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M. Moras et al.
polychromatic stage, of key proteins of mitophagy belonging to different pathways, such as NIX and PINK1.26 NIX upregulation was described by several works studying NIX-mediated mitophagy in murine reticulocytes,12-14 but no clear evidence was found to explain the upregulation of PINK1. Under the condition of VDAC1 downregulation, NIX protein levels were not altered, suggesting that mitochondrial retention was not due to a defect in the NIX pathway but in the PINK1 pathway, as described below. It was previously reported that VDAC is required to promote Parkin recruitment to the mitochondria.18 Our data suggest that this could be due to a lack in PINK1 accumulation at the OMM.
Since VDAC1 is part of a multimeric complex with ATAD3A,23 we speculate that in the absence of VDAC1, ATAD3A is free to form alternative complexes.40 Recently, a new function of ATAD3A was described in hematopoi- etic progenitor cells, promoting the import of PINK1.41 In this context PINK1 is rapidly degraded by the mitochon- drial peptidases MPP and Parl to prevent its accumulation in the surface prevent mitophagy. However, when ATAD3A dissociates from the translocase of the inner membrane (TIM) complex, the import of PINK1 is blocked, causing its accumulation.42 It is noteworthy that also hexokinase 2 (HK2) and adenine nucleotide trans- porter (ANT), both proteins known to be involved in a multimeric mitochondrial membrane protein complex, have been recently shown to be essential for PINK1 accu- mulation at the OMM,43,44 suggesting a role of this com- plex in PINK1/Parkin mitophagy. Further studies are nec- essary to assess this hypothesis and evaluate the crosstalk between VDAC1, ATAD3A, ANT and/or HK2 in the ter- minal phase of erythropoiesis.
In conclusion, our data are the first demonstration of a fundamental role of mitochondrial-selective autophagy at the erythroblast stage. Loss of VDAC1 causes an ineffec- tive elimination of mitochondria in the transition between basophilic and orthochromatic erythroblasts, demonstrat- ing the critical role of VDAC1 in late stage erythropoiesis.
We have taken account of recent reports highlighting the possible unspecific and strain-dependent effects of shRNA in knocking down mouse erythroblasts enucle- ation.45 However, we have observed the same effects after knocking down VDAC1 by two different approaches (shRNA and siRNA) and by using different experimental models, strongly supporting our conclusions.
Over the last few years, several studies have demon- strated the association of mitophagy defects with various hematological syndromes, highlighting the importance of mitochondrial content and mitophagy during human ery- thropoiesis.46-53 Our work provides a better understanding of the mechanisms regulating mitochondrial autophagy in human erythroid progenitors, particularly during the terminal phase of erythropoiesis. Elucidating the different pathways involved in mitochondrial clearance and their kinetics, will promote the development of novel thera- peutic approaches for treating hematological disorders that involve defective erythroid maturation.
Disclosures
No conflicts of interest to disclose
Contributions
MM designed experiments, performed experiments, analyzed data and wrote the paper; CH, PGM, MD, SK and CF per- formed experiments; JL evaluated protocols and supervised blood samples purchasing; CLVK critically evaluated experiments and obtained funding; SK and NT critically evaluated experiments, provided supervision, wrote the paper and obtained funding; SDL and MAO conceived the project, obtained funding, designed and critically evaluated experiments, provided supervi- sion and wrote the paper; SDL and MAO contributed equally to this paper. All authors read and commented on the paper and approved the final version.
Acknowledgments
The authors would like to thank Mrs Corentine Chrysostome et Mr Abdellah Nait for their secretarial and technical assis- tance, respectively. We acknowledge the Cell Sorting facility of the Institut Imagine; the ImagoSeine core facility of the Institut Jacques Monod, member of IBiSA and France-BioImaging (ANR-10-INBS-04) infrastructure; the Laboratory of Excellence GR-Ex (Grant ANR-11-LABX-0051) and the Guests Researcher program from Paris Diderot University.
Funding
MM is funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska- Curie grant agreement No. 665850, the Club du Globule Rouge et du Fer (CGRF) and Société Française d’Hématologie (SFH); PG-M is founded by the CLARIN-COFUND program from the Principado de Asturias and the European Union.
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