Engaging neuron-intrinsic signaling for axon growth after spinal cord injury

脊髓损伤后轴突生长的神经元内在信号传导

• 批准号: 9383972
• 负责人: Jian Zhong
• 金额: $ 66.99万
• 依托单位: WINIFRED MASTERSON BURKE MED RES INST
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9383972
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Animal Model; Axon; Back; Behavioral Assay; Brain; Cells; Clinic; Competence; Contralateral; Corticospinal Tracts; Data; Development; Dorsal; Enterobacteria phage P1 Cre recombinase; Frequencies; Genetic; Genetic Recombination; Goals; Growth; Image; Injury; Interneurons; Intervention; Knowledge; Label; Lectin; Left; Lesion; MAP2K1 gene; Measures; Microscopy; Mission; Mitogen-Activated Protein Kinases; Modeling; Monitor; Motor Neurons; Mus; Natural regeneration; Neurons; Optic Nerve; PTEN gene; Paralysed; Pathway interactions; Peripheral; Photons; Pilot Projects; Process; Proteins; Public Health; Recovery of Function; Reporter; Research; Role; Signal Transduction; Site; Spinal; Spinal Cord; Spinal cord injury; Spinal cord injury patients; Synapses; Tamoxifen; Techniques; Testing; Time; Tracer; Translating; United States National Institutes of Health; Wild Type Mouse; Work; axon growth; axon regeneration; base; central nervous system injury; clinical application; curative treatments; fetal; gain of function; in vivo; injured; innovation; insight; loss of function; motor function recovery; mouse model; neurotransmission; novel; postsynaptic; postsynaptic neurons; preclinical study; regenerative; repaired; repetitive transcranial magnetic stimulation; spinal nerve posterior root; synaptic function; synaptogenesis; transcription factor; treatment strategy

For victims of spinal cord injury (SCI) to recover motor function, large numbers of damaged corticospinal tract (CST) axons would need to regenerate and re-connect with spinal inter- and motor neurons. However, axons do not regenerate in the mature injured spinal cord. Decades of research into this problem have yielded much insight into the mechanisms of axon growth and reasons why they fail in the SCI context, but no strategies enabling long-range axon regeneration have emerged, much less new treatments for SCI. To address this unmet need, my lab focuses on ways to re- activate in mature injured CNS neurons the intracellular axon growth signaling mechanisms that are active in developing neurons. The long-term goal of our research is to enable long-range axon regeneration and the re-establishment of functional circuitry in the injured spinal cord. We have recently observed that activation of RAF – MEK signaling in cortical motor neurons enables substantial regenerative growth of injured CST axons in genetically modified mice. We observed similar effects in wild type mice treated with repetitive transcranial stimulation (rTMS). The overall objective of this application is to thoroughly explore the extent of axon regenerative growth and synaptic re-connection that can be achieved by elevation of RAF – MEK signaling, or by rTMS. We plan to pursue the following three Specific Aims: First, to determine how much CST axon regeneration or sprouting can be stimulated in genetically modified B-RAF gain-of function mice subjected to three different established models of SCI. Second, we have generated a novel anterograde transsynaptic tracer by fusing the lectin WGA with the inducible Cre recombinase CreERT2. Upon activation by tamoxifen, this tracer triggers the expression of a protein of choice in postsynaptic neurons in a reporter mouse. We here plan to express the tracer in cortical motor neurons, to induce expression of a genetically encoded fluorescent Ca2+ indicator in their postsynaptic neurons. This will allow us to label new synapses formed by newly sprouting CST axons, and also to demonstrate their functional activity as reflected in Ca2+ transients. Finally, we plan to explore the power of rTMS to enable CST axon regeneration in wild type mice. Initial data indicate that the level of MEK activity correlates with rTMS-dependent CST axon regeneration. Therefore, we will use MEK1/2 conditional loss-of-function mice to test whether MEK activation is crucial for rTMS- dependent regeneration. The proposed study is innovative, as it takes advantage of new technical approaches (rTMS and the CreERT2WGA fusion tracer) to address the problem of long-range axon regeneration in the spinal cord. This research is also significant because it tests new concepts and strategies that may eventually contribute to axonal repair and functional recovery in SCI patients.

为了使恐怖犯罪（SCI）恢复运动功能，大量受损 皮质脊髓区（CST）轴突将需要与脊柱间和运动重新连接并重新连接 神经元。但是，轴突不会在成熟受伤的脊髓中再生。数十年的研究 进入这个问题已经对轴突生长的机制有很多深入的了解，以及原因 它们在SCI环境中失败，但是没有能够实现远距离轴突再生的策略 SCI的新疗法少得多。为了满足这种未满足的需求，我的实验室专注于重新 在成熟受伤的中枢神经系统神经元中激活细胞内轴突生长信号传导机制 积极发展神经元。我们研究的长期目标是启用远程轴突 在受伤的脊髓中的功能电路的再生和重新建立。我们有 最近观察到皮质运动神经元中RAF - MEK信号传导的激活启用 一般修饰的小鼠中受伤的CST轴突的重大再生生长。我们观察到 在用重复经颅刺激（RTMS）处理的野生型小鼠中的类似作用。总体 该应用的目的是彻底探索轴突再生增长的程度和 可以通过升高RAF - MEK信号传导或RTMS来实现的突触重新连接。我们 计划追求以下三个特定目标：首先，确定多少CST轴突 可以在一般修饰的B-RAF功能鼠标中刺激再生或发芽小鼠 受到三种不同的SCI模型。其次，我们生成了一本小说 通过将讲座WGA与诱导的CRE重组酶融合，顺行透射示踪剂 creert2。他莫昔芬激活后，该示踪剂会触发选择蛋白在 记者小鼠中的突触后神经元。我们在这里计划在皮质电机中表达示踪剂 神经元，诱导在其中诱导一般编码的荧光Ca2+指示器的表达 突触后神经元。这将使我们能够标记新发芽的CST形成的新突触 轴突，还可以证明其功能活性，如Ca2+瞬变所反映的。最后，我们计划 探索RTM的功率，使CST轴突在野生型小鼠中的再生。初始数据指示 MEK活性的水平与依赖RTMS的CST轴突再生有关。因此，我们 将使用MEK1/2有条件的功能丧失小鼠来测试MEK激活对RTMS是否至关重要 - 依赖性再生。拟议的研究是创新的，因为它利用了新的技术 方法（RTMS和CREERT2WGA融合示踪剂）解决了远程轴突的问题 脊髓的再生。这项研究也很重要，因为它测试了新概念和 最终可能有助于SCI患者轴突修复和功能恢复的策略。