TPA Protects the Synapse in the Iscemic Brain

项目摘要 脑缺血引起突触活动的快速损伤，从而引起神经功能缺损。更 具体而言，缺血损伤诱导α-氨基-3-羟基-5-甲基-4-异恶唑丙酸的动员， 酸受体（AMPAR）从突触后致密区（PSD）转移到突触外部位，导致突触后致密区（PSD）的转化。 活动突触[AMPAR和N-甲基-D-天冬氨酸受体（NMDAR）]转化为沉默突触（ 仅NMDAR），导致突触功能丧失。这种有害影响在早期是可逆的。 在缺血性损伤的早期阶段，但在后期阶段会导致不可逆的突触失败， 永久性神经功能缺损的发展。放大是稳态突触可塑性的一种形式， 沉默的突触（只有NMDAR）通过一系列由NMDAR介导的事件将AMPAR募集到PSD。 突触后密度蛋白95（PSD-95）。这一过程受到细胞周期蛋白依赖性激酶5（Cdk 5）的严格调控。 通过其调节PSD-95在PSD中的表达的能力。Cdk 5通过其在膜中的结合而被激活 它的特异性激活剂p35缺血性中风诱导丝氨酸蛋白酶组织型 纤溶酶原激活物（tPA）从内皮细胞进入血管内空间，在那里它具有纤溶作用 通过其将纤溶酶原切割成纤溶酶的能力介导;并从神经元进入突触间隙， 角色不太清楚。基于其血栓溶解特性，静脉内（IV）施用重组体 tPA（rtPA）用于治疗急性缺血性卒中（AIS）患者。然而，在静脉注射rtPA后， 也穿过血脑屏障（BBB）并到达缺血组织中的突触。 值得注意的是，来自我们实验室的数据表明，tPA保护遭受缺血性损伤的突触。 在本申请中，我们将测试新的假设，即tPA，从突触前末梢释放， 对缺血性损伤的反应，或静脉内给药（rtPA），改善神经功能的结果后， 缺血性中风通过促进Cdk 5介导的缺血性损伤沉默的突触的再激活。我们 提出这种作用不需要纤溶酶的产生，因此没有出血并发症。 我们将在三个具体目标中检验我们的假设。在第一个目标中，我们将调查的机制， tPA增加突触中p35（Cdk 5激活剂）的丰度。在第二部分中，我们将研究 由tPA诱导的p35丰度的增加导致Cdk 5活化。在第三个目标中，我们将使用动物 脑缺血模型，以及一系列神经行为学和神经放射学技术，以测试 用蛋白水解无活性tPA治疗改善缺血性脑卒中后神经学结果的假设 通过触发因缺血性损伤而沉默的突触的重新激活来引发中风。我们假设， tPA与纤溶酶诱导的出血性并发症的发生无关。

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.