The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2^ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

人类线粒体 DNA 聚合酶 γ 是一种全酶，参与线粒体 DNA (mtDNA) 的复制和维护，由催化亚基 (POLG) 和赋予持续合成能力的二聚体辅助亚基 (POLG2) 组成。POLG或POLG2的突变会在人类中引起 POLG 相关疾病，导致孟德尔遗传性线粒体疾病的子集，其特征是 mtDNA 耗竭 (MDD) 或多个缺失的积累，呈现多器官缺陷，并常常导致年轻人过早死亡年龄。考虑到POLG2模型的缺乏，我们通过CRISPR/Cas9技术生成了稳定的斑马鱼polg2突变系（polg2 ^ia304），该突变系携带10个核苷酸的移码突变和过早终止密码子缺失。与野生型兄弟姐妹相比，斑马鱼plg2纯合突变体发育较慢，生存能力下降，在幼年阶段之前死亡。突变体表现出一系列POLG相关表型，与受 POLG 相关疾病影响的人类患者的症状相当，包括显着的 MDD、线粒体网络和动力学改变以及线粒体呼吸减少。组织学分析检测到高能量需求组织的形态变化，以及骨骼肌纤维的显着紊乱。与最后的发现一致，运动测定强调了幼虫运动能力的下降。值得注意的是，先前证明对 POLG 模型有效的甲苯磺酸氯非铵药物治疗可以部分缓解 Polg2 突变动物的 MDD。总而言之，我们的结果表明斑马鱼是研究与 POLG2 相关的人类 POLG 相关疾病病因病理学的有效模型，也是筛选 POLG 导向药物在 POLG2 相关形式中功效的合适平台。