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

对于脊髓损伤（SCI）的受害者来说，为了恢复运动功能， 皮质脊髓束（CST）轴突将需要再生并重新连接脊髓内和运动神经， 神经元然而，轴突不能在成熟的损伤脊髓中再生。几十年的研究 对这个问题的深入研究，使我们对轴突生长的机制和为什么 它们在脊髓损伤的情况下失败了，但是还没有出现能够实现长距离轴突再生的策略， 更不用说脊髓损伤的新疗法了为了解决这一未满足的需求，我的实验室专注于如何重新- 激活成熟受损CNS神经元的细胞内轴突生长信号机制， 活跃于发育中的神经元。我们研究的长期目标是使长距离轴突 再生和重建受损脊髓中的功能回路。我们有 最近观察到，皮层运动神经元中RAF-MEK信号的激活使得 在转基因小鼠中损伤的CST轴突的实质性再生生长。我们观察到 在用重复经颅刺激（rTMS）处理的野生型小鼠中具有类似的效果。整体 本申请的目的是彻底探索轴突再生生长的程度， 突触重新连接可以通过RAF-MEK信号传导的升高或通过rTMS来实现。我们 计划追求以下三个具体目标：第一，确定有多少CST轴突 在基因修饰的B-RAF功能获得性小鼠中， 三种不同的SCI模型。第二，我们创造了一部小说 通过融合凝集素WGA与诱导型Cre重组酶的顺行跨突触示踪剂 CreERT 2.在被他莫昔芬激活后，这种示踪剂触发了细胞中选择的蛋白质的表达。 突触后神经元在一个报告小鼠。我们在这里计划表达皮质运动中的示踪剂 神经元，诱导表达基因编码的荧光Ca 2+指示剂， 突触后神经元这将使我们能够标记由新发芽的CST形成的新突触 轴突，并证明其功能活性，反映在Ca 2+瞬变。最后，我们计划 探索rTMS使野生型小鼠中的CST轴突再生的能力。初步数据显示 MEK活性水平与rTMS依赖性CST轴突再生相关。所以我们 将使用MEK 1/2条件性功能丧失小鼠来测试MEK激活是否对rTMS至关重要- 依赖再生这项研究是创新的，因为它利用了新的技术， 方法（rTMS和CreERT 2 WGA融合示踪剂）来解决长距离轴突的问题 脊髓的再生这项研究也很重要，因为它测试了新的概念， 这些策略最终可能有助于SCI患者的轴突修复和功能恢复。

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.