Autophagy deficiency abolishes liver mitochondrial DNA segregation

Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific <i>atg7</i> (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. <i>prkn</i> (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.<b>Abbreviations:</b> <i>Apob</i>: apolipoprotein B; <i>Atg1</i>: autophagy-related 1; <i>Atg7</i>: autophagy related 7; <i>Atp5a1</i>: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; <i>BNIP3</i>: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; <i>mt-Atp8</i>: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; <i>mt-Co3</i>: mitochondrially encoded cytochrome c oxidase III; <i>mt-Cytb</i>: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; <i>Ndufa9</i>: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; <i>Nnt</i>: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; <i>Polg2</i>: polymerase (DNA directed), gamma 2, accessory subunit; <i>Ppara</i>: peroxisome proliferator activated receptor alpha; <i>Ppia</i>: peptidylprolyl isomerase A; <i>Prkn</i>: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; <i>Rpl19</i>: ribosomal protein L19; <i>Rps18</i>: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; <i>Ssbp1</i>: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; <i>Tfb1m</i>: transcription factor B1, mitochondrial; <i>Tfb2m</i>: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.

线粒体基因组（mitochondrial genome，mtDNA）突变在人类中普遍存在，可引发广泛的疾病谱。由于细胞内存在多拷贝mtDNA分子，突变型与野生型（wild-type, WT）mtDNA可共存，即异质性（heteroplasmy），除非突变分子比例达到阈值，否则疾病表型会被掩盖。重要的是，随着个体衰老，突变型mtDNA的水平会发生变化，因为mtDNA会以等位基因和细胞特异性的方式发生分离，这可能引发疾病。mtDNA分离主要在肝细胞中显现，导致mtDNA变异随年龄增长而增加，其中包括非同义且可能具有致病性的突变。
本研究使用一种已被广泛验证的异质性小鼠模型模拟mtDNA分离，该模型的核背景为C57BL/6N，线粒体DNA分别来源于NZB/BINJ与C57BL/6N品系。该小鼠品系的肝脏中会出现显著的年龄依赖性NZB来源mtDNA积累，进而提升每个mtDNA分子的呼吸容量。
值得注意的是，肝细胞特异性敲除<i>atg7</i>（autophagy related 7）可完全阻断NZB来源mtDNA的积累，使mtDNA分离在发育至成年的过程中近乎中性。敲除<i>prkn</i>（parkin RBR E3泛素蛋白连接酶）也能部分阻止肝脏中NZB来源mtDNA的积累，但效果较弱。
因此，我们提出，与年龄相关的肝脏mtDNA分离是低适配性mtDNA被巨自噬（macroautophagy）清除的结果。鉴于NZB/BINJ与C57BL/6N的mtDNA分化水平与人类欧亚人群和非洲人群mtDNA的分化水平相当，这些发现对人类具有潜在的借鉴意义，包括线粒体替代治疗（mitochondrial replacement therapy）的安全应用。
<b>缩写：</b> <i>Apob</i>：载脂蛋白B（apolipoprotein B）；<i>Atg1</i>：自噬相关1（autophagy-related 1）；<i>Atg7</i>：自噬相关7（autophagy related 7）；<i>Atp5a1</i>：ATP合酶H+转运线粒体F1复合物α亚基1（ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1）；BL6：C57BL/6N小鼠品系；<i>BNIP3</i>：BCL2/腺病毒E1B相互作用蛋白3（BCL2/adenovirus E1B interacting protein 3）；FCCP：羰基氰化物4-(三氟甲氧基)苯腙（carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone）；GAPDH：甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；MAP1LC3A：微管相关蛋白1轻链3α（microtubule-associated protein 1 light chain 3 alpha）；MAP1LC3B：微管相关蛋白1轻链3β（microtubule-associated protein 1 light chain 3 beta）；<i>mt-Atp8</i>：线粒体编码ATP合酶8（mitochondrially encoded ATP synthase 8）；MT-CO1：线粒体编码细胞色素c氧化酶亚基I（mitochondrially encoded cytochrome c oxidase I）；MT-CO2：线粒体编码细胞色素c氧化酶亚基II（mitochondrially encoded cytochrome c oxidase II）；<i>mt-Co3</i>：线粒体编码细胞色素c氧化酶亚基III（mitochondrially encoded cytochrome c oxidase III）；<i>mt-Cytb</i>：线粒体编码细胞色素b（mitochondrially encoded cytochrome b）；mtDNA：线粒体DNA（mitochondrial DNA）；MUL1：NFκB激活线粒体泛素连接酶1（mitochondrial ubiquitin ligase activator of NFKB 1）；nDNA：核DNA（nuclear DNA）；<i>Ndufa9</i>：NADH:泛醌氧化还原酶亚基A9（NADH:ubiquinone oxidoreductase subunit A9）；NDUFB8：NADH:泛醌氧化还原酶亚基B8（NADH:ubiquinone oxidoreductase subunit B8）；<i>Nnt</i>：烟酰胺核苷酸转氢酶（nicotinamide nucleotide transhydrogenase）；NZB：NZB/BINJ小鼠品系；OXPHOS：氧化磷酸化（oxidative phosphorylation）；PINK1：PTEN诱导假定激酶1（PTEN induced putative kinase 1）；<i>Polg2</i>：DNA聚合酶γ2辅助亚基（polymerase (DNA directed), gamma 2, accessory subunit）；<i>Ppara</i>：过氧化物酶体增殖物激活受体α（peroxisome proliferator activated receptor alpha）；<i>Ppia</i>：肽基脯氨酰异构酶A（peptidylprolyl isomerase A）；<i>Prkn</i>：parkin RBR E3泛素蛋白连接酶（parkin RBR E3 ubiquitin protein ligase）；P10：出生后第10天（post-natal day 10）；P21：出生后第21天（post-natal day 21）；P100：出生后第100天（post-natal day 100）；qPCR：定量聚合酶链式反应（quantitative polymerase chain reaction）；<i>Rpl19</i>：核糖体蛋白L19（ribosomal protein L19）；<i>Rps18</i>：核糖体蛋白S18（ribosomal protein S18）；SD：标准差（standard deviation）；SEM：标准误（standard error of the mean）；SDHB：琥珀酸脱氢酶复合物亚基B铁硫蛋白（succinate dehydrogenase complex, subunit B, iron sulfur (Ip)）；SQSTM1：隔离体1（sequestosome 1）；<i>Ssbp1</i>：单链DNA结合蛋白1（single-stranded DNA binding protein 1）；TFAM：线粒体转录因子A（transcription factor A, mitochondrial）；<i>Tfb1m</i>：线粒体转录因子B1（transcription factor B1, mitochondrial）；<i>Tfb2m</i>：线粒体转录因子B2（transcription factor B2, mitochondrial）；TOMM20：线粒体外膜转位酶20（translocase of outer mitochondrial membrane 20）；UQCRC2：泛醇细胞色素c还原酶核心蛋白2（ubiquinol cytochrome c reductase core protein 2）；WT：野生型（wild-type）。