Human erythroid differentiation requires VDAC1-mediated mitochondrial clearance

Erythroblast maturation in mammals is dependent on organelle clearance throughout terminal erythropoiesis. We studied the role of the outer mitochondrial membrane protein voltage-dependent anion channel-1 (VDAC1) in human terminal erythropoiesis. We show that short hairpin (shRNA)-mediated downregulation of VDAC1 accelerates erythroblast maturation. Thereafter, erythroblasts are blocked at the orthochromatic stage, exhibiting a significant decreased level of enucleation, concomitant with an increased cell death. We demonstrate that mitochondria clearance starts at the transition from basophilic to polychromatic erythroblast, and that VDAC1 downregulation induces the mitochondrial retention. In damaged mitochondria from non-erythroid cells, VDAC1 was identified as a target for Parkin-mediated ubiquitination to recruit the phagophore. Here, we showed that VDAC1 is involved in phagophore’s membrane recruitment regulating selective mitophagy of still functional mitochondria from human erythroblasts. These findings demonstrate for the first time a crucial role for VDAC1 in human erythroblast terminal differentiation, regulating mitochondria clearance.Fil: Moras, Martina. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Hattab, Claude. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Gonzalez Menendez, Pedro. Laboratoire d’Excellence GR-Ex; Francia. Université Montpellier II; Francia. Centre National de la Recherche Scientifique; FranciaFil: Fader Kaiser, Claudio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Dussiot, Michael. Universite de Paris; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Larghero, Jerome. Hôpital Saint-Louis. Unité de Thérapie cellulaire; FranciaFil: Le Van Kim, Caroline. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Kinet, Sandrina. Laboratoire d’Excellence GR-Ex; Francia. Université Montpellier II; Francia. Centre National de la Recherche Scientifique; FranciaFil: Taylor, Naomi. Laboratoire d’Excellence GR-Ex; Francia. Centre National de la Recherche Scientifique; Francia. Université Montpellier II; Francia. Center for Cancer Research; Estados UnidosFil: Lefevre, Sophie D.. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; FranciaFil: Ostuni, Mariano. Universite de Paris; Francia. Institut National de Transfusion Sanguine; Francia. Laboratoire d’Excellence GR-Ex; Franci

Rôle du complexe VDAC1/TSPO1 au cours de l'érythropoïèse humaine, focus sur la mitophagie

