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。强调了该蛋白质的相关作用,因为改变线粒体AIF特性的突变会导致急性小儿线粒体病和肿瘤转移。通过产生原始的AIF缺陷小鼠品系,这项研究试图分析,在一个单一的范式中,AIF损失和随后的氧化磷酸化(OXPHOS)功能障碍的细胞和发育代谢后果。
我们开发了一种新的缺乏AIF的小鼠品系,使用分子和细胞生物学方法,细胞,胚胎,和成年小鼠表型改变。此外,我们对原代和永生化AIF敲除小鼠胚胎成纤维细胞(MEFs)进行了离体测定,以确定细胞死亡特征和线粒体电子传递链(ETC)破坏引起的代谢适应性反应.
AIF缺乏使线粒体ETC不稳定,并引起超复合体解体,线粒体跨膜电位损失,和高产量的线粒体活性氧(ROS)。AIF-/YMEF通过线粒体网络重组和向厌氧糖酵解的代谢重编程来抵消这些OXPHOS改变,这些代谢重编程由Thr172的AMPK磷酸化,葡萄糖转运蛋白GLUT-4的过表达,随后的葡萄糖摄取增强,和厌氧乳酸的产生。晚期表型的特征在于P53/P21介导的衰老的激活。值得注意的是,大约2%的AIF-/YMEF减少了线粒体质量和ROS水平,并自发增殖。这些循环AIF-/YMEF对半胱天冬酶非依赖性细胞死亡诱导物具有抗性。AIF缺陷小鼠品系在E11.5和E13.5之间具有胚胎致死性,具有能量损失,防扩散,和增加的凋亡水平。与AIF-/YMEF相反,AIFKO胚胎无法将其代谢重新编程为无氧糖酵解。杂合AIF+/-雌性显示进行性骨髓,胸腺,和脾细胞丢失。此外,大约10%的AIF+/-女性发生了以大脑发育障碍为特征的围产期脑积水,脑膜纤维化,和髓质出血;那些小鼠在出生后5周死亡。具有脑积水的AIF/-表现出室管膜层中纤毛上皮的损失。该表型由ROS过量触发。因此,通过向水坝和新生儿提供饮用水中的抗氧化剂,可以减少脑积水AIF/-女性的发生。
在单个敲除模型中,在3个不同的级别(细胞,胚胎,和成年小鼠),我们证明了通过控制线粒体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策略铺平了道路.
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.
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.
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.
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.