因于半胱天冬酶对细胞骨架蛋白的蛋白水解裂解。由于这些限制，更新和更具体的凋亡探针是必要的，以确认TUNEL实验提供的证据。由于胱天蛋白酶是特异性的，在天冬氨酸残基后裂解，这将产生抗原性不同的胱天蛋白酶裂解产物（CCP），因此代表裂解位点定向抗体的期望靶标。使用这种方法，我们设计了一种抗体的CCP的胞衬蛋白，神经元细胞骨架蛋白，并显示这些产品在AD中的广泛积累。在本发明中，我们建议1）进一步表征该抗体以确定细胞凋亡的多个途径是否导致共同效应物半胱天冬酶的活化; 2）开发针对半胱天冬酶-8的活性片段的切割位点定向抗体，并使用细胞凋亡的模型系统表征这些抗体; 3）将该抗体与forbin CCP抗体一起使用，以确定胱天蛋白酶活化和CCP积累与其他与AD相关的事件之间的关系，所述其他与AD相关的事件包括淀粉样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.