ROLE OF CASPASES IN N-METHYL-D-ASPARTATE-INDUCED APOPTOSIS IN CEREBROCORTICAL NEURONS

Overactivation of glutamate receptors mediates neuronal death in sever al acute and chronic neurodegenerative diseases. The intracellular pro cesses underlying this form of death, however, remain poorly understoo d. Depending on the severity of insult, N-methyl-D-aspartate (NMDA) re ceptor activation induces either apoptosis or necrosis, Cysteine prote ases related to interleukin-1 beta-converting enzyme (ICE), recently t ermed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA-induced apop tosis, we used two functionally distinct approaches to decrease substr ate cleavage by caspases. One is a novel peptide (V-ICEinh) that conta ins the caspase catalytic site and acts as a pseudoenzyme that binds c aspase substrates and prevents their cleavage. The other is a pseudosu bstrate peptide (Z-VAD . fmk) that inhibits caspase activity. Pretreat ment with either V-ICEinh or Z-VAD fmk protects cerebrocortical neuron s from NMDA-induced apoptosis, suggesting a role for caspases in NMDA- induced apoptosis. To explore the signaling pathways involved, we look ed at the effects of NMDA receptor activation on Ca2+ influx, producti on of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA-induced Ca2+ influx nor the init ial collapse of mitochondrial membrane potential could be prevented by pretreatment with V-ICEinh or Z-VAD fmk, In contrast, ROS formation a nd lipid peroxidation were completely blocked by both V-ICEinh and Z-V AD . fmk. Taken together, our results suggest that Ca2+ influx and mit ochondrial depolarization occur upstream from caspase activation, wher eas ROS formation and lipid peroxidation may be downstream events in t he cascade leading to cortical neuronal apoptosis.