Wnt-LRP6信号通路激活REST蛋白的表达并发挥神经保护作用

Wnt-LRP6 signaling pathway regulates the expression of REST and its neuroprotective function

为检测以LRP6受体为激活开关的Wnt-β-catenin信号通路与PrP106-126毒性多肽引起的神经元损伤之间的相互关系,并研究该信号通路与神经保护性蛋白REST的相关性,从而进一步阐明Wnt-LRP6-REST对毒性多肽引起的神经元损伤的影响。利用Wnt信号通路的激活剂Licl、抑制剂DKK1或PrP106-126毒性多肽刺激原代神经元,通过免疫印迹检测神经保护性蛋白REST及Wnt信号通路下游相关蛋白的变化情况,通过激光共聚焦观察REST与Wnt信号通路正相关蛋白β-catenin的共定位情况。分别构建LRP6的过表达质粒pCMV-C-HALRP和干扰质粒pGPH1/GFP/Neo-LRP6-Rat shRNA-1和shRNA-2,将已经构建好的质粒转染进入原代神经元。通过免疫印迹检测PrP106-126毒性多肽对Wnt信号通路标志性蛋白(β-catenin及GSK3β)的影响,检测LRP6的过表达或干扰对REST或Wnt信号通路下游蛋白的影响。通过免疫荧光观察LRP6对由毒性多肽诱导的神经纤维损伤的影响,用流式细胞仪检测相应的细胞活力。由LRP6受体激活的Wnt-β-catenin信号通路在一定程度上正向调控神经保护性蛋白REST的表达,LRP6在信号调节的过程中起到了关键作用,并且LRP6对由PrP106-126毒性多肽引起的神经元损伤有缓解作用,LRP6的过表达增加了神经元细胞的活力,起到了神经保护作用。

Aims of this study were to investigate the influence of PrP106-126 on canonical Wnt-LRP6 pathway and the relationship between low-density lipoprotein receptor-related protein 6 （LRP6） and RE1-silencing transcription factor （REST） in PrP106-126-induced neuropathological changes.Wnt-LRP6 inhibitor and activator were used to test the effect of Wnt signaling on REST.By overexpression or knockdown of LRP6,the relationship between LRP6 and REST in PrP106126-induced neuropathological changes was examined by immunofluorescence and Western blot analysis.The function of LRP6 in PrP106-126-stimulated primary neuronal death was verified by Annexin V-FITC/PI apoptosis assay.The results showed that Wnt signaling partly regulated the expression of REST;LRP6 was critical for the activation of REST and protects neurons from PrP106-126-induced synaptic and neurofibrillary degeneration.In conclusion,the LRP6-Wnt-catenin/REST signaling played critical and collaborative roles in neuroprotection.This signaling of neuronal survival regulation could be explored as a viable therapeutic target for prion diseases and associated neurodegenerative diseases.