Promoting targeted reinnervation of phrenic motor neurons and restoration of respiratory function using cell-specific expression of BDNF after cervical spinal cord injury

项目摘要 我们正在测试一种新的策略，以再生受损的下行球脊髓呼吸轴突， 大鼠颈髓损伤（SCI）后膈运动神经元（PhMN）的再神经支配。SCI是由创伤引起的 超过一半的病例发生在颈部，导致呼吸障碍 通过破坏呼吸控制回路SCI引起的功能缺陷的恢复是有限的 由于神经元再生轴突的内在驱动力低以及缺乏向生长轴突发出信号的引导线索 适当的目标，除其他外。C3-C5中颈脊髓水平容纳PhMN，其是 负责隔膜激活。PhMN主要由脊髓上神经支配， 呼吸神经元位于脑干核，称为喙侧呼吸组（rVRG）。我们 寻求通过恢复控制PhMN的关键回路来逆转SCI后的呼吸功能障碍， 隔膜激活。脑源性神经营养因子（BDNF），生长神经营养因子家族的成员 因子，促进轴突生长，并作为一个指导线索。磷酸酶和张力蛋白同源物（PTEN）是一种 哺乳动物雷帕霉素靶蛋白（mTOR）的负调节因子，负责促生长途径 包括轴突生长。已显示下调PTEN诱导轴突再生。我们的目标是 促进PhMN的靶向神经再支配，并通过全身性抑制PTEN来恢复膈肌功能， 拮抗肽通过损伤诱导轴突生长，然后选择性地过表达BDNF， PhMN通过腺相关病毒（AAV）引导生长的轴突。在目标1中，我们将确定 提供轴突导向分子BDNF的PhMN特异性来源，促进靶向PhMN神经再支配 脊髓损伤后rVRG轴突的变化。我们将使用表达双重表达的AAV载体评估rVRG轴突。 顺行/跨突触示踪剂，检查再生和侧支发芽，并识别突触 通过跨突触标志物的突触后积累与备用PhMN重新连接。在目标2中，我们将 确定rVRG-PhMN回路重新连接是否促进颈椎SCI后的脊髓恢复。我们 将通过测试体内膈肌来评估rVRG-PhMN重新连接恢复膈肌功能的能力 通过肌电图（EMG）激活。令人兴奋的是，我们可以区分恢复模式，即同侧 再生与对侧发芽，通过选择性沉默单侧rVRG神经元与抑制设计师 受体专门激活的设计药物（DREADDs），并记录任何后续变化， 肌电图。BDNF过表达与神经性疼痛和运动功能异常有关。我们将测试 BDNF的意外后果，包括疼痛表型和运动增益/缺陷。我们的目标是利用 本文提出的策略是将负责膈肌激活的运动神经元与呼吸中枢重新连接 在脊髓中恢复颈椎脊髓损伤后中断的呼吸功能。潜在的治疗 正在探索的益处将对患有呼吸功能障碍的SCI患者产生深远的影响。

PROJECT SUMMARY We are testing a novel strategy to regenerate damaged descending bulbospinal respiratory axons and reinnervate phrenic motor neurons (PhMN) after cervical spinal cord injury (SCI) in rats. SCI is caused by trauma to the spinal cord, and more than half of all cases occur in the cervical region, leading to breathing compromise by damaging circuits involved in respiratory control. Restoration of functional deficits caused by SCI is limited due to the low intrinsic drive of neurons to regenerate axons and a lack of guidance cues to signal growing axons to appropriate targets, among others. The C3-C5 mid-cervical spinal cord levels house the PhMNs, which are responsible for diaphragm activation. PhMNs are predominately mono-synaptically innervated by supraspinal respiratory neurons located in a brainstem nucleus called the rostral Ventral Respiratory Group (rVRG). We are seeking to reverse respiratory dysfunction after SCI by restoring the crucial circuit controlling PhMNs, and thus diaphragm activation. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, promotes axonal growth and acts as a guidance cue. Phosphatase and tensin homolog (PTEN) is a negative regulator of mammalian target of rapamycin (mTOR), which is responsible for pro-growth pathways including axon growth. Downregulation of PTEN has been shown to induce axon regeneration. We aim to promote targeted reinnervation of PhMNs and restore diaphragm function by systemically inhibiting PTEN with antagonist peptides to induce axon growth through the injury, followed by BDNF overexpression selectively in PhMNs via an adeno-associated virus (AAV) to direct growing axons. In Aim 1, we will determine whether providing a PhMN-specific source of the axon guidance molecule, BDNF, promotes targeted PhMN reinnervation by rVRG axons following cervical SCI. We will assess rVRG axons using an AAV vector expressing a dual anterograde/trans-synaptic tracer, examining regrowth and collateral sprouting, and identify synaptic reconnection with spared PhMNs by post-synaptic accumulation of the trans-synaptic marker. In Aim 2, we will determine whether rVRG-PhMN circuit re-connectivity promotes diaphragmatic recovery after cervical SCI. We will assess the ability of rVRG-PhMN reconnection to restore diaphragm function by testing in vivo diaphragm activation via electromyography (EMG). Excitingly, we can distinguish between modes of recovery, i.e. ipsilateral regrowth versus contralateral sprouting, by selectively silencing unilateral rVRG neurons with inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) and recording any subsequent changes in EMGs. BDNF overexpression is associated with neuropathic pain and abnormal motor function. We will test for unintended consequences of BDNF, including pain phenotypes and motor gains/deficits. We aim to use the strategy proposed here to reconnect motor neurons responsible for diaphragm activation with respiratory centers in the medulla to restore respiratory function following disruption after cervical SCI. The potential therapeutic benefits being explored will have profound implications for SCI patients suffering respiratory dysfunction.