NIX induces mitochondrial autophagy in reticulocytes

The controlled elimination of defective mitochondria is necessary for the health of long-lived post-mitotic cells, like cardiomyocytes and neurons. Mitochondrial elimination also occurs during the course of normal development, in lens epithelial and erythroid cells. Strikingly, at the final stage of erythroid cell maturation, newly formed erythrocytes, also known as reticulocytes, eliminate their entire cohort of mitochondria. We have employed this model to investigate the mechanism of programmed mitochondrial clearance. NIX (BNIP3L) is a Bcl-2-related protein that is upregulated during terminal erythroid differentiation. NIX-deficient reticulocytes have a significant defect of mitochondrial clearance. Consistent with the ability of NIX to cause mitochondrial depolarization, we show that mitochondria are depolarized in wild type but not NIX deficient reticulocytes. NIX does not function through established proapoptotic pathways, nor does it mediate the induction of autophagy in erythroid cells. Rather, NIX is required for the selective incorporation of mitochondria into autophagosomes. Elucidation of the mechanism of this effect will improve our understanding of the role of autophagy in the maintenance of cellular homeostasis.     

Selective mitochondrial autophagy during erythroid maturation

Accumulating evidence suggests that autophagy can be selective in the clearance of organelles in yeast and in mammalian cells. We have observed that the sequestration of mitochondria by autophagosomes was defective in reticulocytes in the absence of Nix. Nix is required for the dissipation of mitochondrial membrane potential (ΔΨm) during erythroid maturation. Moreover, pharmacological agents that induce the loss of ΔΨm can restore the sequestration of mitochondria by autophagosomes and promote mitochondrial clearance in Nix-/- erythroid cells. Our data suggest that mitochondrial depolarization induces recognition and sequestration of mitochondria by autophagosomes. Elucidating the mechanisms underlying selective mitochondrial autophagy not only will help us to understand the mechanisms for erythroid maturation, but also may provide insights into mitochondrial quality control by autophagy in the protection against aging, cancer, and neurodegenerative diseases.

Addendum to: Sandoval H, Thiagarajan P, Dasgupta SK, Schumacher A, Prchal JT, Chen M, Wang J. Essential role for Nix in autophagic maturation of erythroid cells. Nature 2008; 454:232-5.     

Selective mitochondrial autophagy during erythroid maturation

Accumulating evidence suggests that autophagy can be selective in the clearance of organelles in yeast and in mammalian cells. We have observed that the sequestration of mitochondria by autophagosomes was defective in reticulocytes in the absence of Nix. Nix is required for the dissipation of mitochondrial membrane potential (DeltaPsim) during erythroid maturation. Moreover, pharmacological agents that induce the loss of DeltaPsim can restore the sequestration of mitochondria by autophagosomes and promote mitochondrial clearance in Nix(-/-) erythroid cells. Our data suggest that mitochondrial depolarization induces recognition and sequestration of mitochondria by autophagosomes. Elucidating the mechanisms underlying selective mitochondrial autophagy not only will help us to understand the mechanisms for erythroid maturation, but also may provide insights into mitochondrial quality control by autophagy in the protection against aging, cancer and neurodegenerative diseases.     

Reticulocyte mitophagy: Monitoring mitochondrial clearance in a mammalian model

Mitochondria are the primary site of energy production in animal cells. In mitochondria, the flow of electrons through the electron transport chain creates a potential difference across the inner membrane, which is utilized for ATP production. However, due to inherent inefficiencies in electron transport, reactive oxygen species are also produced, which damage mitochondrial proteins and nucleic acids, and impair mitochondrial function.¹ Decreased mitochondrial function causes increased reactive oxygen species generation, a decline in cellular function, and potentially cell death.² Therefore, to maintain cellular homeostasis, mechanisms have evolved to selectively eliminate defective mitochondria.³ Mitochondria are constantly undergoing cycles of fission and fusion, and this process appears to have a role in mitochondrial quality control. Following fission, daughter mitochondria are produced, which can differ in their membrane polarization. Depolarized mitochondria are less likely to undergo subsequent fusion, and more likely to undergo autophagic clearance.⁴ As would be predicted, given the potential for cytochrome c release, depolarization is a powerful stimulus for mitochondrial clearance. Depolarization causes recruitment of the E3 ubiquitin ligase Parkin to mitochondria, which is required for their subsequent engulfment by autophagosomes.⁵ Macroautophagy pathways also appear to have a role, as hepatocytes deficient for the E1-like enzyme Atg7 accumulate abnormal mitochondria.⁶ Finally, recent studies in a developmental model have yielded insight into this process. Newly-formed erythrocytes, also known as reticulocytes, eliminate their entire cohort of mitochondria during development.⁷ This process depends on the mitochondrial protein NIX, is partially dependent on autophagy, and is independent of mitochondrial depolarization.^8-10 Here we describe the use of reticulocytes to study mitochondrial clearance.     

