Programmed mitophagy is essential for the glycolytic switch during cell differentiation

Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis‐associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19‐kDa‐interacting protein 3‐like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX‐deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX‐dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts.     

BNIP3 (BCL2 interacting protein 3) regulates pluripotency by modulating mitochondrial homeostasis via mitophagy

Autophagy-mediated mitochondrial degradation plays pivotal roles in both the acquisition and maintenance of pluripotency, but the molecular mechanisms that link autophagy-mediated mitochondrial homeostasis to pluripotency regulation are unclear. Here, we identified that the mitophagy receptor BNIP3 regulates pluripotency. In mouse ESCs, depletion of BNIP3 caused accumulation of aberrant mitochondria accompanied by decreased mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and reduced ATP generation, which led to compromised self-renewal and differentiation. Impairment of mitophagy by knockdown of BNIP3 inhibited mitochondrial clearance during pluripotency induction, resulting in decreased reprogramming efficiency. These defects were rescued by reacquisition of wild-type but not LIR-deficient BNIP3 expression. Taken together, our findings highlight a critical role of BNIP3-mediated mitophagy in the induction and maintenance of pluripotency.Subject terms: Embryonic stem cells, Mitophagy     

BNIP3L-dependent mitophagy accounts for mitochondrial clearance during 3 factors-induced somatic cell reprogramming

Induced pluripotent stem cells (iPSCs) have fewer and immature mitochondria than somatic cells and mainly rely on glycolysis for energy source. During somatic cell reprogramming, somatic mitochondria and other organelles get remodeled. However, events of organelle remodeling and interaction during somatic cell reprogramming have not been extensively explored. We show that both SKP/SKO (Sox2, Klf4, Pou5f1/Oct4) and SKPM/SKOM (SKP/SKO plus Myc/c-Myc) reprogramming lead to decreased mitochondrial mass but with different kinetics and by divergent pathways. Rapid, MYC/c-MYC-induced cell proliferation may function as the main driver of mitochondrial decrease in SKPM/SKOM reprogramming. In SKP/SKO reprogramming, however, mitochondrial mass initially increases and subsequently decreases via mitophagy. This mitophagy is dependent on the mitochondrial outer membrane receptor BNIP3L/NIX but not on mitochondrial membrane potential (ΔΨ_m) dissipation, and this SKP/SKO-induced mitophagy functions in an important role during the reprogramming process. Furthermore, endosome-related RAB5 is involved in mitophagosome formation in SKP/SKO reprogramming. These results reveal a novel role of mitophagy in reprogramming that entails the interaction between mitochondria, macroautophagy/autophagy and endosomes.     

Human antigen R regulates hypoxia-induced mitophagy in renal tubular cells through PARKIN/BNIP3L expressions

Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we aimed to understand the role of human antigen R (HuR) in hypoxia-induced mitophagy process in the renal tubular cells. Mitophagy marker expressions (PARKIN, p-PARKIN, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. Immunofluorescence studies were performed to analyze mitophagosome, mitolysosome, co-localization of p-PARKIN/TOMM20 and BNIP3L/TOMM20. HuR-mediated regulation of PARKIN/BNIP3L expressions was determined by RNA-immunoprecipitation analysis and RNA stability experiments. Hypoxia induced mitochondrial dysfunction by increased ROS, decline in membrane potential and activated mitophagy through up-regulated PARKIN, PINK1, BNIP3 and BNIP3L expressions. HuR knockdown studies revealed that HuR regulates hypoxia-induced mitophagosome and mitolysosome formation. HuR was significantly bound to PARKIN and BNIP3L mRNA under hypoxia and thereby up-regulated their expressions through mRNA stability. Altogether, our data highlight the importance of HuR in mitophagy regulation through up-regulating PARKIN/BNIP3L expressions in renal tubular cells.     

