Hippocampal Neurons in Culture

We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by /3-amyloid(A~3)(25—35). Both A/1(25—35)and staurosporine-induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA-induced death appeared more complex. Two irreversible inhibitors of the interleukin-1~ converting enzyme (ICE) and related proteases, Z-VaIAIa-Asp-CH2F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by A/9(25—35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calciumregulated protease calpain, also inhibited death induced by all agents. A~3(25—35)and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdowrl was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA-induced death. These results support the role of apoptosis in neuronal death due to A/9(25—35) treatment and also suggest a role for calcium-regulated proteases in this process.