从NADH/NAD+氧化还原状态介导的代谢途径探讨线粒体基因tRNALeu(UUR)A3243G突变致病的机制研究

The most common monogenic diabetes is caused by mitochondrial tRNALeu(UUR)A3243G （mt.3243A>G）mutation. Various clinical manifestations were found to be related to the m.3243A>G mutation, with maternally inherited diabetes and deafness as typical clinical characteristics. The heteroplasmy levels in leukocytes is only correlated with HbA1c and BMD T-score on total hip, and the molecular mechanism of the mutation on different clinical manifestations is still unknown. In our preliminary studies, defects in the oxidative respiratory chain complex I and mitochondrial function were observed in the mutation carriers. Using an intensive fluorescent genetically encoded sensor, we found significant changed NADH/NAD+ in the cytosol of living m.3243A>G urine derived stem cells in vivo. Also, using GC-MS profiling methods, we found significant changed metabolites that were associated with NADH/NAD+. Therefore, based on these previous novel findings, we proposed a hypothesis: the pathogenic mechanism of the mt.3243A>G mutation was based on metabolic pathways purterbations mediated by redox state of NADH/NAD+. Furthermore, we will quantitatively analyze the related metabolites and enzymes by metabolomics and Western blotting. By integrated analysis of metabolites and enzyme changes, we could testify our hypothesis and provide novel therapeutic targets for mitochondrial DNA 3243 A>G mutation.

线粒体tRNALeu(UUR)A3243G突变（mt.3243A>G突变）糖尿病是最常见的单基因突变糖尿病，涉及多器官损伤。mt.3243A>G突变杂胞质性的检测仅能部分反映疾病的严重程度，该突变导致临床表现异质性的分子机制尚不明确。我们前期研究发现，此突变可引起患者氧化呼吸链复合体I损伤为代表的线粒体功能异常，活体单细胞代谢荧光探针发现mt.3243A>G突变的尿源干细胞胞浆内NADH/NAD+发生了明显的改变，代谢组学全谱扫描发现与NADH/NAD+相关通路代谢物异常。本项目拟在上述工作基础上，提出大胆假说：mt.3243A>G突变是基于NADH/NAD+氧化还原状态介导的代谢途径的改变从而导致致病的发生。同时，采用代谢组学和分子生物学的方法定量检测和NADH/NAD+相关的代谢物和酶，以期通过不同层面的综合分析，验证我们的假说，为mt.3243A>G突变提供新的治疗靶点。

线粒体tRNALeu(UUR)A3243G突变（mt.3243A>G突变）糖尿病是最常见的单基因突变糖尿病，涉及多器官损伤，其典型临床特点为伴听力损害、呈母系遗传规律。我们研究发现突变患者的骨密度下降且和血液白细胞的突变率成负相关。课题组建立了mt.3243A>G突变患者尿液干细胞的分离和提取体系，从患者体内培养出高突变和低突变的尿液干细胞，发现高突变细胞从形态到线粒体功能均和正常人的细胞有较大的改变，而低突变的干细胞和正常细胞基本没有差别，为机制研究和后续自体干细胞治疗提供了物质基础。通过对患者自体高突变，低突变尿液干细胞的代谢组学和荧光探针分析，找到了突变影响的差异代谢物，初步确定突变患者细胞的线粒体呼吸链复合体II被激活，作为复合体I损伤的补充的机制。通过对尿液干细胞的转录组学分析，发现Wnt通路和未折叠蛋白反应相关的基因发生改变，经RT-PCR和Western验证，找到了关键因子ATF5。对ATF5因子进行小分子干扰后，发现高突变细胞的线粒体功能得到恢复，对尿液干细胞进行成骨分化后发现，敲低ATF5可以很好的恢复细胞的成骨功能，是一个未来治疗mt.3243A>G突变引发骨质疏松的靶点，拥有转化价值。