Autophagy-dependent and -independent mechanisms of mitochondrial clearance during reticulocyte maturation

Erythrocyte formation involves the elimination of mitochondria at the reticulocyte stage of development. Nix-/- reticulocytes fail to eliminate their mitochondria at this step due to a defect in the targeting of mitochondria to autophagosomes. To determine the role of autophagy in this process, we generated Atg7-/- transplant mice. Atg7-/- reticulocytes exhibit a partial defect in mitochondrial clearance, demonstrating that there are both autophagy-dependent and -independent mechanisms of mitochondrial clearance. We used Atg7-/- autophagy-defective reticulocytes to study temporal events in mitochondrial clearance. Mitochondrial depolarization precedes elimination, but in Atg7-/- reticulocytes the depolarization event is markedly delayed. Since Atg7 regulates autophagosome formation, we infer that mitochondrial depolarization occurs downstream of autophagosome formation in reticulocytes. We propose that there are two mechanisms of mitochondrial clearance: one that is triggered by mitochondrial depolarization, and a second NIX-dependent mechanism, which is not. The NIX-dependent mechanism remains to be elucidated.     

Mitochondrial clearance by autophagy in developing erythrocytes: Clearly important,but just how much so?

Erythrocytes are anucleated cells devoid of organelles. Expulsion of the nucleus from erythroblasts leads to the formation of reticulocytes, which still contain organelles. The mechanisms responsible for the final removal of organelles from developing erythroid cells are still being elucidated. Mitochondria are the most abundant organelles to be cleared for the completion of erythropoiesis. Macroautophagy, referred to as autophagy, is a regulated catabolic pathway consisting of the engulfment of cytoplasmic cargo by a double membraned-vesicle, the autophagosome, which typically then fuses to lysosomal compartments for the degradation of the sequestered material. Early electron microscopic observations of reticulocytes suggested the autophagic engulfment of mitochondria (mitophagy) as a possible mechanism for mitochondrial clearance in these. Recently, a number of studies have backed this hypothesis with molecular evidence. Indeed, the absence of Nix, which targets mitochondria to autophagosomes, or the deficiency of proteins in the autophagic pathway lead to impaired mitochondrial clearance from developing erythroid cells. Importantly, however, the extent to which the absence of mitophagy affects erythroid development differs depending on the model and gene investigated. This review will therefore focus on comparing the different studies of mitophagy in erythroid development and highlight some of the remaining controversial points.     

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.     

Effects of Ca2+ and lipoxygenase inhibitors on hexokinase degradation in rabbit reticulocytes

In rabbit reticulocytes more than half of the total hexokinase activity is mitochondrial bound and shows a fast decay during reticulocyte maturation. During in vitro incubation of rabbit reticulocytes, Ca²⁺ increases the decay of hexokinase while salicylhydroxamate (SHAM), an inhibitor of lipoxygenase, reduces the decay. Swelling of mitochondria, by incubation of the cells in hypotonic solutions, greatly enhances hexokinase decay, but both the Ca²⁺ and SHAM are still appreciable suggesting that Ca²⁺ and the swelling act by additive mechanisms, both able to influence hexokinase decay. This was confirmed by incubation of rabbit brain mitochondria in hypotonic solutions which does not promote any hexokinase decay, while the presence of Ca²⁺ does. Analyses of hexokinase isozymic pattern after incubation of reticulocytes in hypotonic solution both with and without Ca²⁺ and SHAM showed that the decay of hexokinase mainly involves the mitochrondrial bound isozymic forms.Abbreviations SHAM Salicylhydroxamate - HPLC High-Performance Liquid Chromatography     

