Serine proteases are best known for their role in the blood coagulation/fibrinolysis pathways. However, it is becoming apparent that serine proteases and their receptors serve a role in the central nervous system (CNS). Indeed, the CNS expresses many components of serine protease-signaling systems, including precursors to the proteases, thrombin, plasmin, Factor Xa, tissue plasminogen activator (tPaA),urokinase, as well as the brain specific proteases and serpin protease inhibitors. At least one protease activated receptor (PAR1) is expressed in neurons and glia. The presence of elements of the clotting system in the CNS holds intriguing implications for situations in which blood constituents penetrate brain tissue, since entry of blood proteases might cause aberrant activation of signaling pathways in the brain that compromise neuronal survival. Consistent with this idea, thrombin, plasmin, and tPA have been implicated in various models of neurotoxicity. Our preliminary data showing that thrombin/plasmin potential the NMDA receptor by 2-6 fold through relief of Mg2+ blockade is consistent with the idea that serine proteases have harmful effects if they are extravasated during disruption of the blood brain barrier. Given the prominent role of NMDA receptor over-activation in mediating neuronal damage during neurotrauma or ischemia, potentiation of NMDA receptor function could enhance the neurotoxic accumulation of Ca2+ in neurons during stroke or brain injury-two situations in which the blood brain barrier is compromised. Alternatively, enhancement of NMDA receptor function by extravasated serine proteases might play in posttraumatic epilepsy. The goal of the experiments described in this proposal is to better understand the molecular and biophysical mechanisms underlying serine protease actions, as well as the role of serine proteases in animal models of stroke. Five experimental directions are proposed to probe serine protease action on NMDA reception function and neuronal survival in vivo. 1) Electrophysiological recording from neurons in hippocampal slices from knockout mice that lack PAR1 will evaluate the role of this protease receptor in mediating thrombin/plasmin effectors on the NMDA receptor. 2) Experiments in excised patches from neurons in hippocampal slices will determine the biophysical mechanism by which serine protease relieve Mg2+ blockade of NMDA receptors. 3) Dual electrophysiological/Ca2+ imaging experiments will evaluate the role of intracellular Ca2+ and PKC activation in the mediation of thrombin/plasmin actions on the NMDA receptor. 4) Cytochemical experiments will test whether thrombin, prothrombin, or plasminogen are extravasated during ischemia. 5) In vivo experiments will test whether removal of the PAR1 gene alters neuronal survival following transient ischemia. These experiments should provide a better understanding of serine protease signaling in the brain and its role in stroke.
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