ALBERT

Description: Hematopoietic stem and progenitor cells (HSPC) fate is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation pre-conditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers following irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function following irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43 deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular ATP levels activate the purinergic receptor P2RX7 and lead to AMPK reduced activity in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented following mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved due to the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system following transplantation but also support and induce the metabolic recovery of their irradiated-damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery following myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Description: Hematopoietic stem cell/progenitor (HSCP) transplantation (HSCT) is routinely used for the treatment of cancer and inborn hematopoietic defects. The bone marrow (BM) microenvironment (ME) is a major regulator of hematopoietic function and fate. Clinical data supports osteoblastic regeneration after HSCT despite the inability of BM mesenchymal stem cells (BM-MSC) to engraft. Therefore, understanding the hematopoietic-dependent mechanisms controlling ME mesenchymal regeneration is expected to provide molecular targets for intervention in the context of HSCT. Hematopoietic connexin-43 (H-Cx43) mediates HSCP survival and efficient blood formation by scavenging damaging excess reactive oxygen species (ROS) through transfer to BM mesenchymal stromal cells (BM-MSC) after chemotherapy, preventing lethal hematopoietic failure (Taniguchi-Ishikwawa E et al., PNAS 2012), while the expression of Cx43 on BM-MSC regulates CXCL12 secretion and HSCP homeostasis (Schajnovitz A et al., Nat. Immunol., 2011). Since Cx43 is expressed in mitochondria, we hypothesized that H-Cx43 mediated ROS transfer upon stress depends on hematopoietic mitochondria transfer and uptake by the BM-MSC. We created chimeric mice by transplanting Vav1-CreTg/-, Cox8 mitochondrial localization signal-Dendra2Tg/- wild-type (mDendra2/WT) or Cx43fl/fl(mDendra2/Cx43Δ/Δ) HSCP to lethally irradiated, congenic WT mice and assessed the recovery of stromal cell regeneration via transfer of mitochondria to BM-MSC. H-Cx43Δ/Δchimeric mice have delayed lympho-hematopoietic recovery after irradiation or chemotherapy which can be reversed by restoration of hematopoietic Cx43 expression. H-Cx43Δ/Δchimeric mice exhibit decreased (~60-80%) and delayed colony-forming-unit-fibroblast (CFU-F) and osteoblast (CFU-Ob) regeneration and hematopoietic recovery. The delayed hematopoietic response in H-Cx43Δ/Δchimeras associated with ~40% reduction in mitochondrial transfer from HSCP to Lin-/CD45-/PDGFRα+/Sca1- BM stromal cells (MSC/P). Reverse transplantation experiments indicate that stromal Cx43 is dispensable for mitochondrial transfer from BM stroma to HSCP. Impaired mitochondrial uptake in H-Cx43Δ/Δchimeras associated with ~30-40% decreased mitochondrial ROS (mROS), membrane potential (MMP) and proliferation (assessed by in vivo BrdU uptake) of recipient MSC/P, suggesting that the transferred mitochondria reprogram the recipient mesenchymal progenitor metabolism. Defects of mitotransfer from H-Cx43Δ/ΔHSCP to BM MSC/P and in recipient BM MSC/P mitochondrial activity were recapitulated in in vitro co-cultures. Interestingly, intracellular [ATP] is upregulated (~2 fold) in MSC/P from chimeric H-Cx43Δ/ΔBM that received donor-derived mitochondria, as compared to donor mitochondria containing MSC/P from WTchimeras. Hemichannel opening causes loss of ATP, we therefore speculated that ATP released from MSC/P upon irradiation and transplantation is uptaken by HSPC, activating mitochondrial transfer as part of BM regeneration. Forced glycolysis-dependent restoration of [ATP] in MSC/P but not in HSCP enhances transfer of mitochondria from HSCP to MSC/P, suggesting that BM stromal [ATP] is an irradiation-responsive positive regulator of mitochondria transfer. Hemichannel-derived exogenous ATP suppresses AMPK activation, which regulates cellular metabolic homeostasis by modulating mitochondrial ROS, mitochondria dynamics and the fate of mitochondria. We found that MSC/P recipient of H-Cx43Δ/Δ mitochondria have increased AMPK activity as assessed by increased phosphorylation of AMPK and its downstream effectors ULK1 and ACC (~2-fold) when compared with MSC/P recipient of H-WT mitochondria, whereas MSC/P containing no donor-derived mitochondria from either chimeric mice are insensitive to the effect of Cx43 deficiency. In vivo administration of the AMPK inhibitor BML-275 dramatically increased the mitochondria transfer from HSCP to MSC/P in WT and H-Cx43Δ/Δ chimeras, and completely restores the negative effect of H-Cx43 deficiency on BM mesenchymal and hematopoietic regeneration. Our data indicate that hematopoietic mitochondrial Cx43 is required to control both mitochondrial transfer and BM ME energetic balance and regeneration after myeloablative irradiation. Disclosures No relevant conflicts of interest to declare.

