Immunolocalization of caspase proteolysis in situ: evidence for widespread caspase-mediated apoptosis of neurons and glia in the postnatal rat brain.
Activation of a member of the caspase family of cysteine proteases is thought to be required for the execution of apoptosis in neurons and other cell types. We describe here an antibody (Ab127) reactive with a neoantigenic site on caspase substrate proteins degraded during apoptosis, and its characterization as a biochemical and histochemical probe for apoptosis-associated proteolysis in growth factor-deprived neural cells in vitro and the developing postnatal rat brain. Neuronally differentiated PC12 cells became strongly Ab127 immunoreactive only during apoptosis following nerve growth factor withdrawal. Apoptosis-associated caspase proteolysis was detectable on western blots as markedly increased immunoreactivity of a approximately 46,000 mol. wt polypeptide, a product also generated by caspase-3 treatment of cell-free extracts. In the postnatal rat brain, intense immunoreactivity indicative of caspase activation was exhibited by small proportions of neurons and glia in distinct regional and temporal patterns. The degenerating nature of these cells was confirmed by their argyrophilia, cytoplasmic immunoreactivity for c-jun and fragmented processes. Combined immunofluorescence and Hoechst 33342 staining demonstrated that cells immunopositive for caspase activation have apoptotic nuclear morphologies. Caspase proteolysis was observed throughout the neuraxis in a minority of progenitor cells in germinal zones, postmitotic neurons in the parenchyma, and glia in the corpus callosum and other white matter tracts, but was observed rarely in the adult brain. These data characterize a new approach for evaluating apoptosis in physiological and pathological neurodegeneration, and demonstrate that caspase-associated apoptosis is a widespread mechanism for the programmed death of neurons and glia in the postnatal rat brain.
Siman
R
R
Cephalon Inc., West Chester, PA 19380, USA.
Bozyczko-Coyne
D
D
Meyer
S L
SL
Bhat
R V
RV
D000818:Animals; D000831:Animals, Newborn / Q000502:physiology*; D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000502:physiology*; D001921:Brain / Q000166:cytology* / Q000201:enzymology*; D020169:Caspases / Q000378:metabolism* / Q000502:physiology*; D002467:Cell Nucleus / Q000276:immunology / Q000648:ultrastructure; D002478:Cells, Cultured; D019085:Fluorescent Antibody Technique, Direct; D015151:Immunoblotting; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009457:Neuroglia / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D016716:PC12 Cells; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D009419:Nerve Tissue Proteins; D016755:Proto-Oncogene Proteins c-jun; D020169:Caspases