<i>POLG</i> mutations lead to abnormal mitochondrial remodeling during neural differentiation of human pluripotent stem cells via SIRT3/AMPK pathway inhibition

We showed previously that POLG mutations cause major changes in mitochondrial function, including loss of mitochondrial respiratory chain (MRC) complex I, mitochondrial DNA (mtDNA) depletion and an abnormal NAD+/NADH ratio in both neural stem cells (NSCs) and astrocytes differentiated from induced pluripotent stem cells (iPSCs). In the current study, we looked at mitochondrial remodeling as stem cells transit pluripotency and during differentiation from NSCs to both dopaminergic (DA) neurons and astrocytes comparing the process in POLG-mutated and control stem cells. We saw that mitochondrial membrane potential (MMP), mitochondrial volume, ATP production and reactive oxygen species (ROS) changed in similar ways in POLG and control NSCs, but mtDNA replication, MRC complex I and NAD+ metabolism failed to remodel normally. In DA neurons differentiated from NSCs, we saw that POLG mutations caused failure to increase MMP and ATP production and blunted the increase in mtDNA and complex I. Interestingly, mitochondrial remodeling during astrocyte differentiation from NSCs was similar in both POLG-mutated and control NSCs. Further, we showed downregulation of the SIRT3/AMPK pathways in POLG-mutated cells, suggesting that POLG mutations lead to abnormal mitochondrial remodeling in early neural development due to the downregulation of these pathways.我们既往研究证实，POLG（DNA聚合酶γ）突变可引发线粒体功能的显著异常，具体表现为：由诱导多能干细胞（induced pluripotent stem cells, iPSCs）分化得到的神经干细胞（neural stem cells, NSCs）与星形胶质细胞中，存在线粒体呼吸链（mitochondrial respiratory chain, MRC）复合物I缺失、线粒体DNA（mitochondrial DNA, mtDNA）耗竭以及NAD+/NADH比值异常的表型。本研究中，我们针对干细胞跨越多能性阶段，以及神经干细胞向多巴胺能（dopaminergic, DA）神经元与星形胶质细胞分化过程中的线粒体重塑事件展开分析，对比了POLG突变细胞与对照干细胞的该过程差异。观测结果显示，POLG突变与对照NSCs的线粒体膜电位（mitochondrial membrane potential, MMP）、线粒体体积、ATP生成量以及活性氧（reactive oxygen species, ROS）水平均呈现相似的变化趋势，但mtDNA复制、MRC复合物I与NAD+代谢却未能完成正常的重塑程序。在由NSCs分化而来的DA神经元中，POLG突变会导致线粒体膜电位与ATP生成量无法有效提升，并削弱了mtDNA与复合物I的水平上调过程。值得注意的是，在星形胶质细胞分化阶段，POLG突变与对照NSCs的线粒体重塑并无显著差异。此外，我们发现POLG突变细胞中SIRT3/AMPK通路（SIRT3/AMPK pathways）表达下调，这提示POLG突变通过下调上述通路，导致早期神经发育过程中出现异常的线粒体重塑。