TRANSIENT BRAIN ISCHEMIA PROVOKES CA2+, PIP2 AND CALPAIN RESPONSES PRIOR TO DELAYED NEURONAL DEATH IN MONKEYS

To clarify the mechanism of postischaemic delayed cornu Ammonis (CA)-1 neuronal death, we studied correlations among calpain activation and its subcellular localization, the immunoreactivity or phosphatidylinos itol 4,5-bisphosphate (PIP2) and Ca2+ mobilization in the monkey hippo campus by two independent experimental approaches: in vivo transient b rain ischaemia and in vitro hypoxia-hypoglycaemia of hippocampal acute slices. The CA-1 sector undergoing 20 min of ischaemia in vivo showed microscopically a small number of neuronal deaths on day 1 and almost global neuronal loss on day 5 after ischaemia. Immediately after isch aemia, CA-1 neurons ultrastructurally showed vacuolation and/or disrup tion of the lysosomes. Western blotting using antibodies against inact ivated or activated mu-calpain demonstrated mu-calpain activation spec ifically in the CA-1 sector immediately after ischaemia. This finding was confirmed in the perikarya of CA-1 neurons by immunohistochemistry . CA-1 neurons on day 1 showed sustained activation of mu-calpain, and increased immunostaining for inactivated and activated forms of mu- a nd m-calpains and for PIP2. Activated mu-calpain and PIP2 were found t o be localized at the vacuolated lysosomal membrane or endoplasmic ret iculum and mitochondrial membrane respectively, by immunoelectron micr oscopy. Calcium imaging data using hippocampal acute slices showed tha t hypoxia-hypoglycaemia in vitro provoked intense Ca2+ mobilization wi th increased PIP2 immunostaining specifically in CA-1 neurons. These d ata suggest that transient brain ischaemia increases intracellular Ca2 + and PIP2 breakdown, which will activate calpain proteolytic activity . Therefore, we suggest that activated calpain at the lysosomal membra ne, with the possible release of biodegrading enzyme, will cause posti schaemic CA-1 neuronal death.