Over the past two decades biologists have recognized that there are two broad forms of cell death--necrosis and apoptosis. The former is seen after many severe, acute injuries and is associated with rapid cellular destruction. The latter can be triggered by a variety of acute and chronic insults and may take hours to weeks to fully manifest. There is now compelling evidence that a major component of the neuronal death seen in a variety of acute and chronic neurological diseases, including stroke, is apoptotic, and that this type of death is mediated by several endogenous cysteine proteases. The focus of this application is to determine the physiological consequences of aborting apoptotic neuronal death and to determine whether the triggers for apoptotic injury are present in human neocortex deprived of oxygen and glucose. Specifically: (1) Examine the function of voltage-gated and ligand-gated channels in autonomic ganglion neurons and cerebellar granule neurons that have been deprived of trophic factors but rescued from cell death by inhibition of proteases. (2) Determine the time course of protease activation in brain slices from rodents, and then humans (obtained from neocortical biopsy), and then identify interventions that inhibit protease activation. These experiments will utilize standard methods for intracellular recording and voltage clamping of cultured neurons and quantitative confocal imaging of brain slices. They will provide the first direct information about the physiological properties of neurons that have had an apoptotic death program interrupted. They should allow us to determine the functional potential of neurons when apoptosis has either been stabilized or actually reversed. Most important, they will provide direct evidence for the recruitment of apoptotic pathways in ischemic human brain.