The ins and outs of human reticulocyte maturation

The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal rearrangements and changes to cellular architecture. Autophagy is implicated in the sequential removal of erythroid organelles during erythropoiesis, although how this is regulated during late stages of erythroid differentiation, and the potential contribution of autophagy during reticulocyte maturation, remain unclear. Using an optimized ex vivo differentiation system for human erythropoiesis, we have observed that maturing reticulocytes are characterized by the presence of one or few large vacuolar compartments. These label strongly for glycophorin A (GYPA/GPA) which is internalized from the plasma membrane; however, they also contain organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting that they are endocytic/autophagic hybrid structures. Interestingly, we observed the release of these vacuoles by exocytosis in maturing reticulocytes, and speculate that autophagy is needed to concentrate the final remnants of the reticulocyte endomembrane system in autophagosome/endosome hybrid compartments that are primed to undergo exocytosis.     

The ins and outs of human reticulocyte maturation: Autophagy and the endosome/exosome pathway

The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal rearrangements and changes to cellular architecture. Autophagy is implicated in the sequential removal of erythroid organelles during erythropoiesis, although how this is regulated during late stages of erythroid differentiation, and the potential contribution of autophagy during reticulocyte maturation, remain unclear. Using an optimized ex vivo differentiation system for human erythropoiesis, we have observed that maturing reticulocytes are characterized by the presence of one or few large vacuolar compartments. These label strongly for glycophorin A (GYPA/GPA) which is internalized from the plasma membrane; however, they also contain organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting that they are endocytic/autophagic hybrid structures. Interestingly, we observed the release of these vacuoles by exocytosis in maturing reticulocytes, and speculate that autophagy is needed to concentrate the final remnants of the reticulocyte endomembrane system in autophagosome/endosome hybrid compartments that are primed to undergo exocytosis.     

Nix is a selective autophagy receptor for mitochondrial clearance

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.     

A short linear motif in BNIP3L (NIX) mediates mitochondrial clearance in reticulocytes

Elimination of defective mitochondria is essential for the health of long-lived, postmitotic cells. To gain insight into this process, we examined programmed mitochondrial clearance in reticulocytes. BNIP3L is a mitochondrial outer membrane protein that is required for clearance. It has been suggested that BNIP3L functions by causing mitochondrial depolarization, activating autophagy, or engaging the autophagy machinery. Here we showed in mice that BNIP3L activity localizes to a small region in its cytoplasmic domain, the minimal essential region (MER). The MER is a novel sequence, which comprises three contiguous hydrophobic amino acid residues, and flanking charged residues. Mutation of the central leucine residue causes complete loss of BNIP3L activity, and prevents rescue of mitochondrial clearance. Structural bioinformatics analysis predicts that the BNIP3L cytoplasmic domain lacks stable tertiary structure, but that the MER forms an α-helix upon binding to another protein. These findings support an adaptor model of BNIP3L, centered on the MER.     

A short linear motif in BNIP3L (NIX) mediates mitochondrial clearance in reticulocytes

Elimination of defective mitochondria is essential for the health of long-lived, postmitotic cells. To gain insight into this process, we examined programmed mitochondrial clearance in reticulocytes. BNIP3L is a mitochondrial outer membrane protein that is required for clearance. It has been suggested that BNIP3L functions by causing mitochondrial depolarization, activating autophagy, or engaging the autophagy machinery. Here we showed in mice that BNIP3L activity localizes to a small region in its cytoplasmic domain, the minimal essential region (MER). The MER is a novel sequence, which comprises three contiguous hydrophobic amino acid residues, and flanking charged residues. Mutation of the central leucine residue causes complete loss of BNIP3L activity, and prevents rescue of mitochondrial clearance. Structural bioinformatics analysis predicts that the BNIP3L cytoplasmic domain lacks stable tertiary structure, but that the MER forms an α-helix upon binding to another protein. These findings support an adaptor model of BNIP3L, centered on the MER.     

Maternal inheritance of mitochondrial DNA

Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) organelle autophagy as allophagy.     

