Lgi1基因通过控制髓鞘装配和TSC1-mTORC1依赖的脂质生物合成促进外周神经系统髓鞘化

Lgi1 promotes the peripheral nervous system myelination via controlling myelin sheath assembling and TSC1-mTOR dependent lipid biosynthesis

目的 探索富亮氨酸胶质瘤失活基因1(Lgi1)在外周神经系统(PNS)髓鞘化的作用及其分子机制。方法 应用蛋白质印迹法检测Lgi1与髓鞘相关蛋白在产后发育时期SN组(坐骨神经)P3-P60、Brain组(大脑)P0-P180、敲除Lgi1后SN组与Brain组的表达,并进一步探索Lgi1-/-小鼠Laminin-integrin信号及TSC1-mTORC1通路相关蛋白表达情况;利用免疫组织化学与电镜探究敲除Lgi1后引起的髓鞘超微结构改变。结果 蛋白质印迹法显示,Lgi1的缺失会导致PNS中髓鞘相关蛋白MBP、MOG和MAG与特异性髓鞘蛋白MPZ的表达水平下降(P<0.05),且电镜下实验组小鼠SN中无髓鞘的轴突数量增加,而轴突总数不变,g-ratio比率增加,且未折叠髓鞘增加;Lgi1-/-小鼠SN中Laminin、integrinβ-1、integrinβ-4蛋白表达明显减少,integrin α-1表达增加(P<0.05);免疫荧光染色结果显示,MBP与Laminin共染部分较少;此外,与对照组相比,Lgi1-/-小鼠中脂肪酸合成酶FASN蛋白表达量减少,pS6水平增加和TSC1表达减少(P<0.05)。结论 Lgi1基因参与调控PNS髓鞘化过程,且通过Laminin-integrin信号及TSC1-mTORC1通路抑制髓鞘的形成并导致PNS低髓鞘化。

Objective To investigate the function of leucine-rich glioma inactivated 1(Lgi1)and its molecular mechanism in the myelination of the peripheral nervous system(PNS).Methods Western blotting was used to monitor the expression of Lgi1 and myelin-related proteins in the SN group(sciatic nerve)at postnatal day P3-P60,Brain group(brain)at postnatal day P0-P180,SN and Brain groups after knocking out Lgi1 during postnatal development,and to further investigate the mechanism of Lgi1 on PNS myelination and to detect the changes of Laminin-integrin signaling and TSC1-mTOC1 pathway in Lgi1-/-mice.Immunohistochemistry and electron microscopy(EM)were used to examine the changes in myelin-related proteins and the ultrastructural changes of myelin sheath in Lgi1 deficient mice.Results Western blotting revealed that deletion of Lgi1 caused decreased expression level of myelin-associated proteins[Myelin basic protein(MBP),Myelin oligodendrocyte glycoprotein(MOP),Myelin-associated glycoprotein(MAP),and Myelin protein zero(MPZ)]in the PNS(P<0.05).Immunohistochemistry and EM revealed an increased number of unmyelinated axons in the SN of Lgi1-/-mice.EM data also showed that the g-ratio and unfolded myelin sheath were increased in Lgi1-/-PNS.Laminin,integrin-1β,and integrin-4βwere detected in the SN of Lgi1-/-mice using western blotting,and the results showed a significant decrease in protein expression and an increase in integrin-1αexpression(P<0.05).Additionally,immunofluorescence showed less MBP co-staining with Laminin.Furthermore,lower FASN level and unbalanced TSC1-mTORC1 activity were shown by western blotting in Lgi1-/-mice.Conclusion Through Laminin-integrin signaling and the TSC1-mTORC1 dependent lipid biosynthesis,the Lgi1 gene is involved in controlling the process of PNS myelination.