ABSTRACT

Mitochondrial quality control, which is crucial for maintaining cellular homeostasis, has been considered to be achieved exclusively through mitophagy. Here we report an alternative mitochondrial quality control pathway mediated by extracellular mitochondria release. By performing time-lapse confocal imaging on a stable cell line with fluorescent-labeled mitochondria, we observed release of mitochondria from cells into the extracellular space. Correlative light-electron microscopy revealed that majority of the extracellular mitochondria are in free form and, on rare occasions, some are enclosed in membrane-surrounded vesicles. Rotenone- and carbonyl cyanide m-chlorophenylhydrazone-induced mitochondrial quality impairment promotes the extracellular release of depolarized mitochondria. Overexpression of PRKN (parkin RBR E3 ubiquitin protein ligase), which has a pivotal role in mitophagy regulation, suppresses the extracellular mitochondria release under basal and stress condition, whereas its knockdown exacerbates it. Correspondingly, overexpression of PRKN-independent mitophagy regulators, BNIP3 (BCL2 interacting protein 3) and BNIP3L/NIX (BCL2 interacting protein 3 like), suppress extracellular mitochondria release. Autophagy-deficient cell lines show elevated extracellular mitochondria release. These results imply that perturbation of mitophagy pathway prompts mitochondria expulsion. Presence of mitochondrial protein can also be detected in mouse sera. Sera of PRKN-deficient mice contain higher level of mitochondrial protein compared to that of wild-type mice. More importantly, fibroblasts and cerebrospinal fluid samples from Parkinson disease patients carrying loss-of-function PRKN mutations show increased extracellular mitochondria compared to control subjects, providing evidence in a clinical context. Taken together, our findings suggest that extracellular mitochondria release is a comparable yet distinct quality control pathway from conventional mitophagy.

Abbreviations: ACTB: actin beta; ANXA5: annexin A5; ATP5F1A/ATP5A: ATP synthase F1 subunit alpha; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CM: conditioned media; CSF: cerebrospinal fluid; DMSO: dimethyl sulfoxide; EM: electron microscopy; HSPD1/Hsp60: heat shock protein family D (Hsp60) member 1; KD: knockdown; KO: knockout; MAP1LC3A/LC3: microtubule associated protein 1 light chain 3 alpha; MT-CO1: mitochondrially encoded cytochrome c oxidase I; NDUFB8: NADH:ubiquinone oxidoreductase subunit B8; OE: overexpression; OPA1: OPA1 mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PB: phosphate buffer; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDHB: succinate dehydrogenase complex iron sulfur subunit B; TOMM20: translocase of outer mitochondrial membrane 20; TOMM40: translocase of outer mitochondrial membrane 40; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; WT: wild-type

摘要

对维持细胞稳态至关重要的线粒体质量控制被认为是完全通过线粒体自噬来实现的。在这里，我们报告了一种由细胞外线粒体释放介导的替代线粒体质量控制途径。通过对具有荧光标记的线粒体的稳定细胞系进行延时共聚焦成像，我们观察到线粒体从细胞释放到细胞外空间。相关光电子显微镜显示大多数细胞外线粒体处于游离形式，在极少数情况下，一些线粒体被包裹在膜包围的囊泡中。鱼藤酮和羰基氰化物间氯苯腙诱导的线粒体质量损伤促进去极化线粒体的细胞外释放。PRKN（parkin RBR E3 泛素蛋白连接酶）的过表达，它在线粒体自噬调节中起关键作用，在基础和应激条件下抑制细胞外线粒体的释放，而其敲低则加剧了它。相应地，不依赖 PRKN 的线粒体自噬调节剂 BNIP3（BCL2 相互作用蛋白 3）和 BNIP3L/NIX（BCL2 相互作用蛋白 3 样）的过表达抑制细胞外线粒体释放。自噬缺陷细胞系显示细胞外线粒体释放升高。这些结果意味着线粒体自噬通路的扰动促使线粒体排出。还可以在小鼠血清中检测到线粒体蛋白的存在。与野生型小鼠相比，PRKN 缺陷小鼠的血清含有更高水平的线粒体蛋白。更重要的是，来自携带功能丧失的帕金森病患者的成纤维细胞和脑脊液样本与对照受试者相比， PRKN突变显示细胞外线粒体增加，为临床提供了证据。总之，我们的研究结果表明，细胞外线粒体释放是一种与传统线粒体自噬相当但又截然不同的质量控制途径。

缩写：ACTB：肌动蛋白β；ANXA5：膜联蛋白 A5；ATP5F1A/ATP5A：ATP合酶F1亚基α；ATG：自噬相关；BNIP3：BCL2 相互作用蛋白 3；BNIP3L/NIX：BCL2 相互作用蛋白 3 样；CCCP：羰基氰间氯苯腙；CM：条件培养基；CSF：脑脊液；DMSO：二甲亚砜；EM：电子显微镜；HSPD1/Hsp60：热休克蛋白家族 D (Hsp60) 成员 1；KD：击倒；KO：淘汰赛；MAP1LC3A/LC3：微管相关蛋白 1 轻链 3 α；MT-CO1：线粒体编码的细胞色素 c 氧化酶 I；NDUFB8：NADH：泛醌氧化还原酶亚基 B8；OE：过度表达；OPA1：OPA1 线粒体动力蛋白，如 GTPase；OXPHOS：氧化磷酸化；PBS：磷酸盐缓冲液；PB：磷酸盐缓冲液；PD：帕金森病；PINK1：PTEN 诱导激酶 1；PRKN：parkin RBR E3 泛素蛋白连接酶；RB1CC1/FIP200：RB1 感应线圈1；SDHB：琥珀酸脱氢酶复合物铁硫亚基B；TOMM20：线粒体外膜转位酶 20；TOMM40：线粒体外膜 40 的转位酶；UQCRC2：泛醇-细胞色素 c 还原酶核心蛋白 2；WT：野生型