Novel approaches to stimulating neurotrophin signaling for stroke recovery

项目总结/摘要 中风是成人神经功能障碍的主要原因，幸存者通常会留下永久性缺陷， 由缺血诱导的脑损伤引起的神经元损失。目前还没有成功的治疗方法， 一旦发生脑损伤，恢复中风患者的正常功能，康复除外 治疗，这是有限的，在其能力，以促进完全恢复。在临床前研究中， 用来自未受伤神经组织的新的神经解剖学连接替代受损的通路， 被称为神经可塑性的过程，已被证明是改善功能恢复的有前途的策略 伤后我们的实验室已经使用抗体疗法来阻断神经突抑制蛋白Nogo， A促进神经可塑性并显著改善受 实验性中风然而，抗Nogo-A抗体的细胞和分子机制- 介导的恢复在很大程度上仍然未知。我们之前已经证明Nogo-A可以改变神经 生长因子（NGF）依赖的神经营养因子信号传导对神经元存活和神经突产生负面影响 结果该提案的目标是验证TrkA信号传导的中断是中风的结果- 诱导Nogo-A表达，并验证旨在重新建立可塑性促进的方法， 通过NGF受体（TrkA和p75 NTR）的神经营养因子信号传导。我们的中心假设是， 规避或阻断Nogo-A对TrkA的抑制以刺激TrkA介导的神经营养因子信号传导途径 将增强神经可塑性并改善缺血性中风后的恢复。在目标1中，我们将使用primary 在培养的神经元中检测受Nogo-A影响的TrkA和p75 NTR神经营养因子信号传导机制， 设计用于规避或阻断Nogo-A对TrkA信号传导的抑制作用的测试策略。在目标2中， 将确定Nogo-A在体内缺血性卒中后TrkA介导的神经营养因子信号传导中的作用 使用神经元特异性Nogo-A敲除小鼠。将使用生物化学和免疫组织化学技术 评估中风对NGF受体及其下游效应物的影响，以及它们之间的联系。 野生型和敲除小鼠中Nogo-A受体的表达。在目标3中，我们将评估治疗干预措施， 规避或阻断Nogo-A对TrkA受体的抑制作用以增强中风后的恢复。我们 将使用我们研究良好的体内中风模型来确定抗Nogo-A治疗的程度， 抗体或新型选择性TrkA激动剂藤黄酰胺影响NGF信号传导和中风恢复。 总之，这项提议的结果将增加我们对潜在的神经营养因子介导的 中风损伤导致的信号传导机制改变。因此，拟议的工作将提供一个基础， 新的和有前途的治疗中风康复，这是至关重要的设计和 未来临床试验的成功结果。

Project Summary/Abstract Stroke is a leading cause of adult neurologic disability with survivors often left with permanent deficits due to neuronal loss resulting from ischemia-induced brain injury. There are currently no successful treatments to restore normal function to stroke patients once brain damage has occurred, with the exception of rehabilitation therapy, which is limited in its ability to promote full recovery. In preclinical studies, treatments that stimulate the replacement of damaged pathways with new neuroanatomical connections from uninjured neural tissue, a process known as neuroplasticity, have proven to be promising strategies for improving recovery of function after injury. Our laboratory has used antibody therapy directed at blocking the neurite inhibitory protein Nogo- A to promote neuroplasticity and significantly improve the functional outcome of animals affected by experimental stroke. However, the cellular and molecular mechanisms responsible for anti-Nogo-A antibody- mediated recovery remain largely unknown. We have previously demonstrated that Nogo-A alters nerve growth factor (NGF)-dependent neurotrophin signaling to negatively influence neuronal survival and neurite outgrowth. The goal of this proposal is to verify that disruption of TrkA signaling is a consequence of stroke- induced Nogo-A expression, and to validate approaches designed to re-establish plasticity-promoting neurotrophin signaling by NGF receptors (TrkA and p75NTR). Our central hypothesis is that treatments that circumvent or block Nogo-A inhibition of TrkA to stimulate TrkA-mediated neurotrophin signaling pathways will enhance neuroplasticity and improve recovery following ischemic stroke. In Aim 1 we will use primary neurons in culture to examine TrkA and p75NTR neurotrophin signaling mechanisms affected by Nogo-A and test strategies designed to circumvent or block the inhibitory effect of Nogo-A on TrkA signaling. In Aim 2 we will determine the role of Nogo-A on TrkA-mediated neurotrophin signaling following ischemic stroke in vivo using neuron-specific Nogo-A knockout mice. Biochemical and immunohistochemical techniques will be used to assess the effect of stroke on NGF receptors, their downstream effectors and the association of these receptors with Nogo-A in wild type and knockout mice. In Aim 3 we will evaluate therapeutic interventions that circumvent or block the inhibitory action of Nogo-A on the TrkA receptor to enhance recovery after stroke. We will use our well-studied in vivo stroke model to determine the extent to which treatment with anti-Nogo-A antibody or the novel selective TrkA agonist gambogic amide affects NGF signaling and stroke recovery. Together, the results from this proposal will increase our understanding of underlying neurotrophin-mediated signaling mechanisms altered as a result of stroke injury. Thus, the proposed work will provide a foundation for novel and promising therapies for stroke rehabilitation, which is critically important for the design and successful outcome of future clinical trials.