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M. Moras et al.
(Figure 5B), there was a significant accumulation of p62, an adaptor protein that allows bridging between the ubiq- uitinated cargos and the phagophore membrane35 (Figure 5A to C). Furthermore, p62 expression was detected by flow cytometry in different stages on fixed cells stained with a4-integrin and Band3 antibodies. As well as for mitochondrial content, a significant increase in p62 was observed at polychromatic and orthochromatic stages in shVDAC1-transduced cells (Figure 5D).
After engulfment of the mitochondria, the autophago- some usually fuses with a lysosome, leading to the degra- dation of the mitochondria along with the adaptor pro- teins (Figure 5A). In order to decipher whether p62 accu- mulation was due to a stabilization at MOM on ubiquiti- nated proteins or to a defect in mitophagy completion by lysosomal degradation, we studied the autophagosomal membrane recruitment to mitochondria by immunofluo- rescence. We observed a significant decreased co-localiza- tion between autophagosomal LC3-II and the mitochon- drial marker TOM22 in shVDAC1-transduced cells (P<0.01; Figure 5E), suggesting a defect in the phagophore membrane recruitment and consequently a defect in mitophagy completion.
In order to assess the specificity of our results, we con- structed shSCR-K562 and shVDAC1-K562 stable cell lines and transfected shSCR-K562 cells with a small inter- fering RNA (siRNA) against VDAC1 (siVDAC1-K562). We confirmed VDAC1 downregulation by western blot (Online Supplementary Figure S5A), and showed, as for dif- ferentiating erythroblasts, no difference in LC3 activation (Online Supplementary Figure S5B) and an increase in p62 protein level (Online Supplementary Figure S5C). We also observed a decrease of LC3/TOM22 co-localization in shVDAC1-K562 and siVDAC1-K562, which indicates that the recruitment of the phagophore membrane to the mitochondria was impaired upon VDAC1 downregula- tion (Online Supplementary Figure S5D). These data con- firmed that VDAC1 downregulation in K562 erythroid cells recapitulates the phenotype observed on VDAC1- downregulated erythroblasts.
PINK1 protein level is reduced upon VDAC1 downregulation
The phagophore recruitment is dependent on the stabi- lization of some specific MOM proteins and direct or indirect interaction with lipidated LC3 (Figure 5A). We first evaluated NIX levels, previously identified in mouse reticulocytes as being critical for the recruitment of autophagosomal membranes and appropriate mitophagy.36 Notably, we detected no significant decrease in neither NIX dimers nor NIX transcripts in human ery- throblasts with decreased VDAC1 expression (Online Supplementary Figure S6). We then studied the second main mitophagy induction pathway and observed a sig- nificant decrease of full-length PINK1 protein level in shVDAC1-transduced bulk erythroblasts at D10 by west- ern blotting (Figure 6A). PINK1 expression was then eval- uated by flow cytometry on basophilic, polychromatic and orthochromatic erythroblasts based on the a4-inte- grin and Band3 expression pattern. We confirmed a sig- nificant decrease of PINK1 level starting from the poly- chromatic stage (Figure 6B), in line with the differences of mitochondrial mass and p62 expression.
We next confirmed the reduction of PINK1 protein level upon VDAC1 downregulation in K562 cell lines (Figure
6C). Furthermore, we observed a reduction of the mito- chondrial localization of PINK1, as shown by the reduc- tion of PINK1/TOM22 co-localization in shVDAC1-K562 cells (Figure 6D). Finally, we observed a decrease of the phagophore recruitment to the mitochondria when downregulating PINK1 in shSCR-K562, as observed with the significant reduction of LC3/TOM22 co-localization (Figure 6E). In contrast, LC3/TOM22 co-localization did not worsen upon PINK1 downregulation in shVDAC1- K562 cells (Figure 6E).
Discussion
Mitochondrial clearance is a crucial process for the pro- duction of mature red blood cells and several studies have shown that mitophagy occurs at the reticulocyte stage, allowing the complete clearance of mitochondria and leading to final murine erythrocyte maturation.13,14 Mitophagy is a mitochondrial-specific form of general macroautophagy, which can be mediated by different well characterized pathways involving protein signaling as the PINK1/Parkin pathway, NIX and BNIP3. While autophagy related genes (ATG) have been shown to be critical for mitochondrial clearance during erythropoiesis,6-11 and the NIX-driven pathway has been identified as plying a role in murine erythropoiesis,12-14 the importance of these mitophagy pathways in human ery- throid terminal differentiation is unknown.
Here we focused on the role of VDAC1, an OMM pro- tein, known to be involved in mitophagy in non-ery- throid cells.
We found that VDAC1 downregulation accelerates human erythroblast differentiation until the orthochro- matic erythroblast stage, but then blocks enucleation and cumulates mitochondria with altered ultrastructure and decreased function, resulting in apoptosis.
In agreement with previous data on the role of VDAC1 in the structuration of mitochondria,23 we observed altered mitochondria ultrastructure and a decrease in ER- mitochondria contact sites in shVDAC1-transduced ery- throblasts by electron microscopy. Notably, complexes between VDAC and hexokinase-1 at ER-mitochondrial contact sites have been shown to regulate mitochondrial metabolism.34 In our study, mitochondria in VDAC1- downregulated erythroblasts showed a decrease in OCR and intracellular ATP content, a signature of reduced mitochondrial activity. Thus, perturbations in mitochon- drial metabolism may influence the kinetics of erythrob- last differentiation and their enucleation profiles.
We demonstrated that VDAC1 is required for progres- sive mitochondrial clearance during the terminal phase of differentiation. This is in line with a previous study showing a decrease in mitochondrial mass after the proerythroblastic stage.37 We found that this is regulated through the PINK1/Parkin pathway, with VDAC1 modu- lating PINK1 accumulation at the mitochondria outer membrane and the recruitment of the phagophore. Accordingly, the attenuation of mitochondrial clearance was associated with increased levels of p62, an adaptor protein that allows bridging between the ubiquitinated cargos and the phagophore membrane.35
Our data therefore highlight the critical role of VDAC1 function in the recognition of mitochondria that are “to be degraded” during the process of red cell maturation.
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haematologica | 2022; 107(1)