Brain ischemia caused by cardiac arrest and stroke kills 300,000 people and disables another 150,000 each year in the United States. The general goal of my research effort is to characterize the molecular events that cause postischemic neuronal death and develop clinically effective therapies to reduce brain damage after cardiac arrest and stroke. This proposal focuses on the causal role of calpain-mediated proteolysis. Calpains are a family of Ca2+-dependent cytosolic proteases. Brain calpain activity is increased by focal and global ischemia, and calpain inhibitors are neuroprotective. However, the mechanism by which calpains contribute to post-ischemic neuronal death has not been determined. Several Ca2+ regulatory proteins are known to be calpain substrates. These include plasma membrane Ca2+-ATPase, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, the ryanodine receptor Ca2+ channel, and the IP3 receptor Ca2+ channel. The hypothesis is that calpain-mediated proteolysis of Ca2+ regulatory proteins disrupts Ca2+ homeostasis in post-ischemic neurons. The result is a sustained elevation in cytosolic Ca2+ and persistent calpain activation in a positive feedback pathway that is potentially irreversible and ultimately leads to delayed neuronal death. This hypothesis will be tested using an established in vivo model of transient forebrain ischemia in rats.
Specific Aim 1 will determine if post-ischemic calpain inhibition prevents delayed death of hippocampal CA1 pyramidal neurons.
Specific Aim 2 will characterize calpain-mediated cleavage of Ca2+ regulatory proteins in the postischemic hippocampus by Western blot.
Specific Aim 3 will localize calpain-cleaved Ca2+ regulatory proteins by immunohistochemistry using cleavage site-specific antibodies.
Specific Aim 4 will analyze the functional consequences of Ca2+ regulatory protein cleavage in microsomes and synaptosomes after 1) calpain-mediated proteolysis in vitro or 2) transient ischemia in vivo. In addition, plasma membrane Ca2+-ATPase dysfunction in the post-ischemic hippocampus will be localized by in situ histochemistry. These studies will provide important insights into the causal mechanisms of 1) calpain-mediated neuronal injury, 2) disrupted neuronal Ca2+ homeostasis, and 3) delayed neuronal death. Elucidating these mechanisms is essential for the development of effective therapies for patients suffering from cardiac arrest and stroke.
