Lysosomal release of cathepsins in ischemic brain damage

Much progress has been made in elucidating the molecular mechanisms that underlie ischemic brain damage, a leading killer in the United States and elsewhere. In many common animal models, a period of ischemia is followed by reperfusion of the affected brain areas. Neuronal death is often delayed until long after reperfusion, and glutamate-dependent increases of intracellular calcium and of reactive oxygen species (ROS) contribute to delayed death. However, the damaging targets of calcium and ROS are not well-established. The goal this thesis was to determine whether lysosomes are damaging targets of these agents following ischemia.; The data presented indicate that lysosomes are permeabilized by calcium and ROS following ischemia. Cathepsin B is a cysteine protease that is normally present in lysosomes, but it accumulates in the cytosol following ischemia in the rat hippocampal slice, probably due to lysosomal membrane permeabilization (LMP). Antioxidants and inhibitors of NMDA-mediated calcium influx block this ischemia-induced accumulation of cathepsin B in the cytosol. Additionally, pharmacological inhibition of cathepsin B and the lysosomal aspartic protease, cathepsin D, attenuates delayed ischemic damage, indicating that these proteases are perpetrators of damage.; The molecular mechanism leading from NMDA-mediated calcium influx to LMP was partially elucidated. Permeabilization depends on activation of cPLA2 (calcium-dependent cytosolic phospholipase A2). Arachidonic acid, but not prostaglandin E2 or superoxide, causes release of acid phosphatase from isolated lysosomes. These results indicate that free arachidonic acid likely causes LMP following ischemia. However, cytosolic accumulation of cathepsin B following ischemia depends on the activity of COX-2 (cyclooxygenase 2), indicating that superoxide or prostaglandins must indirectly contribute to LMP. In a model developed to explain these observations, arachidonic acid causes LMP following ischemia, but other agents, such as superoxide produced by COX-2, contribute by activating cPLA2. Consistent with this model, COX-2 is the major source of superoxide following ischemia. Other enzymes that activate cPLA2 were found to contribute to LMP and to superoxide production, including ERK (extracellular signal-regulated kinase) and nitric oxide synthase. Together, these results indicate that arachidonic acid metabolism downstream of NMDA-mediated calcium influx plays a major role in lysosomal membrane permeabilization following ischemia.