SPATIAL-RESOLUTION OF FODRIN PROTEOLYSIS IN POSTISCHEMIC BRAIN

One of the major obstacles in investigating in vivo proteolytic phenomena has been the inaccessibility to spatial information as to where in the tissue the reaction proceeds because the orthodox method employing electrophoretic analysis requires homogenization of samples and thus results in loss of such spatial information. To overcome this technical drawback, we have developed methodology to produce antibodies that specifically distinguish a proteolyzed form of a given protein from its intact form. Here we describe our immunohistochemical observation of calpain-catalyzed fodrin proteolysis in postischemic gerbil hippocampus, using an antibody exclusively specific to the proteolyzed 150-kDa form of fodrin alpha subunit. Our data establish a novel discovery that transient (10 min) global forebrain ischemia followed by reperfusion induces at least two distinct phases of fodrin proteolysis in hippocampus: an early phase in molecular layer and in stratum oriens of CA3 and CA1 sectors within 15 min and a late drastic and persistent phase in the entire CA1 after 4-24 h. The former may be one of the early events initiating the complex cascade leading to the delayed neuronal death, while the latter should be considered as a more direct cause for the actual degeneration in CA1.