外周神经损伤后背根节表达甘丙肽的神经元的来源、功能和分子调控机制

We identified ten neuron types in primary sensory neurons of dorsal root ganglia (DRG) by single cell RNA-seq. Galanin (Gal) is a biomarker of one type of small DRG neuron. We performed single cell RNA-seq on DRG individual neurons after peripheral nerve injury. Bioinformatical analysis of the transcriptomes indicated that peripheral nerve injury could cause the change in Gal-contained neurons. There was a group of genes upregulated in Gal-contained neurons at medium-late phase of peripheral nerve injury, which was closely related to the chronicity of neuropathic pain. In this project, we would identify the origin of new Gal-contained neurons after peripheral nerve injury, and uncover the mechanisms underlying the change of the Gal-contained neurons induced by peripheral nerve injury. And we would determinate the role of Gal-contained neurons in the somatosensory sensations under normal condition and after peripheral nerve injury. Firstly, we perform cell-lineage analysis of Gal-contained neurons by Gal-CreER transgenic mice. Thus we could track back to the origin of new Gal-neurons after peripheral nerve injury. Secondly, we would measure the electrophysiological characteristics of Gal-contained neurons after peripheral nerve injury by transgenic mice. We would also define the role of Gal-contained neurons in the regulation of peripheral neuropathic pain. Last but not least, we would perform single cell RNA-seq of more DRG neurons from early phase of peripheral nerve injury to screen the transcriptional factors for the potential candidates regulating the change of Gal-contained neuron by bioinformatical analysis. We would identify the transcriptional factors by experiments. This project would provide new data for the study on the molecular and cellular mechanisms underlying neuropathic pain. This project would also present a new method to study the change of neuron types under pathologic condition.

背根节中的感觉神经元可分为十个细胞类群，甘丙肽是其中一类小神经元的标记分子。甘丙肽神经元的功能目前尚未被研究。单细胞RNA测序结果显示外周神经损伤后甘丙肽细胞类群有显著的扩大。有一组基因在神经损伤中晚期的甘丙肽神经元中表达显著上调，这可能与神经病理性痛的慢性化过程相关。本项目将阐明外周神经损伤后新增甘丙肽神经元的来源并解析相关的分子机制，本项目还将揭示甘丙肽神经元在生理或病理条件下的作用。首先，通过转基因小鼠进行遗传标记，做细胞谱系分析追溯新增甘丙肽神经元的来源。第二，通过转基因小鼠检测外周神经损伤后甘丙肽神经元的电生理特性变化，以及在神经病理性痛调控中的作用。最后，对外周神经损伤早期的背根节神经元做单细胞RNA测序分析，筛查和分析参与调控甘丙肽的转录因子及分子作用网络，并通过实验确认。本项目将为神经病理性痛的研究提供新的分子和细胞机制，也为研究病理条件下背根节神经元类群变化提供新的方法。

甘丙肽是背根神经节（DRG）中一类小神经元的标记分子，目前为止甘丙肽神经元的功能尚未被研究。外周神经损伤可引起神经病理性痛。外周神经损伤甘丙肽在DRG中显著上调，但是新增甘丙肽神经元的来源、甘丙肽的转录调控机制、甘丙肽神经元在神经病理性痛中的作用都不清楚。项目中，我们对神经病理性痛模型下的小鼠DRG做了单细胞RNA测序分析。我们发现了有三个SNI诱导的神经元类型（SNIIC），这三类SNIICs分别来源于Cldn9+/ Gal+，Mrgprd+和Trappc3l+ 的DRG神经元类型。其中Mrgprd起源的SNIIC2 只短暂出现在SNI 24h。神经损伤2天后，由于Gal表达的增加和Mrgprd表达的降低，SNIIC2转变成了SNIIC1。通过转录调控网络分析和实验验证，我们揭示转录因子Atf3和Egr1可以增强Gal的表达，而激活的Cpeb1可能在SNI后2天内抑制Mrgprd的表达。此外，通过伪时间线的分析，我们重构了神经损伤引起的神经元类型的转变。我们筛选了神经病理性痛发展过程中的转录组的动态变化，以寻找潜在的镇痛靶点。我们发现，SNIIC1神经元中心肌营养因子样细胞因子1（Clcf1）以及细胞因子受体样因子1（Crlf1）的表达量增加。鞘内注射CLC/CLF可以激活脊髓背角的星形胶质细胞而不是小胶质细胞，并引起机械性痛敏。而且SNI模型后敲除DRG 中Clcf1的表达可以减缓SNI引起的机械性痛敏。我们还通过转基因小鼠研究Gal阳性DRG神经元的功能，结果提示特异性杀死Gal阳性DRG神经元只影响小鼠对伤害性热的感受，不影响对机械刺激的感受。综上，本项目揭示了神经损伤后甘丙肽神经元的起源、转录调控机制、以及分泌的细胞因子在神经病理性痛中的调控作用。我们的单细胞测序数据为神经病理性痛的研究提供了一个新的框架，以了解神经元类型的变化以及神经病理性痛发展背后的分子和细胞机制。