Elsevier

Neuroscience

Volume 92, Issue 4, June 1999, Pages 1425-1442
Neuroscience

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

https://doi.org/10.1016/S0306-4522(99)00034-2Get rights and content

Abstract

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 ∼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.

Section snippets

Materials

An antiserum reactive with a neoantigenic site created by caspase proteolysis was prepared using procedures we established for other processed proteins.54., 59. The strategy takes advantage of the unique preference of caspases for cleaving substrates' COOH-terminal to aspartate residues.10., 37., 48., 67. Briefly, the heptapeptide CKGDEVD was synthesized by solid-phase methods by the Cephalon Peptide Chemistry group, and its structure confirmed by mass spectrometry. The peptide was conjugated

Design of an antibody probe for caspase-mediated proteolysis

To generate an antibody that reacts with a neoantigenic site on proteins degraded in apoptotic cells, a hexapeptide corresponding to a domain from poly(ADP-ribose) polymerase (PARP), KGDEVD, was chosen as immunogen (Fig. 1A). The hexapeptide is immediately upstream of the caspase cleavage locus within PARP in apoptotic cells,37 is conserved in avian, murine, bovine and human PARP,9., 28., 58. and forms the basis of potent synthetic substrates and inhibitors of caspases,42., 67. all of which

Discussion

This report describes the antibody-based detection of caspase-mediated proteolysis in situ, and presents evidence that caspase-associated apoptosis is a widespread mechanism for the programmed death of neuroblasts, differentiating neurons and white matter glia in the developing CNS. The specificity of Ab127 for detecting caspase activation during apoptosis is demonstrated by immunoblot and immunohistochemical analyses of PC12 cells neuronally differentiated with NGF and then deprived of trophic

Conclusions

Ab127 is a biochemical and histochemical probe for apoptosis-associated caspase proteolysis. In situ localization of caspase proteolysis and cytoplasmic c-jun immunoreactivity have provided evidence that caspase-mediated apoptosis is a widespread mechanism for the programmed death of progenitor cells, differentiating neurons and white matter glia in the postnatal rat brain. Ab127 may be a useful tool for evaluating the contribution of apoptosis to neurodegenerative processes in the developing

Acknowledgements

We thank Dr Mohamed Iqbal and the Cephalon Peptide Chemistry group for preparation and characterization of the peptide immunogen, and BethAnn McKenna and Chrysanthe Spais for excellent technical assistance.

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