TrkA促进KIF1A去甲基化参与神经病理性疼痛的机制研究

The nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling pathway plays an important role in the pathogenesis of neuropathic pain. We have previously found that kinesin superfamily member 1A-KIF1A is a major axonal transport motor protein that is involved in delivering TrkA from cell body to the nerve ending. The depletion of kifla gene can result in deficiency in the pain sensation and in an attenuated NGF/TrkA signaling indicating a membership of KIF1A in the NGF/TrkA signaling pathway. NGF treatment increased the expression of KIF1A at the protein level, whereas the expression level of KIF1A was decreased by means of TrkA antagonist, highlighting the possibility that NGF increases the expression of KIF1A via TrkA. Epigenetically, DNA demethylation in CpG sequence causes the high expression of related genes. It is reported that demethylation regulation is implicated in pain hypersensitivity, our preliminary results showed that the genomic DNA methylation was significantly down-regulated in sciatic nerve chronic constriction injury (CCI) mice. In this project, we hypothesize that the increased levels of KIF1A by NGF treatment may associate with phospho-TrkA dependent DNA demethylation in CpG sequence of kif1a promoter and may produce pain. However, it remains unclear how phospho-TrkA affects DNA demethylation of kif1a promoter and transcription. Therefore, a deeper insight into the molecular mechanism of kif1a DNA demethylation and transcription may yield a new therapeutic target for neuropathic pain.

NGF/TrkA信号转导通路在神经病理性疼痛的发生中起重要作用。驱动蛋白家族成员KIF1A将TrkA从神经元胞体转运至神经末梢，kif1a基因剔除小鼠表型为痛觉缺失而且NGF/TrkA信号转导减弱，表明KIF1A是NGF/TrkA信号转导通路的一环。我们发现外源性NGF使KIF1A蛋白表达增多，拮抗TrkA作用则KIF1A蛋白表达减少，提示NGF通过TrkA增加KIF1A蛋白表达。表观遗传学研究表明DNA启动子CpG位点去甲基化可使相关基因高表达并且参与痛觉超敏，我们预实验证实神经病理性疼痛模型小鼠的基因组DNA整体甲基化水平明显降低，因而我们假设TrkA使KIF1A蛋白表达增多的机制是活化的pTrkA促进kif1a基因启动子CpG序列去甲基化。本课题拟以表观遗传学为切入点，探索pTrkA调控kif1a基因启动子DNA去甲基化及转录活性的分子机制，为神经病理性疼痛的治疗寻找新靶点。

研究背景：.我们的早期研究目标是探索p-TrkA是否与kif1a基因相结合，但是经过前期研究与验证，我们发现p-TrkA蛋白并不适合做ChIP实验。根据之前我们的研究，因为已经证实了PI3K信号的激活增强了辣椒素的敏感性，并改善了KIF1A缺失引起的神经元的表型变化，表明PI3K是DRG中感觉系统调节不可或缺的一部分。课题组查阅到CREB作为PI3K常见的下游分子蛋白，是一种细胞核内调控因子，自身磷酸化的CREB（p-CREB）可实现调节转录功能，并且近年来一些研究表明DNA甲基化与NPP的病理密切相关，这种独特的基因表达谱可能会是未来疼痛治疗的方向，因此我们着力进行了相关的研究。.研究目的：.激活的PI3K/Akt可以刺激神经元细胞中CREB的表达。因此，课题组希望通过CCI模型，验证PI3K/Akt/CREB通路激活后，p-CREB可以募集TET1并与kif1a基因的启动子结合，影响kif1a的DNA甲基化进而调节其转录这样一个假设。本研究通过CCI模型，研究NPP中 PI3K通路促进KIF1A表达的机制，证明KIF1A在疼痛致病中的重要性。.研究结论：.（1）CCI术后可以激活PI3K/Akt/CREB通路，影响TET1的表达。NPP状态下kif1a基因发生DNA去甲基化并调高KIF1A的表达。.（2）（2）p-CREB募集TET1，作用于kif1a基因启动子上。我们研究发现，p-CREB蛋白表达的降低伴发TET1蛋白的降低。而TET1的抑制或者过表达会伴发p-CREB同步性的表达降低或者升高。.（3）抑制PI3K/Akt/CREB通路可以改善NPP表型并且可以抑制TET1的表达，逆转kif1a DNA去甲基化导致的疼痛。.（4）TET1的抑制或者过表达可以相应地影响p-CREB。并且可以通过影响kif1a基因的DNA甲基化程度进而调整KIF1A的表达。