Much attention has been paid to proteinases derived from not only neurons but also microglia in relation to neuronal death. There is accumulating evidence that intra and extracellular proteinases in these cells are part of the basic machinery of neuronal death pathways. Some members of the ced-3/interleukin-1β converting enzyme (ICE) (caspase) family of cysteine proteinases have been thought to play a major role in apoptosis of not only non-neuronal cells but also neurons. Calpain has also been demonstrated to be a mediator of the neurodegenerative response. Recent studies have shown that excitotoxic and ischemic neuronal injury could be attenuated by inhibitors of caspases and calpain. Several recent studies have suggested the involvement of endosomal/lysosomal proteinases, including cathepsins B, D and E, in neuronal death induced by excitotoxins and ischemia. Furthermore, it has been reported that the extracellular tissue-type plasminogen activator/plasmin proteolytic cascade is involved in excitotoxic injury of the hippocampal neurons. In addition to such neuronal proteinases, microglial proteinases are believed to be important for the modification of neuronal functions positively or negatively. Cathepsins E and S derived from microglia have been suggested to contribute to neuronal survival through degradation and removal of β-amyloid, damaged neurons and cellular debris. On the other hand, 6-hydroxydopamine-induced microglial cell death was inhibited by inhibitors of aspartic proteinases and caspases, suggesting the involvement of cathepsins E and D and caspases in microglial cell death. Therefore, identification of which proteinases play a causative role in neuronal death execution and clarification of the regulators and substrates for such proteinases is very important for understanding the molecular basis of the neuronal death pathways and to develop novel neuroprotective agents.
Recently, the technique for high time- and spatial-resolution imaging of intracellular calcium ion concentration ([Ca2+]i) has been developed using rapid scanning confocal microscopy to investigate calcium dynamics in restricted areas of cells. Here, the techniques are summarized and the images obtained are introduced. For such imaging, the development of fast scanning confocal microscopes was indispensable. Therefore, the UV-applicable video-rate (30 frames/sec) scanning confocal microscope in which a resonant galvanometer scanner is used for fast horizontal scans has been developed. The microscope enabled us to obtain ratio-images of [Ca2+]i using indo-1, a ratio-imaging probe. The key to success in high time- and temporal-resolution imaging is to find the best probe-loading condition, which depends on the probes and the cells. It is also very important to increase the signal-to-noise ratio value without any effects on the cells. Using the technique, we have succeeded in kinetic analysis of calcium waves in cultured hepatocytes. We have also succeeded in determining the relationship between calcium sparks to calcium transients in excitation-contraction coupling.