Mutations affecting the mitochondrial fusion protein Optic Atrophy 1 (OPA1) cause autosomal dominant optic atrophy (DOA) – one of the most common form of mitochondrial disease. The majority of patients develop isolated optic atrophy, but about 20% of OPA1 mutation carriers manifest more severe neurological deficits as part of a “DOA+” phenotype. OPA1 deficiency causes mitochondrial fragmentation and also disrupts cristae organization, oxidative phosphorylation, mitochondrial DNA (mtDNA) maintenance, and cell viability. It has not yet been established whether phenotypic severity can be modulated by genetic modifiers of OPA1. To better understand the genetic regulation of mitochondrial dynamics, we established a high-throughput imaging pipeline using supervised machine learning (ML) to perform unbiased, quantitative mitochondrial morphology analysis that was coupled with a bespoke siRNA library targeting the entire known mitochondrial proteome (1531 genes), providing a detailed phenotypic screening of human fibroblasts. In control fibroblasts, we identified known and novel genes whose depletion promoted elongation or fragmentation of the mitochondrial network. In DOA+ patient fibroblasts, we identified 91 candidate genes whose depletion prevents mitochondrial fragmentation, including the mitochondrial fission genes DNM1L, MIEF1, and SLC25A46, but also genes not previously linked to mitochondrial dynamics such as Phosphatidyl Glycerophosphate Synthase (PGS1), which belongs to the cardiolipin (CL) synthesis pathway. PGS1 depletion reduces CL content in mitochondria and rebalances mitochondrial dynamics in OPA1-deficient fibroblasts by inhibiting mitochondrial fission, which improves defective respiration, but does not rescue mtDNA depletion, cristae dysmorphology or apoptotic sensitivity. Our data reveal that the multifaceted roles of OPA1 in mitochondria can be functionally uncoupled by modulating mitochondrial lipid metabolism, providing novel insights into the cellular relevance of mitochondrial fragmentation. This study illustrates the power of a first-in-kind objective automated imaging approach to uncover genetic modifiers of mitochondrial disease through high-throughput phenotypic screening of patient fibroblasts.

中文翻译：

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患者来源的OPA1突变成纤维细胞中遗传修饰子的高通量表型筛选将PGS1鉴定为线粒体片段化的功能抑制剂

影响线粒体融合蛋白视神经萎缩1（OPA1）的突变会导致常染色体显性视神经萎缩（DOA）-线粒体疾病的最常见形式之一。大多数患者发生孤立性视神经萎缩，但约有20％的OPA1突变携带者表现出更严重的神经功能缺损，这是“ DOA +”表型的一部分。OPA1缺乏会导致线粒体断裂，还会破坏cr的组织，氧化磷酸化，线粒体DNA（mtDNA）维持和细胞活力。尚未确定是否可以通过OPA1的遗传修饰因子调节表型严重性。为了更好地了解线粒体动力学的遗传调控，我们建立了一个高通量成像管道，使用监督机器学习（ML）进行无偏的，定量的线粒体形态分析，并结合针对整个已知线粒体蛋白质组（1531个基因）的定制siRNA库），提供了人类成纤维细胞的详细表型筛选。在对照成纤维细胞中，我们鉴定出了已知和新颖的基因，它们的消耗促进了线粒体网络的伸长或断裂。在DOA +患者的成纤维细胞中，我们鉴定了91种候选基因，这些基因的耗竭阻止线粒体片段化，包括线粒体裂变基因DNM1L，MIEF1和SLC25A46，还有以前未与线粒体动力学相关的基因，例如磷脂酰甘油磷酸磷酸合成酶（PGS1），属于心磷脂（CL）合成途径。PGS1耗竭通过抑制线粒体裂变而减少了线粒体中的CL含量，并重新平衡了OPA1缺乏的成纤维细胞中的线粒体动力学，从而改善了呼吸不良，但不能挽救mtDNA耗竭，cr状畸形或凋亡敏感性。我们的数据表明，OPA1在线粒体中的多面性作用可以通过调节线粒体脂质代谢在功能上脱钩，从而为线粒体片段化的细胞相关性提供新的见解。这项研究表明，通过对患者成纤维细胞进行高通量表型筛选，首创的客观自动成像方法可揭示线粒体疾病的遗传修饰因子。