跨膜蛋白tmem63b对小鼠外周神经髓鞘发育的调控作用及机制研究

Axonal myelination is essential for nerve impulse transmission and other related functions of peripheral nervous system (PNS), however, the involved molecular mechanisms are still uncertain. Myelin sheath is a particular membrane structure which contains diverse transmembrane proteins. Numerous studies have shown that transmembrane proteins are important for the structure and function of myelin sheath. Tmem63 family proteins are transmembrane proteins include members of tmem63a, b and c. In order to investigate the effect of tmem63 on myelin sheath development, we have constructed tmem63 a, b and c knockout mice respectively. Initial experiment results showed that loss of tmem63b but not tmem63a or c lead to declined grip strength and balancing ability in mice. Furthermore, tmem63b deficiency also caused myelin sheath abnormality in mice showed investigated by transmission electron microscope. Therefore, we infer that tmem63b might participate in regulating mice PNS development process. In this study, praxiology, morphology and molecular biology tests will be employed to investigate the effect of tmem63 on mouse myelin sheath development and functionary mechanisms. Finally, the study will further enrich our understanding about PNS development mechanisms and also provide new drug targets for treating PNS myelination related disorders.

外周神经髓鞘是神经系统传递神经冲动，执行功能的保障，但其形成及发育的调控机制尚不清楚。髓鞘为一种含有多种膜蛋白的特殊膜结构，大量研究表明膜蛋白对其结构与功能具有重要影响。tmem63家族是一类跨膜蛋白，包括tmem63a/b/c三种家族成员。为研究跨膜蛋白tmem63对髓鞘发育的影响，我们分别构建了tmem63a、b及c基因敲除小鼠。初步研究结果发现tmem63b缺失可导致小鼠四肢抓力及运动平衡能力下降，而tmem63a和c敲除小鼠未见异常。并且，透射电镜结果显示tmem63b基因缺失可引起小鼠坐骨神经髓鞘结构畸形化。据此我们推测tmem63b参与了小鼠外周神经髓鞘发育过程。本课题拟通过行为学、形态学及分子水平研究，明确tmem63b对小鼠坐骨神经髓鞘发育的影响及其作用的分子机制。本项目研究结果有助于完善外周神经髓鞘的发育机理，并可能为外周神经系统髓鞘化紊乱相关疾病的治疗提供新的靶点。

神经髓鞘的形成是神经系统传递神经冲动信号，执行其功能的基础保障，髓鞘化过程的异常会导致多种疾病的发生。由于对外周神经髓鞘化进程的分子机制了解还不够深入，导致临床上缺乏有效治疗脱髓鞘或外周神经损伤修复的措施。前人研究指出跨膜蛋白在外周神经髓鞘形成及损伤修复过程中发挥重要作用。我们发现跨膜蛋白tmem63b蛋白的缺失造成了小鼠坐骨神经髓鞘结构严重畸形。为了探究其作用机制，我们构建了tmem63b敲除小鼠模型，tmem63b抗体拮抗小鼠模型及坐骨神经夹伤模型，从行为学，形态学，分子生物学几个方面，运用转棒实验，透射电镜，免疫印记，荧光定量PCR，蛋白质组学，绝对定量脂质组学等方法进行研究。结果显示tmem63b蛋白的缺失引起小鼠运动能力下降，抓握力减弱，神经传导速率下降及髓鞘相关基因PMP22，Krox20，MPZ表达下调。同时，坐骨神经损伤模型中，敲除小鼠运动能力恢复较野生型小鼠下降。通过蛋白组学测序得知，tmem63b表达的异常引起脂肪酸合成关键酶棕榈酰转移酶表达的异常，推测可能造成髓鞘脂质组成改变引起其结构畸形。随后绝对定量脂质组学结果指出tmem63b蛋白缺失造成PC、PE、SM、PS、DG和TG等含量的下降，随后通过高脂饮食喂养敲除小鼠，其髓鞘结构畸形得以缓减。已有研究指出，tmem63b是Ca2+通道蛋白，经抑制剂Neomycin，GaCl及Gsmtx4处理后的小鼠髓鞘结构均出现与敲除小鼠类似的畸形。我们推测tmem63b敲除后引起离子通道关闭，从而导致脂质合成转运的异常，造成髓鞘膜脂分布异常，进而引起髓鞘结构和神经运动功能的异常。本项目探索了tmem63b蛋白新的生物学功能及其分子调控机制，完善了外周神经髓鞘发育的机理。Tmem63b可能成为外周神经系统髓鞘化紊乱相关疾病治疗的新的药物靶点，为今后的治疗方案提供新的思路和策略。