Direct Reprogramming of the brain after ischemic stroke in the aged mouse

缺血性中风是人类死亡和残疾的主要原因。除了神经保护之外 在之前的临床试验中失败的策略，再生疗法已经获得了对大脑越来越多的关注 中风后的修复和功能恢复。最近，一项突破性的发现表明，转导 非神经元细胞，如反应性星形胶质细胞与泛神经元转录因子NeuroD1(ND1)和 少数其他人可以将这些细胞直接重新编程为神经前体细胞，甚至是成熟的、有功能的神经元 通过称为直接重编程/转换的过程绕过干细胞阶段。慢病毒载体传递 ND1对反应性星形胶质细胞的作用导致永久重新编程的功能神经元，而不需要 维持基因的异位表达。因此，谱系内直接重新编程意味着 通过利用现有的增殖性星形胶质细胞，获得前所未有的内源性神经发生资源。这个 Proposal是缺血性卒中后直接重编程的一种新应用，并探索了其在 衰老的小鼠。这种方法利用了损伤诱导的星形胶质细胞在脑内激活和积聚的优势 梗死区周围。在损伤部位重新编程的新神经元，称为诱导神经元(iNeurons或INS) 是自体的和有丝分裂后的，这消除了移植的排斥反应和肿瘤形成的风险 外源细胞。在我们的初步实验中，我们成功地将星形胶质细胞转化为成熟的神经元 体外和在局灶性缺血性卒中小鼠模型中。甚至在大脑中也发现了许多转化的INS 中风和中转后一个月。基于我们的体外和体内数据和新出现的证据 从其他组，我们建议在老年局灶性缺血性卒中模型中测试这种再生疗法 老鼠。特定目标1将研究星形胶质细胞在体外和体内重新编程为INS的情况 缺氧/缺血状态。使用带有GFAP启动子和mCherry标记的ND1慢病毒，我们 将验证将反应性星形胶质细胞重新编程为内源性 大脑修复所需的神经供应。《特定目标2》将检验这样一种假设，即在右侧引导重新编程 时间可以减少神经发生的物理和化学障碍。利益的机制和一种 平衡的微环境既具有神经保护作用，又允许再生，将进行测试。 特指3将研究直接转换与增加外周的康复策略相结合 老年小鼠的活动，旨在克服老年神经再生和神经可塑性受损的问题 大脑。我们假设，组合方法促进了活动依赖的神经可塑性，电路 中风后的修复和功能恢复。这三个目标以协调但不同的再生为目标 机制，有令人信服的证据和最先进的技术支持。我们预测每一个目标 单独和/或联合使用将为再生疗法提供新的策略。

Ischemic stroke is a leading cause of human death and disability. In addition to neuroprotective strategies that have failed previous clinical trials, regenerative therapies have gain escalating attention for brain repair and functional recovery after stroke. Recently, a breakthrough discovery demonstrates that transduction of non-neuronal cells such as reactive astrocytes with the panneuronal transcription factor NeuroD1 (ND1) and a few others can reprogram these cells directly into neural progenitors or even mature and functional neurons via a process called direct reprogramming/conversion that bypasses stem cell stage. Lentiviral vector delivery of ND1 to reactive astrocytes results in permanently reprogrammed functional neurons without the need for maintained ectopic expression of the gene. Thus, intra-lineage direct reprogramming implicates an unprecedented resource of endogenous neurogenesis by leveraging existing proliferative astrocytes. The proposal is a novel application of the direct reprogramming after ischemic stroke and explores its application in aged mice. This approach takes the advantages of injury-induced astrocyte activation and accumulation in the peri-infarct region. Reprogrammed new neurons, termed induced neurons (iNeurons or iNs), at the injury site are autologous and post-mitotic, which eliminate the risk of rejection and tumorigenesis of transplanted exogenous cells. In our preliminary experiments, we successfully converted astrocytes into mature neurons in vitro and in focal ischemic stroke models of the mouse. Many converted iNs were identified in the brain even one months after stroke and the conversion. Based on our in vitro and in vivo data and emerging evidence from other groups, we propose to test this regenerative therapy in a focal ischemic stroke model of aged mouse. Specific Aim 1 will study the in vitro and in vivo reprogramming of astrocytes into iNs under hypoxic/ischemic conditions. Using ND1 lentivirus packaged with the GFAP promoter and mCherry marker, we will validate the efficacy, efficiency and time windows of reprogramming reactive astrocytes as an endogenous neuronal supply for brain repair. Specific Aim 2 will test the hypothesis that direct reprogramming at the right time can reduce the physical and chemical barriers for neurogenesis. The mechanism of the benefits and a balanced microenvironment that is neuroprotective as well as permissive for regeneration will be tested. Specific 3 will study the direct conversion combined with a rehabilitative strategy of increased peripheral activities in aged mice, designed to overcome impaired neuroregeneration and neural plasticity in the aged brain. We hypothesize that the combinatorial approach promotes activity-dependent neural plasticity, circuitry repair, and functional recovery after stroke. These three Aims target coordinated but distinct regenerative mechanisms, endorsed by compelling evidence and state-of-the-art technologies. We predict that each Aim alone and/or together will provide novel strategies for a regenerative therapy.