Mitochondrial Free Radical Signal in Ceramide-Dependent Apoptosis: A Putative Mechanism for Neuronal Death in Parkinson's Disease

Abstract: We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by β-amyloid(Aβ) (25–35). Both Aβ(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-1β converting enzyme (ICE) and related proteases, Z-Val-Ala-Asp-CH₂F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by Aβ(25–35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium-regulated protease calpain, also inhibited death induced by all agents. Aβ(25–35) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown 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 Aβ(25–35) treatment and also suggest a role for calcium-regulated proteases in this process.