White matter restoration and functional recovery after experimental stroke

 描述（由申请人提供）：白色物质（WM）损伤，以脱髓鞘和轴突完整性丧失为特征，是卒中后长期感觉运动和认知缺陷的重要原因。WM修复包括轴突再生、少突胶质细胞生成和脱髓鞘或新生轴突的髓鞘形成，将有助于重建神经元连接和重建轴突信号传导。不幸的是，成人脑具有有限的髓鞘再生能力，至少部分是由于少突胶质细胞前体细胞（OPC）分化为成熟的髓鞘化少突胶质细胞（OL）的失败。因此，促进OPC分化的干预措施可能有助于卒中患者的轴突髓鞘再生、WM修复和长期神经功能恢复。 重组组织型纤溶酶原激活剂（tPA）是FDA批准的唯一用于缺血性卒中溶栓治疗的药物。然而，最近的研究发现，tPA的各种神经保护作用是独立于其溶栓活性。此外，tPA的鼻内给药可改善功能恢复，促进卒中后轴突发芽。目前，tPA的使用仅限于中风发作后的前4.5小时，因为超过这个时间窗，它通过BBB损伤极大地增加了脑出血（ICH）的风险。该提案将研究tPAm（一种缺乏溶栓活性（因此不会诱导ICH）的tPA突变形式）在缺血性卒中啮齿动物模型中的疗效。我们已经在原代培养物中发现，tPAm有效地促进OPC分化为成熟OL。tPAm的这种作用取决于其激活PPAR，这是一种调节细胞分化并具有抗氧化和抗炎功能的主转录因子。体内研究表明，缺乏内源性tPA加重功能缺陷和WM损伤远端MCAO后长达35天。相反，短暂MCAO后6小时给予tPAm可改善长期神经行为和WM完整性。我们的研究结果表明，tPAm增强中风后WM的完整性，至少部分，通过促进OPC分化和轴突髓鞘形成。本研究将探讨tPAm的新的WM修复增强作用及其相关机制。我们将测试以下总体假设：治疗与蛋白酶失活tPAm促进WM修复和中风后的长期神经功能恢复，至少部分通过促进OPC分化和轴突髓鞘通过PPAR。将对年轻成年小鼠和老年小鼠进行试验。提出了三个具体目标。目的1：确定卒中后用蛋白酶失活的tPAm治疗是否能增强WM的完整性并促进长期神经功能恢复。目的2：验证tPAm通过激活PPAR β诱导OPC分化/成熟并促进轴突髓鞘形成的假设。原代OPC和神经元-OPC共培养系统将被应用于测试这一假设。目标3：检验tPAm诱导的OPC分化和轴突髓鞘形成/髓鞘再生对于脑卒中后WM完整性和长期神经恢复的有益作用至关重要的假设。

 DESCRIPTION (provided by applicant): White matter (WM) injury, characterized by demyelination and loss of axonal integrity, is an important cause of long-term sensorimotor and cognitive deficits after stroke. WM repair, including axonal regrowth, oligodendrogenesis and the myelination of demyelinated or newly generated axons, would help rebuild neuronal connectivity and reestablish axonal signal conduction. Unfortunately, the adult brain has limited capacity for remyelination, at least in part due to the failure of differentiation of oligodendrocyte precursor cells (OPCs) into mature, myelinating oligodendrocytes (OLs). Thus, interventions that promote OPC differentiation may facilitate axonal remyelination, WM repair and long-term neurological recovery in stroke patients. Human recombinant tissue plasminogen activator (tPA) is the only FDA approved drug for the thrombolytic treatment of ischemic stroke. However, recent research has discovered various neuroprotective effects by tPA that are independent of its thrombolytic activity. Moreover, intranasal administration of tPA improves functional recovery and promotes axonal sprouting after stroke. Currently, the use of tPA is limited to the first 4.5 hr after the oset of stroke, as beyond this time window it tremendously increases the risk of intracerebral hemorrhage (ICH) via BBB damage. This proposal will investigate the efficacy of tPAm, a mutant form of tPA that lacks thrombolytic activity (thus would not induce ICH), in rodent models of ischemic stroke. We have found in primary cultures that tPAm potently promotes the differentiation of OPCs into mature OLs. This effect of tPAm depends on its activation of PPAR, a master transcriptional factor that regulates cell differentiation and possesses antioxidant and anti-inflammatory functions. In vivo studies suggest that lack of endogenous tPA exacerbates functional deficits and WM injury up to 35 days after distal MCAO. In contrast, tPAm administration 6 hr after transient MCAO improved long-term neurological behavior and WM integrity. Our results suggest that tPAm enhances post-stroke WM integrity, at least in part, by promoting OPC differentiation and axonal myelination. This proposal will investigate the novel WM repair-enhancing role of tPAm and the underlying mechanisms. We will test the following overarching hypothesis: Treatment with protease-inactive tPAm facilitates WM repair and long-term neurological recovery after stroke, at least in part by promoting OPC differentiation and axonal myelination through PPAR. Both young adult and aged mice will be tested. Three Specific Aims are proposed. Aim 1: Determine whether post-stroke treatment with the protease-inactive tPAm enhances WM integrity and promotes long-term neurological recovery. Aim 2: Test the hypothesis that tPAm induces OPC differentiation/maturation and promotes axonal myelination via PPAR activation. Primary OPCs and a neuron-OPC co-culture system will be applied to test this hypothesis. Aim 3: Test the hypothesis that tPAm-induced OPC differentiation and axonal myelination/remyelination are essential for its beneficial effects on WM integrity and long-term neurological recovery after stroke.