Development of Site-Directed Caspase-Cleavage Antibodies

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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.