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 虽然这些策略在以前的临床试验中失败了，但再生疗法已经获得了越来越多的关注， 中风后的修复和功能恢复。最近，一项突破性的发现表明， 非神经元细胞，如具有泛神经元转录因子NeuroD 1（ND 1）的反应性星形胶质细胞， 其他一些人可以将这些细胞直接重编程为神经祖细胞，甚至是成熟的功能性神经元。 通过一个称为直接重编程/转换的过程，绕过干细胞阶段。慢病毒载体递送 ND 1与反应性星形胶质细胞的结合导致永久重编程的功能神经元，而不需要 维持基因的异位表达。因此，谱系内直接重编程暗示了 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。的 该提案是缺血性卒中后直接重编程的一种新应用，并探讨了其在 老老鼠这种方法利用了损伤诱导的星形胶质细胞活化和积聚的优势， 梗死周围区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发生的风险。 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。 小鼠的体外和局灶性缺血性中风模型。许多转化的iN在大脑中被识别出来， 中风和转变后一个月。基于我们的体外和体内数据以及新出现的证据 从其他小组，我们建议测试这种再生疗法在局灶性缺血性中风模型的老年人， 老鼠.具体目标1将研究星形胶质细胞在体外和体内重编程为iN， 缺氧/缺血状况。使用包装有GFAP启动子和mCherry标记的ND 1慢病毒， 将验证将反应性星形胶质细胞重编程为内源性星形胶质细胞的功效、效率和时间窗。 神经元供应来修复大脑具体目标2将检验以下假设： 时间可以减少神经发生的物理和化学障碍。利益机制和 将测试具有神经保护性以及允许再生的平衡微环境。 具体3将研究直接转换结合外周增加的康复策略 活动，旨在克服老年人受损的神经再生和神经可塑性 个脑袋我们假设，组合方法促进活动依赖的神经可塑性，电路 修复和中风后的功能恢复。这三个目标的目标协调，但不同的再生 这些机制得到了令人信服的证据和最先进的技术的支持。我们预测，每个目标 单独和/或一起将为再生治疗提供新的策略。

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.