综述: 线粒体自噬在低氧适应中的作用

低氧是一种典型的应激环境,细胞在低氧条件下能量和氧化代谢发生改变,其中线粒体产生的大量活性氧严重威胁细胞的存活．线粒体自噬是近年来被发现的细胞适应低氧的一种适应性代谢反应．细胞在低氧条件下能通过上调低氧诱导因 子1(HIF-1),激活BNIP3/BNIP3L及Beclin-1介导的通路诱导线粒体自噬,最终减少ROS的产生,促进细胞的存活,使机体产生低氧适应．综述了线粒体自噬在低氧适应中的作用及其机制．     

Receptor-mediated mitophagy in yeast and mammalian systems

Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease.     

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.     

线粒体与多潜能干细胞功能

线粒体是细胞内重要的细胞器,主要功能是通过氧化磷酸化为细胞生命活动提供能量。近年来,研究表明,在多潜能干细胞（Pluripotent stem cells, PSCs）中线粒体表现出独有的特征,即在多能性状态下,PSCs主要依靠糖酵解提供能量,其分化期间线粒体氧化磷酸化代谢能力逐渐增强。相反,体细胞重编程为多潜能干细胞期间,线粒体氧化磷酸化向糖酵解途径的转变是其成功重编程必需的代谢过程。另外,线粒体通过生物合成和形态结构的动态重塑维持了PSCs多能性、诱导分化及诱导多能干细胞（Induced pluripotent stem cells, iPSCs）的重编程。因此,本文综述了PSCs线粒体形态结构及其在调控PSCs多能性、合成代谢、氧化还原状态的平衡、分化及重新编程中的作用,为深入了解线粒体调控PSCs功能的作用提供理论基础。     

BNIP3L/NIX-mediated mitophagy: molecular mechanisms and implications for human disease

Mitophagy is a highly conserved cellular process that maintains the mitochondrial quantity by eliminating dysfunctional or superfluous mitochondria through autophagy machinery. The mitochondrial outer membrane protein BNIP3L/Nix serves as a mitophagy receptor by recognizing autophagosomes. BNIP3L is initially known to clear the mitochondria during the development of reticulocytes. Recent studies indicated it also engages in a variety of physiological and pathological processes. In this review, we provide an overview of how BNIP3L induces mitophagy and discuss the biological functions of BNIP3L and its regulation at the molecular level. We further discuss current evidence indicating the involvement of BNIP3L-mediated mitophagy in human disease, particularly in cancer and neurological disorders.Subject terms: Cancer, Mitophagy     

NIX‐ing mitochondria: from development to pathology

Hypoxia occurs physiologically in the developing body, and changing oxygen tensions are known to direct tissue differentiation; however, in the context of pathology, the same hypoxia‐activated mechanisms may negatively affect tissue function. In this issue of The EMBO Journal, Esteban‐Martínez et al (2017) report that programmed mitophagy, dependent on hypoxia‐induced NIP‐3‐like protein X (BNIP3L, best known as NIX), is an essential step in differentiation of both retinal neurons and inflammatory macrophages.     

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.     

Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways

Retinal ganglion cells (RGCs), which exist in the inner retina, are the retinal neurons which can be damaged in the early stage of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, exerts biological functions by binding the receptor (GLP-1R), the expression of which in RGC-5 cells was first shown by our team in 2012. It was reported that liraglutide prevented retinal neurodegeneration in diabetic subjects. However, the involvement of mechanisms such as autophagy and mitochondrial balance in liraglutide-induced retinal protection is unknown. Here, we aimed to investigate the protective effects of liraglutide and explore the potential mechanisms of liraglutide-induced retinal RGC protection. RGC-5 cells were treated with H₂O₂ and/or liraglutide. Cell viability was detected with the CCK-8 kit. The axon marker GAP43, autophagy and mitophagy indicators LC3A/B, Beclin-1, p62, Parkin, BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) and the key regulator of mitochondrial biogenesis PGC-1α were examined via western blot analysis. Autophagy was also evaluated using the ImageXpress Micro XLS system and transmission electron microscopy (TEM). Reactive oxygen species (ROS), mitochondrial membrane potential and fluorescent staining for mitochondria were also measured using the ImageXpress Micro XLS system. Our results showed that pretreatment with liraglutide significantly prevented H₂O₂-induced cell viability decline, mitochondrial morphological deterioration and induction of autophagy, which appeared as increased expression of LC3 II/I and Beclin-1, along with p62 degradation. Moreover, liraglutide suppressed the H₂O₂-induced decline in GAP43 expression, thus protecting cells. However, rapamycin induced autophagy and blocked the protective process. Liraglutide also provided mitochondrial protection and appeared to alleviate H₂O₂-induced ROS overproduction and a decline in mitochondrial membrane potential, partially by promoting mitochondrial generation and attenuating mitophagy. In conclusion, liraglutide attenuates H₂O₂ induced RGC-5 cell injury by inhibiting autophagy through maintaining a balance between mitochondrial biogenesis and mitophagy.     

BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2

Cerebral ischemia induces massive mitochondrial damage. These damaged mitochondria are cleared, thus attenuating brain injury, by mitophagy. Here, we identified the involvement of BNIP3L/NIX in cerebral ischemia-reperfusion (I-R)-induced mitophagy. Bnip3l knockout (bnip3l^?/?) impaired mitophagy and aggravated cerebral I-R injury in mice, which can be rescued by BNIP3L overexpression. The rescuing effects of BNIP3L overexpression can be observed in park2^?/? mice, which showed mitophagy deficiency after I-R. Interestingly, bnip3l and park2 double-knockout mice showed a synergistic mitophagy deficiency with I-R treatment, which further highlighted the roles of BNIP3L-mediated mitophagy as being independent from PARK2. Further experiments indicated that phosphorylation of BNIP3L serine 81 is critical for BNIP3L-mediated mitophagy. Nonphosphorylatable mutant BNIP3L^S81A failed to counteract both mitophagy impairment and neuroprotective effects in bnip3l^?/? mice. Our findings offer insights into mitochondrial quality control in ischemic stroke and bring forth the concept that BNIP3L could be a potential therapeutic target for ischemic stroke, beyond its accepted role in reticulocyte maturation.     

Glucose Metabolism, Hyperosmotic Stress, and Reprogramming of Somatic Cells

The availability of glucose and oxygen are important regulatory elements that help directing stem cell fate. In the undifferentiated state, stem cells, and their artificially reprogrammed equivalent-induced pluripotent stem cells (iPS) are characterized by limited oxidative capacity and active anaerobic glycolysis. Recent studies have shown that pluripotency—a characteristic of staminality—is associated with a poorly developed mitochondrial patrimony, while differentiation is accompanied by an activation of mitochondrial biogenesis. Besides being an important energy source in hypoxia, high glucose level results in hyperosmotic stress. The identification of specific metabolic pathways and biophysical factors that regulate stem cell fate, including high glucose in the extracellular medium, may therefore facilitate reprogramming efficiency and control the differentiation and fate of iPS cells, which are increasingly being explored as therapeutic tools. In this article, we review recent knowledge of the role of glucose metabolism and high glucose level as major anaerobic energy source, and a determinant of osmolarity as possible tools for reprogramming therapies in clinical applications. As in the diabetic setting hyperglycemia negatively affect the stem/progenitor cell fate and likely somatic reprogramming, we also discuss the in vivo potential transferability of the available in vitro findings.     

BNIP3 and Nix: Atypical regulators of cell fate

Since their discovery nearly 25 years ago, the BCL-2 family members BNIP3 and BNIP3L (aka Nix) have been labelled ‘atypical’. Originally, this was because BNIP3 and Nix have divergent BH3 domains compared to other BCL-2 proteins. In addition, this atypical BH3 domain is dispensable for inducing cell death, which is also unusual for a ‘death gene’. Instead, BNIP3 and Nix utilize a transmembrane domain, which allows for dimerization and insertion into and through organelle membranes to elicit cell death. Much has been learned regarding the biological function of these two atypical death genes, including their role in metabolic stress, where BNIP3 is responsive to hypoxia, while Nix responds variably to hypoxia and is also down-stream of PKC signaling and lipotoxic stress. Interestingly, both BNIP3 and Nix respond to signals related to cell atrophy. In addition, our current view of regulated cell death has expanded to include forms of necrosis such as necroptosis, pyroptosis, ferroptosis, and permeability transition-mediated cell death where BNIP3 and Nix have been shown to play context- and cell-type specific roles. Perhaps the most intriguing discoveries in recent years are the results demonstrating roles for BNIP3 and Nix outside of the purview of death genes, such as regulation of proliferation, differentiation/maturation, mitochondrial dynamics, macro- and selective-autophagy. We provide a historical and unbiased overview of these ‘death genes’, including new information related to alternative splicing and post-translational modification. In addition, we propose to redefine these two atypical members of the BCL-2 family as versatile regulators of cell fate.     

