Earlier findings reported in the grant period from 1999 to 2004 were related to hypoxic, toxic, and reperfusion injury to hepatocytes. These studies led to a substantial number of peer-reviewed publications, book chapters and abstracts and demonstrated that hypoxic, toxic and reperfusion injury to hepatocytes is mediated by pH-dependent ATP depletion and mitochondrial failure. Over the last four years, our research focus has shifted to examining the relationship between apoptosis and aging. We demonstrated that older hepatocytes were more sensitive to oxidative stress-induced, apoptosis generated in and targeted to the mitochondrial compartment. Importantly, inhibition of caspase-2 or caspase-9 reversed the age-dependent increase in sensitivity of hepatocytes to oxidative stress. We also showed that the regulation of oxidative stress-induced mitochondrial apoptosis was altered in hepatocytes isolated from old liver and that the extent of endogenous apoptosis in Hver, lung, spleen and kidney, (as evidenced by age-dependent increases in caspase activity- in particular caspase-2 and -9) increased at advanced age. Caspase-8 (the major caspase involved in the extrinsic pathway of apoptosis) was not activated in these same tissues as a function of age, suggesting the preferential activation of the intrinsic apoptotic pathway accompanies aging in multiple regenerative tissues. Furthermore, we showed that activation of caspase-2, -3 and -9 was augmented in liver obtained from the hSOD2+/- mice relative to the age-matched wild type (WT) controls (unpublished data). In fact, caspase-2 was the first caspase seen to increase during aging tn hS0D2+/- mice. Most importantly, we showed that fibroblasts isolated from caspase-2-/- mice were resistant to the mitochondrial oxidant stressor rotenone, but not to the kinase inhibitor staurosporine (STS) (Reprint 1). Finally, we demonstrated that caspase-2-/- mice are resistant to 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons (figure 7). Collectively our data suggest that caspase-2 is a putative regulator/effector of oxidant stress-induced apoptosis and that this caspase may also regulate alterations in apoptotic activity that may occur during an organism's health span.
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