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M. Moras et al.
were deleted for different proteins involved in phagophore formation (i.e., Ulk1, Atg4, Atg7). In these latter models, the importance of autophagy in murine erythroid differentiation was demonstrated by the find- ing that the delayed clearance of mitochondria and ribo- somes is associated with anemia.7-11
Mitophagy results from the binding of adaptors to LC3 (microtubule-associated protein 1 light channel 3B, also known as ATG8) within the growing autophagosome. Once associated with phosphatidyl ethanolamine (PE), LC3-II (PE-conjugated LC3) enables the elongation and maturation of the phagophore that is recruited to ubiqui- tin-covered mitochondria through interaction with the LIR (LC3-interacting region) motif of mitophagy adap- tors. An autophagosome is then built around mitochon- dria that are finally degraded following autophagosome- lysosome fusion. Mitophagy can also occur in an ubiqui- tin-independent manner following accumulation of OMM proteins such as NIX/BNIP3L,12 bringing together autophagosomal membranes and mitochondria via their LIR motif.4 In this regard, it is interesting to note that the absence of NIX results in mitochondrial retention and anemia in mice.12-14
The PINK1/Parkin pathway is one of the best-charac- terized pathways of mitophagy. In healthy functional mitochondria, PTEN-induced putative kinase 1(PINK1) is addressed to the OMM. Once translocated to the inner membrane, this protein is cleaved and targeted for degra- dation by the proteasome. However, on defective mito- chondria, there is an accumulation of PINK1 which results in the translocation of Parkin, an E3 ubiquitin lig- ase, and the subsequent clearance of damaged mitochon- dria by mitophagy.15-17
At the surface of damaged mitochondria, Parkin inter- acts with OMM proteins among which voltage-depen- dent anion channels (VDAC). Furthermore, in the absence of all three VDAC, mitophagy has been shown to be impaired, at least in certain cell types.18-20
VDAC1 is a ubiquitous OMM protein involved in dif- ferent pathways including apoptosis,21,22 mitochondrial cristae scaffold,23 and nucleotide transport.24,25 Its role in erythropoiesis is unknown, but transcriptomic and pro- teomic data could suggest a role at the terminal differen- tiation.26,27 Using a short hairpin RNA (shRNA) approach, we find that downregulation of VDAC1 results in a blockage in erythroid progenitor differentiation at the orthochromatic erythroblast stage, exhibiting a signifi- cantly decreased level of enucleation and cell death.
Furthermore, we show that the clearance of mitochon- dria from terminal erythroblasts is dependent on VDAC1, starting at the polychromatic erythroblast stage of differ- entiation. VDAC1 plays a crucial role to initiate the recruitment of phagophore’s membrane, necessary to achieve an efficient maturation of human red blood cells.
Methods
CD34+ cells isolation and ex vivo erythroid differentiation
CD34+ cells were isolated from cord blood and cultured fol- lowing a human ex vivo differentiation protocol as previously described28 (see the Online Supplementary Appendix). The experi- mental protocol was approved by ethical committees from the Inspire H2020 program (agreement 655850) and from INTS
(2019-1), additional information is included in the Online Supplementary Appendix.
Transduction of CD34+ progenitors
Cells undergoing erythroid differentiation were transduced at day 4 of erythroid differentiation with lentiviral vectors (MIS- SION® pLKO.1) containing scrambled shRNA (shSCR) or shRNA targeting VDAC1 (shVDAC1) (TRCN0000297481) upstream of the EGFP transgene, at a MOI of 10 (Online Supplementary Figure S1A). Transduction efficiency was evaluat- ed by the percentage of EGFP+ cells after 72 hours (h) and EGFP+ cells were sorted using the cell sorter SONY SH800.
Flow cytometry-based analysis of erythroid differentiation
Cells were analyzed for surface markers expression, mito- chondrial content and the presence of a nucleus, starting at day 7. Briefly, 105 cells were stained with 250 nM MitoFluor and Hoechst 34580 in media for 30 minutes (min) at 37°C. Cells were washed and subsequently stained with fluorochrome-con- jugated antibodies against glycophorin A (GPA), Band3 and a4- integrin in phosphate-buffered saline (PBS) 2% bovine serum albumin (BSA), for 30 min at 4°C. Cells were washed once with PBS and 7-AAD was added prior to acquisition to exclude dead cells. Cells were analyzed using a BD FACSCantoTM (BD Biosciences), data were acquired with Diva software and ana- lyzed using FCS express 6 Flow Research Edition software.
Enucleation assay by imaging flow cytometry
Orthochromatic erythroblasts were sorted at day 12 as previ- ously described.28 Cells were maintained in culture for 1 day in phase III medium before staining with 1 mg/mL Hoechst 34580 and anti-GPA antibody. Nucleus polarization was analyzed by Amnis® Imaging Flow Cytometer. Approximately 10,000 events were collected and the “delta centroid GPA-Hoechst” was calculated as the distance of the center of the GPA-labeled ery- throblast from the center of the Hoechst-labeled nucleus. A threshold of 2 for the delta centroid (DC) was fixed in order to discriminate between a polarized and non-polarized nucleus. Analysis was performed using the IDEAS 6.2 software.29
Other standard methods
Quantitative real-time polymerase chain reaction (qRT-PCR), antibodies and dyes, May-Grünwald-Giemsa (MGG)-based identification of erythroblasts stage, apoptosis assay, reactive oxygen species (ROS) detection, mitochondrial respiration analysis, immunoblotting, immunolabeling for confocal microscopy, ATP measurement, electron microscopy, K562 cell culture, image stream co-localization and statistical analysis are fully described in the Online Supplementary Appendix.
Results
shRNA-mediated knockdown of VDAC1 results in an accelerated erythropoiesis until the orthochromatic erythroblast stage of differentiation
In order to assess the specific effects of VDAC1 during erythroid differentiation, we pursued a shRNA-mediated knockdown approach. CD34+ cells were transduced at day 4 of differentiation with a shSCR or a shVDAC1 lentiviral vector, both harboring the EGFP transgene (Online Supplementary Figure S1A), and EGFP+ cells were sorted at day 7 (Figure 1A). Knockdown efficiency was assessed at day 10 and a significant downregulation of
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