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

为了让脊髓损伤(SCI)的受害者恢复运动功能，大量的损伤 皮质脊髓束(CST)轴突需要再生并与脊髓间和运动重新连接 神经元。然而，轴突不会在成熟的损伤脊髓中再生。几十年的研究 对这一问题的研究为轴突生长的机制和原因提供了更多的见解 它们在脊髓损伤的背景下失败了，但还没有出现能够实现远程轴突再生的策略， 更不用说治疗脊髓损伤的新疗法了。为了解决这一未得到满足的需求，我的实验室专注于如何重新 在成熟的受损中枢神经系统神经元中激活细胞内轴突生长信号机制，这是 活跃于发育中的神经元。我们研究的长期目标是使远程轴突 损伤脊髓的再生和功能回路的重建。我们有 最近观察到，激活皮质运动神经元中的RAF-MEK信号使 转基因小鼠损伤的cst轴突的大量再生生长。我们观察到 在重复经颅刺激(RTMS)治疗的野生型小鼠中也有类似的效果。整体而言 本应用的目的是深入探讨轴突再生生长的程度和 突触重新连接可以通过提升RAF-MEK信号或rTMS来实现。我们 计划追求以下三个具体目标：第一，确定多少CST轴突 转基因B-RAF功能增强小鼠的再生或萌发 应用三种不同的脊髓损伤模型。第二，我们创作了一部小说 凝集素WGA与可诱导的Cre重组酶融合形成顺行跨突触示踪剂 CreerT2。一旦被他莫昔芬激活，这个示踪剂就会触发选择的蛋白质在 报道小鼠的突触后神经元。我们计划在皮质马达中表达示踪剂。 诱导神经元表达一种遗传编码的荧光钙离子指示剂 突触后神经元。这将使我们能够标记由新萌发的CST形成的新突触 也是为了证明它们在钙瞬变过程中的功能活性。最后，我们计划 探讨rTMS对野生型小鼠CST轴突再生的作用。初步数据显示 MEK活性水平与依赖rTMS的CST轴突再生相关。因此，我们 将使用MEK1/2条件功能丧失小鼠来测试MEK激活是否对rTMS至关重要- 依赖再生。这项拟议的研究具有创新性，因为它利用了新的技术 解决远距离轴突问题的方法(rTMS和CreERT2WGA融合示踪剂) 脊髓的再生。这项研究还具有重要意义，因为它测试了新的概念和 最终可能有助于脊髓损伤患者轴突修复和功能恢复的策略。

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.