Testosterone increases apoptotic cell death and decreases mitophagy in Leber’s hereditary optic neuropathy cells

Leber’s hereditary optic neuropathy (LHON) is one of the most common mitochondrial diseases caused by point mutations in mitochondrial DNA (mtDNA). The majority of diagnosed LHON cases are caused by a point mutation at position 11,778 in the mitochondrial genome. LHON mainly affects young men in their 20s and 30s with usually poor visual prognosis. It remains unexplained why men are more likely to develop the disease and why only retinal ganglion cells are affected. In this study, a cell model was used for the first time to investigate the influence of testosterone on the cell death mechanism apoptosis and on an autophagy/mitophagy. Cells with m.11778G > A were found to be significantly more susceptible to nucleosome formation and effector caspase activation that serve as hallmarks of apoptotic cell death. Cells having this mutation expressed higher levels of mitophagic receptors BNIP3 and BNIP3L/Nix in a medium with testosterone. Moreover, cells having the mutation exhibited greater mitochondrial mass, which suggests these cells have a decreased cell survival. The observed decrease in cell survival was supported by the observed increase in apoptotic cell death. Autophagy was analyzed after inhibition with Bafilomycin A1 (Baf A1). The results indicate impairment in autophagy in LHON cells due to lower autophagic flux supported by observed lower levels of autophagosome marker LC3-II. The observed impaired lower autophagic flux in mutant cells correlated with increased levels of BNIP3 and BNIP3L/Nix in mutant cells.     

Mitophagy protein PINK1 suppresses colon tumor growth by metabolic reprogramming via p53 activation and reducing acetyl-CoA production

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the US. Understanding the mechanisms of CRC progression is essential to improve treatment. Mitochondria is the powerhouse for healthy cells. However, in tumor cells, less energy is produced by the mitochondria and metabolic reprogramming is an early hallmark of cancer. The metabolic differences between normal and cancer cells are being interrogated to uncover new therapeutic approaches. Mitochondria targeting PTEN-induced kinase 1 (PINK1) is a key regulator of mitophagy, the selective elimination of damaged mitochondria by autophagy. Defective mitophagy is increasingly associated with various diseases including CRC. However, a significant gap exists in our understanding of how PINK1-dependent mitophagy participates in the metabolic regulation of CRC. By mining Oncomine, we found that PINK1 expression was downregulated in human CRC tissues compared to normal colons. Moreover, disruption of PINK1 increased colon tumorigenesis in two colitis-associated CRC mouse models, suggesting that PINK1 functions as a tumor suppressor in CRC. PINK1 overexpression in murine colon tumor cells promoted mitophagy, decreased glycolysis and increased mitochondrial respiration potentially via activation of p53 signaling pathways. In contrast, PINK1 deletion decreased apoptosis, increased glycolysis, and reduced mitochondrial respiration and p53 signaling. Interestingly, PINK1 overexpression in vivo increased apoptotic cell death and suppressed colon tumor xenograft growth. Metabolomic analysis revealed that acetyl-CoA was significantly reduced in tumors with PINK1 overexpression, which was partly due to activation of the HIF-1α-pyruvate dehydrogenase (PDH) kinase 1 (PDHK1)-PDHE1α axis. Strikingly, treating mice with acetate increased acetyl-CoA levels and rescued PINK1-suppressed tumor growth. Importantly, PINK1 disruption simultaneously increased xenografted tumor growth and acetyl-CoA production. In conclusion, mitophagy protein PINK1 suppresses colon tumor growth by metabolic reprogramming and reducing acetyl-CoA production.Subject terms: Tumour-suppressor proteins, Cancer metabolism