Chez les mammifères, les organelles sont éliminées au cours de la différentiation érythroïde pour produire des globules rouges matures. Les mitochondries sont dégradées par une voie d’autophagie spécifique, connue sous le nom de mitophagie. VDAC1 (Voltage Dependent Anion Channel) et TSPO1 (TranSlocator PrOtein) sont deux protéines de la membrane externe de la mitochondrie (MEM), associées pour former un complexe pour lequel il a été décrit, entre autres, un rôle dans l’induction de la mitophagie. Cette fonction, décrite dans des cellules non érythroïdes, est dépendante de la voie de signalisation PINK1/Parkin. En réprimant l’expression de VDAC1 ou TSPO1 par ARN interférence ou en utilisant un ligand synthétique, Ro5-4864, pour moduler les fonctions du complexe, nous avons étudié le rôle du complexe VDAC1/TSPO1 sur la différenciation érythroïde humaine. Quelle que soit la technique utilisée, nous avons observé une réduction de la prolifération et une baisse de l'énucléation des érythroblastes, différenciés à partir de cellules CD34+ issues de sang de cordon. Ceci souligne le rôle essentiel de ce complexe sur la maturation terminale humaine. De plus, et uniquement lors de la diminution de l’expression de VDAC1, nous avons pu observer une accélération de la différenciation. Ces travaux ont permis de mettre en évidence une nouvelle fonction intrinsèque de VDAC1 au cours de la différenciation érythroïde, qui n'est pas liée au complexe formé avec TSPO1. Au cours de ce travail, nous avons également décrit pour la première fois, l’élimination progressive des mitochondries dans les érythroblastes, à partir du stade basophile jusqu’au stade orthochromatique. Ces résultats confirment chez l'homme des données récemment décrites chez la souris. Nous avons également observé que la diminution de l’expression de VDAC1 ou de TSPO1 ou l’ajout de Ro5-4864 inhibe l’élimination des mitochondries. Nos résultats montrent que la diminution de l’expression de VDAC1 réduit l'accumulation de PINK1 au niveau de la MEM et, par conséquent, bloque le recrutement du phagophore, ce qui entraîne une inhibitionde la mitophagie. Nous avons également montré que, dans ces conditions, la réduction de l’élimination des mitochondries est indépendante de NIX, une protéine de la MEM connue pour initier la dégradation des mitochondries dans les réticulocytes de souris. Nos données mettent en évidence pour la première fois l'importance de la voie PINK1/Parkin dans l'élimination des mitochondries au cours de l'érythropoïèse humaine. Nous décrivons également un nouveau mécanisme modulant l'induction de la mitophagie par le contrôle de la quantité de PINK1 à la MEM sous la dépendance de VDAC1.In mammals, organelles must be eliminated from maturing red blood cell to produce functional erythrocytes. Many evidences suggest that mitochondria are removed by a specific autophagic pathway, known as mitophagy. The voltage-dependent anion channel (VDAC1) and the 18-kDa Translocator protein (TSPO1) are two mitochondrial outer membrane (OMM) proteins known to form a protein complex and some studies have reported a role of this complex in the modulation of PINK1/Parkin mitophagy in non-erythroid cells. Using genetic invalidation of genes as well as a synthetic ligand, Ro5-4864, to modulate thecomplex functions, we investigated the role of VDAC1/TSPO1 complex on human erythroiddifferentiation. Whatever the way of modulating VDAC1/TSPO1 function, we observed a reduced proliferation capacity and a failure in enucleation of erythroblasts differentiated fromcord blood-derived CD34+ cells. This highlights the essential role of this specific complex on human terminal maturation. Interestingly however, only upon VDAC1 downregulation we could observed an acceleration of the differentiation. This points out a new intrinsic function of VDAC1 during erythroid differentiation that is not related to the complex formed with TSPO1.Here for the first time, we described a progressive clearance of mitochondria from basophilic to orthochromatic erythroblasts, confirming in human, data recently reported in mice. We also showed that the downregulation of VDAC1 or TSPO1, or the use of Ro5-4864, cause a defect in the progressive mitochondrial clearance that is occurring in the terminal phase of human erythropoiesis. Our results suggest that VDAC1 downregulation reduces PINK1accumulation at OMM and subsequently the recruitment of the phagophore, resulting in inhibition of mitophagy. We also demonstrated that reduction of mitochondria clearance upon VDAC1 downregulation is independent of NIX, an OMM protein known to induce mitochondria clearance in mice reticulocytes.Not only our data pointed out for the first time the importance of the PINK1/Parkin pathway in human erythropoiesis for the elimination of mitochondria, but we also discovered a new pathway governing mitophagy initiation, i.e. the VDAC1-dependent modulation of PINK1 OMM accumulation

From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals

Erythropoiesis occurs mostly in bone marrow and ends in blood stream. Mature red blood cells are generated from multipotent hematopoietic stem cells, through a complex maturation process involving several morphological changes to produce a highly functional specialized cells. In mammals, terminal steps involved expulsion of the nucleus from erythroblasts that leads to the formation of reticulocytes. In order to produce mature biconcave red blood cells, organelles and ribosomes are selectively eliminated from reticulocytes as well as the plasma membrane undergoes remodeling. The mechanisms involved in these last maturation steps are still under investigation. Enucleation involves dramatic chromatin condensation and establishment of the nuclear polarity, which is driven by a rearrangement of actin cytoskeleton and the clathrin-dependent generation of vacuoles at the nuclear-cytoplasmic junction. This process is favored by interaction between the erythroblasts and macrophages at the erythroblastic island. Mitochondria are eliminated by mitophagy. This is a macroautophagy pathway consisting in the engulfment of mitochondria into a double-membrane structure called autophagosome before degradation. Several mice knock-out models were developed to identify mitophagy-involved proteins during erythropoiesis, but whole mechanisms are not completely determined. Less is known concerning the clearance of other organelles, such as smooth and rough ER, Golgi apparatus and ribosomes. Understanding the modulators of organelles clearance in erythropoiesis may elucidate the pathogenesis of different dyserythropoietic diseases such as myelodysplastic syndrome, leukemia and anemia

An Overview of Different Strategies to Recreate the Physiological Environment in Experimental Erythropoiesis

