Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells

Abstract

Fanconi Anemia (FA) is an inherited bone marrow (BM) failure disorder commonly diagnosed during school age. However, in murine models, disrupted function of FA genes leads to a much earlier decline in fetal liver hematopoietic stem cell (FL HSC) number that is associated with increased replication stress (RS). Recent reports have shown mitochondrial metabolism and clearance are essential for long-term BM HSC function. Intriguingly, impaired mitophagy has been reported in FA cells. We hypothesized that RS in FL HSC impacts mitochondrial metabolism to investigate fetal FA pathophysiology. Results show that experimentally induced RS in adult murine BM HSCs evoked a significant increase in mitochondrial metabolism and mitophagy. Reflecting the physiological RS during development in FA, increase mitochondria metabolism and mitophagy were observed in FANCD2-deficient FL HSCs, whereas BM HSCs from adult FANCD2-deficient mice exhibited a significant decrease in mitophagy. These data suggest that RS activates mitochondrial metabolism and mitophagy in HSC.

Keywords: FANCD2; Hematopoietic stem cell; fetal liver; mitochondria metabolism; mitophagy; replication stress.

Figure 1

5-FU-induced RS increases MMP and mitophagy. (A, B) Relative mean fluorescence intensity (MFI) of MMP (TMRE) (A) and mtROS (MSR) (B) in HSPCs (EPCR+ CD150+ CD48- c-Kit+ Lin-) at untreated (d0), day 6 (d6) and day 12 (d12) after 5-FU injection (d0 n=3 mice, d6 n=3 mice, d12 n=3 mice). (C) Mitochondrial area, mitochondrial concentration, and mitochondrial polarity of HSPCs from mitoDendra mouse, at d0, d6 and d12 after 5-FU injection (d0 n=931 cells, d6 n=58 cells, d12 n=867 cells). Data are analyzed by IFM. (D, E) Relative MFI of mitophagy (D) and lysosome acidification (LA, LTR) (E) in HSPCs at d0, d6 and d12 after 5-FU injection (d0 n=3 mice, d6 n=3 mice, d12 n=3 mice). (F, G) Relative MFI of MMP (F) and mtROS (G) in HSPCs at 1 month (1Mo) after 5-FU injection (d0 n=3 mice, d6 n=3 mice, d12 n=3 mice). (H) Mitochondrial area, mitochondrial concentration and mitochondrial polarity in HSPCs at 1 month (1Mo) after 5-FU injection (d0 n=47 cells, 1Mo n=32 cells). Data are analized by IFM. All relative data are divided with average value of d0 and show mean ± SD. *P<0.05, **P<0.01, ****P<0.0001, ns, not significant.

Figure 2

APH-induced RS increases MMP and mitophagy. (A-D) Relative cell count (A), MFI of MMP (B), MFI of mtROS (C) and MFI of mitophagy (D) of cultured HSCs (CD150+ c-Kit+ Lin-) at day 2 (left panel) and day 7 (right panel), with or without APH (UT) (day 2 UT n=3-6 wells, APH n=3-6 wells, day 7 UT n=6 wells, APH n=6 wells). All relative data are divided with average of UT and show mean ± SD. *P<0.05, **P<0.01, ns, not significant.

Figure 3

Mitophagy activity was decreased in Fancd2-deficient BM HSC. (A) Relative MFI (left panel) and representative flow cytometric plots (right panel) showing MMP in adult (8-12 weeks) HSCs (Fancd2 WT n=6 mice, HET n=2 mice, KO n=8 mice, left panel). (B) Relative MFI of mtROS in adult HSCs (Fancd2 WT n=5 mice, HET n=2 mice, KO n=5 mice). (C) Mitochondrial area, mitochondrial concentration and mitochondrial polarity in HSCs. HSCs were stained with anti-Tomm20 and analyzed by IFM (Fancd2 WT n=559 cells, KO n=431 cells). (D) Relative MFI (left panel) and representative flow cytometric plots (right panel) of mitophagy in BM HSCs (Fancd2 WT n=5 mice, HET n=2 mice, KO n=4 mice). (E) Relative MFI (left panel) and representative FACS plots (right panel) of LA in BM HSCs (Fancd2 WT n=6 mice, KO n=5 mice, left panel). All relative data are divided with average of controls (WT or HET) and show mean ± SD. *P<0.05, ****P<0.0001, ns, not significant.

Figure 4

Fancd2 deficiency increases MMP and mitophagy in FL HSC. (A) MFI of AF-488 ssDNA in HSPCs (LSK) at E14.5 (Fancd2 WT n=7003 cells, Het n=12954 cells, KO n=2673 cells). IF of ssDNA was performed and data are analysed by IFM. (B) Relative MFI (left panel) and representative flow cytometric plots (right panel) of MMP in FL HSCs at E13.5 (CD150+ CD48- c-Kit+ Sca-1 Lin- (LSK)) (Fancd2 WT n=10 mice, HET n=19 mice, KO n=11 mice, left panel). (C) MFI (left panel) and representative flow cytometric plots (right panel) of mtROS in FL HSCs at E13.5 (Fancd2 WT n=8 mice, HET n=12 mice, KO n=10 mice, left panel). (D) Mitochondrial area, mitochondrial concentration and mitochondrial polarity in HSPCs (LSK) at E14.5 (Fancd2 WT n=4446 cells, KO n=987 cells). IF of Tomm20 was performed and data are analysed by IFM. (E) Relative MFI (left panel) and representative flow cytometric plots (right panel) of mitophagy in FL HSCs at E13.5 (Fancd2 WT n=3 mice, HET n9 mice, KO n=9 mice, left panel) (F) Relative MFI (left panel) and representative flow cytometric plots (right panel) of LA in FL HSCs at E13.5 (Fancd2 WT n=5 mice, HET n=4 mice, KO n=4 mice). (G) MFI of p4EBP in E13.5 FL HSCs (Fancd2 WT n=3 mice, KO n=4 mice). (H) CFU assay of HSCs with Rapamycin (imTOR) or SD-208 (iTgf-β) or untreatment (UT). Some independent experiments were performed and data are shown as relative numbers to the average of each WT UT (Fancd2 WT UT n=15 imTOR n=9 dishes, iTgf-β n=9 dishes, Fancd2 KO UT n=15 imTOR n=9 dishes, iTgf-β n=9 ishes). All relative data are divided with average of controls (WT or HET) and show mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant.