Novel approaches to stimulating neurotrophin signaling for stroke recovery

项目摘要/摘要 中风是成人神经残疾的主要原因，幸存者通常因以下原因而留下永久性的残疾 缺血诱导的脑损伤导致的神经元丢失。目前还没有成功的治疗方法 脑损伤发生后，中风患者恢复正常功能，但康复除外 治疗，其促进完全康复的能力有限。在临床前研究中，刺激 用未损伤的神经组织的新神经解剖连接取代受损的通路 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 受伤后。我们实验室已经使用抗体疗法来阻断轴突抑制蛋白Nogo- A促进神经可塑性并显著改善受影响的动物的功能结局 实验性中风。然而，抗Nogo-A抗体的细胞和分子机制- 调解的复苏在很大程度上仍是未知的。我们之前已经证明了Nogo-A可以改变神经 生长因子(NGF)依赖的神经营养因子信号对神经元存活和突起的负面影响 外延生长。这项建议的目标是验证TrkA信号的中断是中风的结果- 诱导Nogo-A表达，并验证旨在重建促进可塑性的方法 神经营养因子通过神经生长因子受体(TrkA和p75NTR)的信号传导。我们的中心假设是，治疗 绕过或阻断Nogo-A抑制TrkA刺激TrkA介导的神经营养因子信号通路 将增强神经可塑性，促进缺血性中风后的恢复。在目标1中，我们将使用主要 Nogo-A和Nogo-A对培养神经元TrkA和p75NTR神经营养因子信号机制的影响 旨在规避或阻断Nogo-A对TrkA信号的抑制作用的测试策略。在《目标2》中，我们 将确定Nogo-A在体内对TrkA介导的缺血性中风后神经营养因子信号转导的作用 使用神经元特异性Nogo-A基因敲除小鼠。将使用生化和免疫组织化学技术 目的：评估中风对神经生长因子受体及其下游效应器的影响及其相互关系。 野生型和基因敲除小鼠的Nogo-A受体。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，以促进中风后的康复。我们 将使用我们研究充分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Gamboicamine影响NGF信号转导和卒中康复。 总之，这一提议的结果将增加我们对潜在的神经营养因子介导的理解。 信号机制因中风损伤而改变。因此，拟议的工作将为 新的和有希望的中风康复疗法，这对设计和 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.