Objectives

Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and programmed cell death. The relevant role of this protein is underlined because mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, this study attempted to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction.

Methods

We developed a novel AIF-deficient mouse strain and assessed, using molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we conducted ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown.

Results

AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF^-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming toward anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Notably, approximately 2% of AIF^-/Y MEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF^-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF^-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism toward anaerobic glycolysis. Heterozygous AIF^+/- females displayed progressive bone marrow, thymus, and spleen cellular loss. In addition, approximately 10% of AIF^+/- females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died 5 weeks after birth. AIF^+/- with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype was triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF^+/- females by supplying dams and newborns with an antioxidant in drinking water.

Conclusions

In a single knockout model and at 3 different levels (cell, embryo, and adult mice) we demonstrated that by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by providing new insights into the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies.

中文翻译：

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线粒体 AIF 损失会导致代谢重编程、半胱天冬酶依赖性细胞死亡阻断、胚胎致死和围产期脑积水。

 目标

细胞凋亡诱导因子 (AIF) 是一种参与线粒体电子传递链组装/稳定性和程序性细胞死亡的蛋白质。强调该蛋白的相关作用是因为改变线粒体 AIF 特性的突变会导致急性儿科线粒体病和肿瘤转移。通过生成原始 AIF 缺陷小鼠品系，本研究试图在单一范式中分析 AIF 缺失和随后的氧化磷酸化 (OXPHOS) 功能障碍对细胞和发育代谢的影响。

 方法

我们开发了一种新型 AIF 缺陷小鼠品系，并使用分子和细胞生物学方法评估了细胞、胚胎和成年小鼠的表型改变。此外，我们对原代和永生化 AIF 敲除小鼠胚胎成纤维细胞 (MEF) 进行了体外测定，以确定细胞死亡特征和由线粒体电子传递链 (ETC) 破坏引起的代谢适应性反应。

 结果

AIF 缺乏会破坏线粒体 ETC 的稳定性，并引发超复合物解体、线粒体跨膜电位损失和线粒体活性氧 (ROS) 的大量产生。 AIF ^-/Y MEF 通过线粒体网络重组和向无氧糖酵解的代谢重编程来平衡这些 OXPHOS 改变，具体表现为 Thr172 处的 AMPK 磷酸化、葡萄糖转运蛋白 GLUT-4 的过度表达、随后葡萄糖摄取的增强以及无氧乳酸的产生。晚期表型的特征是 P53/P21 介导的衰老的激活。值得注意的是，大约 2% 的AIF ^-/Y MEF 减少了线粒体质量和 ROS 水平，并自发增殖。这些循环的AIF ^-/Y MEF 对不依赖 caspase 的细胞死亡诱导剂具有抗性。 AIF 缺陷小鼠品系在 E11.5 和 E13.5 之间是胚胎致死的，伴有能量损失、增殖停滞和细胞凋亡水平增加。与AIF- ^/Y MEF相反，AIF KO胚胎无法将其代谢重新编程为无氧糖酵解。杂合子AIF ^+/-女性表现出进行性骨髓、胸腺和脾细胞损失。此外，大约 10% 的AIF ^+/-女性患有围产期脑积水，其特征是大脑发育障碍、脑膜纤维化和髓质出血；这些小鼠在出生后 5 周后死亡。患有脑积水的AIF ^+/-表现出室管膜层纤毛上皮的损失。 这种表型是由 ROS 过量引发的。因此，通过在饮用水中为母猪和新生儿提供抗氧化剂，可以减少女性脑积水AIF ^+/-的发生。

 结论

在单一敲除模型和 3 个不同水平（细胞、胚胎和成年小鼠）中，我们证明通过控制线粒体 OXPHOS/代谢，AIF 是调节细胞分化和命运的关键因素。此外，通过提供对线粒体 OXPHOS 功能障碍的病理后果的新见解，我们的新发现为新的药理学策略铺平了道路。