Molecular Therapies to Promote White Matter Restoration After Traumatic Brain Injury

 描述（由申请人提供）：白色物质（WM）损伤，其特征为轴突变性和髓鞘丢失（脱髓鞘），对于创伤性脑损伤（TBI）后的长期功能缺陷很重要。中枢神经系统表现出有限的WM修复能力，如轴突再生和髓鞘再生。在TBI后WM修复期间，已知少突胶质细胞前体细胞（OPC）活跃增殖。然而，许多新产生的OPC不能分化成成熟的髓磷脂。本申请将检查鼻内递送无蛋白酶活性的纤溶酶原激活物突变体（tPAm）以增强寡树突细胞的分化和成熟从而促进TBI后的白色物质完整性的作用。 产生少突胶质细胞（OL），导致髓鞘再生不足。髓鞘再生失败不仅减少了信号转导，而且导致轴突变性和神经退行性疾病的临床结果。因此，促进OPC分化和成熟的干预措施是增强WM修复和改善功能恢复的有希望的策略。 重组组织型纤溶酶原激活剂（tPA）是FDA批准的缺血性卒中治疗药物，通过丝氨酸蛋白酶作用催化血栓溶解。最近的研究发现，tPA的直接神经修复作用独立于蛋白酶活性。然而，tPA作为TBI的治疗剂的临床用途引起了几个问题，因为它也可以引起血脑屏障损伤和脑水肿。 在这项提案中，我们将探索蛋白酶失活突变tPA（tPAm），取代一个单一的氨基酸（S478 A），以消除蛋白酶的作用，作为一种新的恢复性治疗，以促进髓鞘再生和WM修复TBI后。我们发现纳摩尔浓度的tPAm促进培养的原代OPCs分化为成熟的OLs。此外，tPA敲除可加重TBI后持续至少35天的行为缺陷和WM损伤，而损伤后鼻内给予tPAm可改善小鼠TBI后的长期神经功能和WM完整性。初步数据进一步表明，tPAm通过促进OPC分化和轴突髓鞘再生来增强TBI后的WM修复，并且该作用可能由过氧化物酶体增殖物激活受体γ（PPARγ）核受体介导。在这里，我们将重点关注tPAm的新的髓鞘再生作用，并测试以下假设：用蛋白酶失活的tPAm治疗促进TBI后的WM修复和长期神经恢复，至少部分是通过诱导OPC分化和轴突髓鞘形成，通过激活PPARγ。将测试三个具体目标。目的1：确定TBI后用tPAm治疗是否增强WM完整性并促进长期恢复。目的2：验证tPAm通过激活PPAR γ诱导OPC分化/成熟并促进轴突髓鞘形成的假设。目标3：检验tPAm诱导的OPC分化和轴突髓鞘形成对于保护WM完整性和TBI后长期恢复至关重要的假设。这些研究是首次研究tPAm促进TBI髓鞘再生的潜力，并将确定潜在的作用机制，从而为tPAm在临床中的潜在应用奠定基础。

 DESCRIPTION (provided by applicant): White matter (WM) injury, which is characterized by axonal degeneration and loss of the myelin sheath (demyelination), is important for the long-term functional deficits after traumatic brain injury (TBI). The central nervous system exhibits limited capacity for WM repair, such as axonal regeneration and remyelination. During post-TBI WM repair, oligodendrocyte precursor cells (OPCs) are known to actively proliferate. However, many newly generated OPCs fail to differentiate into mature, myelin- This application will examine the effect of intranasal delivery of protease inactive plasminogen activator mutant (tPAm) to enhance differentiation and maturation of oligodendyocytes to promote white matter integrity following TBI. producing oligodendrocytes (OLs), resulting in inadequate remyelination. Failed remyelination not only diminishes signal transduction, but also leads to axon degeneration and worsens clinical outcome. Thus, interventions that promote OPC differentiation and maturation are promising strategies to enhance WM repair and improve functional recovery. Recombinant tissue plasminogen activator (tPA) is an FDA-approved treatment for ischemic stroke and catalyzes thrombolysis through serine protease action. Recent studies have discovered direct neurorestorative effects of tPA independent of protease activities. However, the clinical use of tPA as a therapeutic agent in TBI raises several concerns, as it can also cause blood-brain barrier damage and brain edema. In this proposal, we will explore protease-inactive mutant tPA (tPAm), with substitution of a single amino acid (S478A) to eliminate protease action, as a novel restorative therapy to promote remyelination and WM repair after TBI. We discovered that nanomolar concentrations of tPAm promote the differentiation of cultured primary OPCs into mature OLs. In addition, tPA knockout exacerbates behavioral deficits and WM injury lasting at least 35 days after TBI whereas post-injury intranasal administration of tPAm improves long-term neurological function and WM integrity after TBI in mice. Pilot data further suggest that tPAm enhances WM repair after TBI by promoting OPC differentiation and axon remyelination and that the effect may be mediated by the peroxisome proliferator-activated receptor γ (PPARγ) nuclear receptor. Here we will focus on the novel remyelinating actions of tPAm and test the following hypothesis: Treatment with protease inactive tPAm facilitates WM repair and long-term neurological recovery after TBI, at least in part, by inducing OPC differentiation and axonal myelination through PPARγ activation. Three Specific Aims will be tested. Aim 1: Determine whether post-TBI treatment with tPAm enhances WM integrity and promotes long-term recovery. Aim 2: Test the hypothesis that tPAm induces OPC differentiation/maturation and promotes axonal myelination through PPAR activation. Aim 3: Test the hypothesis that tPAm-induced OPC differentiation and axonal myelination are essential for the protection of WM integrity and long-term recovery after TBI. These studies are the first to investigate the potential for tPAm to foster remyelination in TBI an will identify the underlying mechanism of action, thereby setting the stage for the potential use o tPAm in the clinic.