Examining mRNA localization in central sensory axons after peripheral nerve injury

在周围神经系统中的神经损伤后，再生容易发生，通常具有取决于损伤的严重性的一些功能恢复;然而，神经损伤后的异常再生反应可能导致神经性疼痛。研究表明，周围神经损伤后，脊髓痛觉感受板中的中枢感觉轴突发生侧支发芽，这可能有助于神经病理性疼痛的发展。我们实验室和其他人最近的工作表明，外周神经内直接翻译的mRNA是损伤后再生所必需的。然而，外周刺激可以改变mRNA的运输或翻译在中央投射DRG轴突的可能性尚未探讨。我推测，周围神经损伤触发感觉神经元基因表达的变化和随后的转运到中央投射轴突的mRNA，导致这些轴突的生长能力的变化。我将使用RT-PCR和原位杂交来量化周围神经损伤前后中枢投射轴突中编码生长相关和神经元信号蛋白的轴突mRNA。为了确定中枢投射DRG轴突中轴突mRNA水平的任何变化是否由损伤诱导的转录驱动，我将使用转录调控减弱的Importin β 1 3'-UTR敲除小鼠。我还将使用病毒介导的基因转移来增加或减少编码生长相关蛋白的mRNA的轴突水平，以研究这如何影响中枢投射感觉轴突的发芽和神经性疼痛的发展。总的来说，这项提案中的工作将提供分子神经生物学技术的培训，并将告诉我们，mRNA转运到中枢感觉轴突中并局部产生蛋白质是否有助于脊髓发芽并进展为神经性疼痛。

Following injury to nerves in the peripheral nervous system, regeneration readily occurs oftentimes with some functional recovery depending on the severity of the injury; however, aberrant regenerative responses after nerve injury could lead to neuropathic pain. Research has suggested that collateral sprouting of central sensory axons in pain receptive lamina of the spinal cord occurs after peripheral nerve injury and this may contribute to the development of neuropathic pain. Recent works from our laboratory and others have shown that mRNAs translated directly within peripheral nerves are needed for regeneration after injury. However, the possibility that peripheral stimuli can alter mRNA transport or translation in centrally projecting DRG axons has not been explored. I hypothesize that injury to peripheral nerves triggers changes in sensory neuron gene expression and subsequent transport of mRNAs into centrally projecting axons that result in changes in the growth capacity of those axons. I will use RT-PCR and in situ hybridization to quantify axonal mRNAs encoding growth-associated and neuronal signaling proteins in centrally projecting axons before and after peripheral nerve injury. To determine if any changes in axonal mRNA levels in centrally projecting DRG axons are driven by injury-induced transcription I will use Importin β1 3’-UTR knockout mice in which transcriptional regulation is attenuated. I will also use viral-mediated gene transfer to increase or decrease the axonal levels of mRNAs encoding growth associated proteins to examine how this influences sprouting of centrally projecting sensory axons and the development of neuropathic pain. Overall, the work in this proposal will provide training in molecular neurobiology techniques and will serve to tell us if transport of mRNAs into central sensory axons with localized generation of proteins contributes to sprouting in the spinal cord and progression to neuropathic pain.