Effects of NIX‐mediated mitophagy on ox‐LDL‐induced macrophage pyroptosis in atherosclerosis

Pyroptosis is a form of cell death that is uniquely dependent on caspase‐1. Pyroptosis involved in oxidized low‐density lipoprotein (ox‐LDL)‐induced human macrophage death through the promotion of caspase‐1 activation is important for the formation of unstable plaques in atherosclerosis. The mitochondrial outer membrane protein NIX directly interacts with microtubule‐associated protein 1 light chain 3 (LC3). Although we previously showed that NIX‐mediated mitochondrial autophagy is involved in the clearance of damaged mitochondria, how NIX contributes to ox‐LDL‐induced macrophage pyroptosis remains unknown. Here, immunoperoxidase staining Nix expression decreased in human atherosclerosis. When we silenced NIX expression in murine macrophage cell, active caspase‐1, and mature interleukin‐1β expression levels were increased and LC3 was reduced. In addition, LDH release and acridine orange and ethidium bromide staining indicated that damage to macrophage cell membranes induced by ox‐LDL was substantially worse. Moreover, intracellular reactive oxygen species and NLRP3 inflammasome levels increased. Taken together, these results demonstrated that NIX inhibits ox‐LDL‐induced macrophage pyroptosis via autophagy in atherosclerosis.     

Characterization of somatic Ca2+ clearance mechanisms in young and mature hippocampal granule cells

Calcium is a key regulator for expression of genes relevant to survival and maturation of newborn neurons. Mammalian hippocampal dentate gyrus generates new granule cells (GCs) throughout adult life. We identified young and mature GCs in hippocampi of young adult mice according to their electrical properties, and investigated contributions of Na/Ca exchanger (NCX), sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA), plasma membrane Ca²⁺-ATPase (PMCA) and mitochondria to Ca²⁺ clearance in somata of GCs. Somatic Ca²⁺ clearance was increased by about 50% as GCs matured. NCX activity increased proportionally during maturation with its relative contribution kept about 40% both in young and mature GCs. On the other hand, the developmental increases in activities of mitochondria and SERCA resulted in higher contributions to Ca²⁺ clearance in mature GCs than in young GCs. Especially mitochondrial function was most highly enhanced during maturation. PMCA activity, however, did not increase during maturation. Low Ca²⁺ clearance in immature GCs might facilitate higher Ca²⁺ accumulation during network activity, which in turn help survival of young GCs.     

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.     

Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A₁, Atg7 knockdown or in atg5^?/? MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.     

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria

Maintenance of healthy mitochondria prevents aging, cancer, and a variety of degenerative diseases that are due to the result of defective mitochondrial quality control (MQC). Recently, we discovered a novel mechanism for MQC, in which Mieap induces intramitochondrial lysosome-like organella that plays a critical role in the elimination of oxidized mitochondrial proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria). However, a large part of the mechanisms for MQC remains unknown. Here, we report additional mechanisms for Mieap-regulated MQC. Reactive oxygen species (ROS) scavengers completely inhibited MALM. A mitochondrial outer membrane protein NIX interacted with Mieap in a ROS-dependent manner via the BH3 domain of NIX and the coiled-coil domain of Mieap. Deficiency of NIX also completely impaired MALM. When MALM was inhibited, Mieap induced vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulfed and degraded the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria. These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.     

MicroRNA-137 Is a Novel Hypoxia-responsive MicroRNA That Inhibits Mitophagy via Regulation of Two Mitophagy Receptors FUNDC1 and NIX

Mitophagy receptors mediate the selective recognition and targeting of damaged mitochondria by autophagosomes. The mechanism for the regulation of these receptors remains unknown. Here, we demonstrated that a novel hypoxia-responsive microRNA, microRNA-137 (miR-137), markedly inhibits mitochondrial degradation by autophagy without affecting global autophagy. miR-137 targets the expression of two mitophagy receptors NIX and FUNDC1. Impaired mitophagy in response to hypoxia caused by miR-137 is reversed by re-expression of FUNDC1 and NIX expression vectors lacking the miR-137 recognition sites at their 3′ UTR. Conversely, miR-137 also suppresses the mitophagy induced by fundc1 (CDS+3′UTR) but not fundc1 (CDS) overexpression. Finally, we found that miR-137 inhibits mitophagy by reducing the expression of the mitophagy receptor thereby leads to inadequate interaction between mitophagy receptor and LC3. Our results demonstrated the regulatory role of miRNA to mitophagy receptors and revealed a novel link between miR-137 and mitophagy.