Description: Lifelong production of blood by hematopoietic stem and progenitor cells (HSPC) depends on stem cells self-renewal and their maintenance by bone marrow (BM) microenvironment. BM chemotherapy or whole body irradiation frequently causes hematological abnormalities in cancer patients. HSC within the BM exhibit quiescent state with low mitochondrial reactive oxygen species (ROS) and hyperpolarizing membrane potential, however upon stress hematopoiesis HSC exit quiescence and either self-renew or differentiate in mature cells. Understanding the mechanism controlling hematopoiesis regeneration upon replicative stress is expected to provide molecular targets for amelioration of chemotherapy induced toxicity. Mitochondria recognize and respond to many stresses by modifying mitochondrial dynamics and macroscopic autophagy (mitophagy), and maintains HSC function both at intrinsic and extrinsic level (Ho et al., Nature 2017). Upon myeloablative stress, hematopoietic Connexin 43 (H-Cx43) preserves HSPC survival and efficient blood formation by transfer of damaging excess ROS to BM stromal cells (BMSC), preventing HSC apoptosis and lethal hematology failure (Taniguchi-Ishikwawa E et al., PNAS 2012), and its intermediate C-loop domain has been shown to act as negative regulator of the beclin complex of microautophagy (Eloy Bejarano et al, Nat Cell Biol. 2014). Since HSC apoptosis depends on mitochondrial activation and mitochondrial homeostasis is maintained by coordinated regulation of mitochondrial fission/fusion and mitophagy, we hypothesized that mitochondrial Cx43 may exert a cell-autonomous activity over the mitochondrial turnover upon cell division in the regeneration phase after myelotoxic chemotherapy. To analyze the role of Cx43 in HSC mitochondrial dynamics and fate, we created HSC mitochondrial reporter mice by crossing Cox8 mitochondrial signal localization peptide-Dendra2 fusion protein transgenic (mito-Dendra2) mice with Vav-Cre; Cx43f/f (H-Cx43Δ/Δ)mice. While quiescent Cx43Δ/Δ HSC do not demonstrate any major phenotype, snapshot high resolution microscopy demonstrated that Cx43 deficiency enhances mitochondrial superoxide levels, depolarizes membrane potential and results in larger mitochondria accumulation (~2 fold) after forced division of HSC. In addition, time lapsed cinematography of red-fluorescent, photo-converted mito-Dendra2 demonstrated that Cx43-deficient mitochondria in cycling HSC split into multiple smaller fragments. Phosphorylation of Drp1 (Ser616) and its accumulation with mitochondria is higher in dividing Cx43Δ/ΔHSC, suggesting that Cx43 is a negative regulator of mitochondrial fission and its deficiency facilitates mitochondrial fragmentation. Inhibition of mitochondrial fission with overexpression of the dominant-negative Drp1 mutant (K38A) or the Drp1 inhibitor Mdivi1 prevents the effect of Cx43 deficiency on mitochondrial hyper-fragmentation. The fate of the smaller fragments is mitophagy as demonstrated by increased co-localization of Cx43 deficient mitochondria with the ubiquitin kinase Pink1, the autophagy reporter's p62 and LC3 punctae, and the lysosomal membrane proteins Lamp1/2 in HSC. Genetic silencing of Atg7 or addition of the autophagolysosome inhibitor bafilomycin A1 to dividing HSC prevents the induction of mitophagy in dividing Cx43Δ/Δ HSC. Overexpression of structure-function mutants in Cx43Δ/Δ HSC (Cys-less, with 6 point mutations in Cysteine residues replaced by Alanine, resulting in impaired hemichannel docking or the truncated C-terminus mutant D257 resulting in impaired signaling and intramolecular interactions needed for channel gating) demonstrated that both the formation of whole intercellular channel and the presence of C-terminus once Cx43 channel formed are indispensable for the negative regulatory role of Cx43 on mitophagy. Our results thus identify gap junction protein Cx43 as an important regulator of mitochondrial fission and mitophagy in dividing HSC which may be a target for preservation of hematopoietic stem cell regenerative capacity. Disclosures No relevant conflicts of interest to declare.