TPA Protects the Synapse in the Iscemic Brain

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PROJECT SUMMARY Cerebral ischemia causes a rapid impairment of synaptic activity that cause neurological deficits. More specifically, the ischemic injury induces the mobilization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) from the postsynaptic density (PSD) to extrasynaptic sites, leading to the conversion of active synapses [with AMPARs and N-Methyl-D-aspartic acid receptors (NMDARs)] into silent synapses (with only NMDARs), with the resultant loss of synaptic function. This deleterious effect is reversible in the early phases of the ischemic injury, but at later stages causes irreversible synaptic failure that leads to the development of permanent neurological deficits. Scaling up is a form of homeostatic synaptic plasticity whereby silent synapses (with only NMDARs) recruit AMPARs to the PSD by a sequence of events mediated by the postsynaptic density protein-95 (PSD-95). This process is tightly regulated by cyclin-dependent kinase 5 (Cdk5) by its ability to regulate the expression of PSD-95 in the PSD. Cdk5 is activated by its binding in the membrane to its specific activator, p35. Ischemic stroke induces the release of the serine proteinase tissue-type plasminogen activator (tPA) from endothelial cells into the intravascular space, where it has a fibrinolytic effect mediated by its ability to cleave plasminogen into plasmin; and from neurons into the synaptic cleft, where its role is less well understood. Based on its thrombolytic properties, intravenous (IV) administration of recombinant tPA (rtPA) is used to treat acute ischemic stroke (AIS) patients. However, following its IV administration rtPA also crosses through the blood-brain barrier (BBB) and reaches the synapses in the ischemic tissue. Significantly, data from our laboratory indicate that tPA protects the synapse that has suffered an ischemic injury. In this application we will test the novel hypothesis that tPA, either released from the presynaptic terminal in response to the ischemic insult, or intravenously administered (rtPA), improves neurological outcome after an ischemic stroke by promoting Cdk5-mediated reactivation of synapses silenced by the ischemic injury. We propose that this effect does not require plasmin generation, and thus is devoid of hemorrhagic complications. We will test our hypothesis in three Specific Aims. In the first Aim we will investigate the mechanism whereby tPA increases the abundance of p35 (Cdk5 activator) in the synapse. In the second we will study whether the increase in p35 abundance induced by tPA leads to Cdk5 activation. In the third Aim we will use an animal model of cerebral ischemia, and a battery of neurobehavioral and neuroradiological techniques, to test the hypothesis that treatment with proteolytically inactive tPA improves neurological outcome after an ischemic stroke by triggering the reactivation of synapses silenced by an ischemic injury. We postulate that this effect of tPA is not associated with the development of plasmin-induced hemorrhagic complications.