Immunolocalization of caspase proteolysis in situ: Evidence for widespreadcaspase-mediated apoptosis of neurons and glia in the postnatal rat brain

Activation of a member of the caspase family of cysteine proteases is thoug ht to be required for the execution of apoptosis in neurons and other cell types. We describe here an antibody (Ab127) reactive with a neoantigenic si te on caspase substrate proteins degraded during apoptosis, and its charact erization as a biochemical and histochemical probe for apoptosis-associated proteolysis in growth factor-deprived neural cells in vitro and the develo ping postnatal rat brain. Neuronally differentiated PC12 cells became stron gly Ab127 immunoreactive only during apoptosis following nerve growth facto r withdrawal. Apoptosis-associated caspase proteolysis was detectable on we stern blots as markedly increased immunoreactivity of a similar to 46,000 m ol, wt polypeptide, a product also generated by caspase-3 treatment of cell -free extracts. In the postnatal rat brain, intense immunoreactivity indica tive of caspase activation was exhibited by small proportions of neurons an d glia in distinct regional and temporal patterns. The degenerating nature of these cells was confirmed by their argyrophilia, cytoplasmic immunoreact ivity for c-jun and fragmented processes. Combined immunofluorescence and H oechst 33342 staining demonstrated that cells immunopositive for caspase ac tivation have apoptotic nuclear morphologies. Caspase proteolysis was obser ved throughout the neuraxis in a minority of progenitor cells in germinal z ones, postmitotic neurons in the parenchyma, and glia in the corpus callosu m and other white matter tracts, but was observed rarely in the adult brain . These data characterize a new approach for evaluating apoptosis in physiolo gical and pathological neurodegeneration, and demonstrate that caspase-asso ciated apoptosis is a widespread mechanism for the programmed death of neur ons and glia in the postnatal rat brain. (C) 1999 IBRO. Published by Elsevi er Science Ltd.