Human erythropoiesis is a complex process leading to the production of mature, enucleated erythrocytes (RBCs). It occurs mainly at bone marrow (BM), where hematopoietic stem cells (HSCs) are engaged in the early erythroid differentiation to commit into erythroid progenitor cells (burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E)). Then, during the terminal differentiation, several erythropoietin-induced signaling pathways trigger the differentiation of CFU-E on successive stages from pro-erythroblast to reticulocytes. The latter are released into the circulation, finalizing their maturation into functional RBCs. This process is finely regulated by the physiological environment including the erythroblast-macrophage interaction in the erythroblastic island (EBI). Several human diseases have been associated with ineffective erythropoiesis, either by a defective or an excessive production of RBCs, as well as an increase or a hemoglobinization defect. Fully understanding the production of mature red blood cells is crucial for the comprehension of erythroid pathologies as well as to the field of transfusion. Many experimental approaches have been carried out to achieve a complete differentiation in vitro to produce functional biconcave mature RBCs. However, the various protocols usually fail to achieve enough quantities of completely mature RBCs. In this review, we focus on the evolution of erythropoiesis studies over the years, taking special interest in efforts that were made to include the microenvironment and erythroblastic islands paradigm. These more physiological approaches will contribute to a deeper comprehension of erythropoiesis, improve the treatment of dyserythropoietic disorders, and break through the barriers in massive RBCs production for transfusion

Induction of ATP Release, PPIX Transport, and Cholesterol Uptake by Human Red Blood Cells Using a New Family of TSPO Ligands

Two main isoforms of the Translocator Protein (TSPO) have been identified. TSPO1 is ubiquitous and is mainly present at the outer mitochondrial membrane of most eukaryotic cells, whereas, TSPO2 is specific to the erythroid lineage, located at the plasma membrane, the nucleus, and the endoplasmic reticulum. The design of specific tools is necessary to determine the molecular associations and functions of TSPO, which remain controversial nowadays. We recently demonstrated that TSPO2 is involved in a supramolecular complex of the erythrocyte membrane, where micromolar doses of the classical TSPO ligands induce ATP release and zinc protoporphyrin (ZnPPIX) transport. In this work, three newly-designed ligands (NCS1016, NCS1018, and NCS1026) were assessed for their ability to modulate the functions of various erythrocyte’s and compare them to the TSPO classical ligands. The three new ligands were effective in reducing intraerythrocytic Plasmodium growth, without compromising erythrocyte survival. While NCS1016 and NCS1018 were the most effective ligands in delaying sorbitol-induced hemolysis, NCS1016 induced the highest uptake of ZnPPIX and NCS1026 was the only ligand inhibiting the cholesterol uptake. Differential effects of ligands are probably due, not only, to ligand features, but also to the dynamic interaction of TSPO with various partners at the cell membrane. Further studies are necessary to fully understand the mechanisms of the TSPO’s complex activation

Influence of 4f filling on electronic and magnetic properties of rare earth-Au surface compounds

One-atom-thick rare-earth/noble metal (RE-NM) compounds are attractive materials to investigate two-dimensional magnetism, since they are easy to synthesize into a common RE-NM2 structure with high crystal perfection. Here we perform a comparative study of the GdAu2, HoAu2, and YbAu2 monolayer compounds grown on Au(111). We find the same atomic lattice quality and moiré superlattice periodicity in the three cases, but different electronic properties and magnetism. The YbAu2 monolayer reveals the characteristic electronic signatures of a mixed-valence configuration in the Yb atom. In contrast, GdAu2 and HoAu2 show the trivalent character of the rare-earth and ferromagnetic transitions below 22 K. Yet, the GdAu2 monolayer has an in-plane magnetic easy-axis, versus the out-of-plane one in HoAu2. The electronic bands of the two trivalent compounds are very similar, while the divalent YbAu2 monolayer exhibits different band features. In the latter, a strong 4f–5d hybridization is manifested in neatly resolved avoided crossings near the Fermi level. First principles theory points to a residual presence of empty 4f states, explaining the fluctuating valence of Yb in the YbAu2 monolayer.This work was supported in part by the Spanish MINECO (MAT-2017-88374-P, MAT2016-78293-C6, FIS2016-75862-P, PGC2018-098613-B-C21, SEV-2017-0706 and SEV-2016-0686), the Spanish Research Agency (PID2019-107338RB-C65), Basque Government Project IT-1255-19, and University of the Basque Country (UPV/EHU) grant GIU18/138 and the European Regional Development Fund (ERDF) under the program Interreg V-A España-Francia-Andorra (Contract No. EFA 194/16 TNSI). The experiments at Elettra were supported by the EU Calipso project and computational resources were provided by the DIPC computing center. L. F. acknowledges financial support from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement MagicFACE No 797109. A. K. K., M. J., P. M. S. and P. M. acknowledge the project EUROFEL-ROADMAP ESFRI.Peer reviewe