Essential role for Nix in autophagic maturation of erythroid cells

Abstract

Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation 1-3 . Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation 2-6 , the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L) 7-9 , in the regulation of erythroid maturation through mitochondrial autophagy. Nix −/− mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix −/− mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (ΔΨ m ), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of ΔΨ m and restored the sequestration of mitochondria into autophagosomes in Nix −/− erythroid cells. These results suggest that Nix-dependent loss of ΔΨ m is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.Nix, a BH3-only member of the Bcl-2 family, is upregulated in erythroid cells undergoing terminal differentiation 10 . To determine the potential function for Nix in erythroid maturation, we generated Nix −/− mice using embryonic stem (ES) cells with a gene trap insertion between exons 3 and 4 of Nix ( Supplementary Fig. 2). We first examined red blood cells in the peripheral blood (RBCs), including reticulocytes and erythrocytes, in Nix −/− mice. Although RBC counts were decreased (Supplementary Table 1), polychromasia and increased reticulocytes were observed in Nix −/− mice ( Fig. 1a and Supplementary Fig. 3a). We also examined RBCs for the expression of an erythroid cell marker, glycophorin-A-associated Ter119, and for transferrin receptor CD71, which is downregulated during terminal erythroid differentiation 11,12 . Although Ter119 low CD71 high and Ter119 + CD71 high early erythroblasts 13 were absent in the peripheral blood, a significant increase in Ter119 + CD71 + reticulocytes was observed in Nix −/− mice (Fig. 1b). Electron microscopy also showed more irregularly shaped cellsCorrespondence and requests for materials should be addressed to M.C. (minc@bcm.tmc.edu) or J.W. (jinwang@bcm.tmc.edu). Author Contributions H.S. conducted the majority of the experiments, supervised by J.W. and M.C.; P.T. stained spleen sections and blood smears; S.K.D. measured osmotic fragility and assisted with biotin and CMFDA labelling; A.S. performed RT-PCR for Epo; J.T.P. and P.T. provided experimental advice; M.C. and J.W. generated the Nix −/− mice, designed experiments and...

Cardiac reanimation: targeting cardiomyocyte death by BNIP3 and NIX/BNIP3L

“…However, NIX/BNIP3L also causes programmed necrosis by localizing to ER/SR, where it increases calcium stores and therefore the mitochondrial delivery of calcium that causes the pore transition. In the context of recent studies identifying a role for NIX/BNIP3L (and probably BNIP3 as well (Hamacher-Brady et al, 2007;Kubli et al, 2008)) in mitochondrial autophagy (Schweers et al, 2007;Sandoval et al, 2008), these findings position NIX/BNIP3L at the apex of multiple parallel pathways leading to programmed cell death (Figure 2).…”

“…However, in contrast with BNIP3 where germ-line gene ablation showed no baseline phenotype, the germ-line NIX/ BNIP3L knockout mouse produced in the Dorn lab exhibited markedly increased blood reticulocyte counts associated with decreased in vivo and in vitro apoptosis of splenic erythroid precursers, and extreme hypersensitivity to erythropoietin (Diwan et al, 2007a). A similar phenotype that included hemolytic anemia was subsequently described by two other groups who independently produced NIX/BNIP3L knockout mice (Schweers et al, 2007;Sandoval et al, 2008). Because hematopoietic abnormalities such as these had the potential to confound cardiac studies, a Cre-lox strategy was used to create mice in which the NIX/BNIP3L gene was selectively ablated in cardiac myocytes, which were subjected to surgical transverse aortic constriction (TAC) (Diwan et al, 2008b).…”

NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy

“…46 Increasing evidence, however, supports the notion that mitophagy can also occur independently of Parkin/Pink1. [47][48][49][50][51] The initial report showing that selective mitophagy occurs in PINK1-deficient neuroblastoma cells 51 was followed by studies showing that Parkin may be recruited through PINK1-independent mechanisms both in vitro and in vivo. 52,53 Pathways independent of both PINK1 and Parkin have also emerged, often involving mitophagy stimuli that cause lesser degrees of or no mitochondrial depolarization.…”

Autophagy at the crossroads of catabolism and anabolism

“…For example, damaged and superfluous mitochondria are targeted to autophagosomes in a process termed mitophagy 24,25 . Numerous studies have led to the identification of mitophagy receptors such as Atg32 in yeast, as well as BCL2/adenovirus E1B 19 kDa proteininteracting protein 3 (BNIP3), NIP3-like protein X (NIX) and FUN14 domain-containing protein 1 (FUNDC1) in mammals [26][27][28][29][30][31][32][33] . These receptors all possess an LIR motif to directly target mitochondria to autophagosomes.…”