Examining mRNA localization in central sensory axons after peripheral nerve injury

周围神经系统损伤后，根据损伤的严重程度，再生往往容易发生，并伴有一定程度的功能恢复；然而，神经损伤后异常的再生反应可导致神经性疼痛。研究表明，周围神经损伤后，脊髓疼痛感受层中枢感觉轴突的侧枝萌发可能与神经性疼痛的发生有关。我们的实验室和其他人最近的工作表明，损伤后再生需要直接在周围神经中翻译的mrna。然而，外周刺激是否能改变DRG轴突中mRNA转运或翻译的可能性尚未被探索。我假设外周神经的损伤触发了感觉神经元基因表达的变化，随后mrna转运到中央突出的轴突，导致轴突生长能力的变化。我将使用RT-PCR和原位杂交来量化周围神经损伤前后中央突出轴突中编码生长相关蛋白和神经元信号蛋白的轴突mrna。为了确定中央突出DRG轴突轴突mRNA水平的任何变化是否由损伤诱导的转录驱动，我将使用转录调节减弱的Importin β1 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央突出的感觉轴突的发芽和神经性疼痛的发展。总的来说，本提案中的工作将提供分子神经生物学技术的培训，并将有助于告诉我们mrna转运到中枢感觉轴突并局部生成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.