PLP乙酰化修饰调控创伤性脑白质损伤后髓鞘形成的作用和机制

The incidence of white matter injury is very high, but there is a lack of effective treatment. Oligodendrocyte maturation and myelination play an important role in white matter injury and repair. We previously found that the level of myelin proteolipid protein (PLP) acetylation is significantly upregulated in SIRT2 knockout (KO) mouse, and that SIRT2 depletion increased axonal injury and impacted myelination following traumatic white matter injury, compared with the wild type group. We postulated that SIRT2 promote oligodendrocyte maturation and myelination by deacetylating PLP after traumatic white matter injury. To verify this hypothesis, we check the level of PLP acetylation in SIRT2 KO mouse, and then determine which lysine residue was acetylated by high-resolution LC-MS/MS analysis. Co-immunoprecipitations is undertaken to elucidate whether PLP directly interacted with SIRT2. Stretch induced cell injury or traumatic white matter injury model was applied to further explore the effects of SIRT2 KO on PLP expression, acetylation, membrane translocation, SREBP signal pathway, myelination and behavioral changes following traumatic white matter injury. Our study aim to uncover a previously unknown mechanism by which SIRT2 mediated deacetylation of PLP to protect white matter induced by trauma, and provide new treatment strategies for neurological function restoration after traumatic white matter injury.

创伤性脑白质损伤发生率高且缺乏有效治疗手段。少突胶质细胞（OL）成熟和髓鞘形成是白质损伤修复的关键。课题组前期发现SIRT2基因敲除后，PLP乙酰化水平上调，同时髓鞘再生障碍，轴突变性增加。我们假设SIRT2通过去乙酰化修饰PLP促进创伤性脑白质损伤后OL细胞成熟及髓鞘再生修复。为验证该作用及机制，本课题首先在SIRT2基因敲除小鼠中检测PLP的乙酰化水平，利用修饰蛋白质质谱技术鉴定PLP乙酰化修饰位点，免疫共沉淀检验SIRT2与PLP相互关系。通过小鼠创伤性脑白质损伤模型结合OL细胞牵张损伤模型，分别从器官和细胞水平观察SIRT2基因敲除后PLP在OL细胞内表达、乙酰化水平、膜转运以及SREBP代谢信号通路的改变，同时评估创伤性脑白质损伤后髓鞘再生和神经功能改变。期望揭示SIRT2介导的PLP去乙酰化修饰在创伤性脑白质损伤修复中的作用和机制，为临床治疗提供新的思路和科学依据。

课题组成功建立了创伤性脑白质损伤模型，构建了一套脑白质损伤和修复评估体系，为临床脑外伤后白质损伤进一步深入了研究构建了良好的动物模型。在体外细胞实验中，通过成功转染SIRT2和PLP及相关突变质粒，证实SIRT2结合并去乙酰化修饰PLP，K105是SIRT2调控的乙酰化位点，KAT9是PLP的乙酰化酶。初步阐明了PLP乙酰化调控分子机制。通过体内和体外水平观察OL细胞，发现PLP过度乙酰化影响了OL细胞分化和成熟。在SIRT2敲除小鼠中，创伤性脑白质损伤后髓鞘再生修复受损和延缓，同时神经损伤功能评价提示SIRT2敲除小鼠运动及记忆能力受损更为严重。为创伤性脑白质损伤的预防和治疗提供新思路。深入探讨相关机制表明SIRT2敲除导致的PLP乙酰化水平升高和髓鞘再生障碍可能与SREBP代谢信号通路改变相关。初步阐明了脑白质损伤修复的可能机制。