FOXO3A directs a protective autophagy program in haematopoietic stem cells - PubMed (original) (raw)

FOXO3A directs a protective autophagy program in haematopoietic stem cells

Matthew R Warr et al. Nature. 2013.

Abstract

Blood production is ensured by rare, self-renewing haematopoietic stem cells (HSCs). How HSCs accommodate the diverse cellular stresses associated with their life-long activity remains elusive. Here we identify autophagy as an essential mechanism protecting HSCs from metabolic stress. We show that mouse HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy after ex vivo cytokine withdrawal and in vivo calorie restriction. We demonstrate that FOXO3A is critical to maintain a gene expression program that poises HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FOXO3A-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system.

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Conflict of interest statement

Authors Information. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to E.P. (PassegueE@stemcell.ucsf.edu).

Figures

Figure 1

Figure 1. HSCs induce autophagy following metabolic stress

a, b, Fluorescent microscopy of cultured Gfp-Lc3 GMPs (a) and HSCs (b). c, Electron microscopy images of WT HSCs. Arrowheads indicate autophagic vesicles. d, GFP-LC3 loss in cultured Gfp-Lc3 HSCs (n = 3). Results are expressed as percentage of GFP-LC3 MFI in -BafA compared to +BafA conditions. e, GFP-LC3 levels in HSCs of fed and starved Gfp-Lc3 mice (n = 3). Results are expressed as percent of GFP-LC3 MFI in fed mice. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 2

Figure 2. Autophagy protects HSCs from starvation-induced apoptosis

a, Apoptosis levels in cultured WT HSCs (n = 3–5). Results are expressed as percent caspase activation compared to +cytokines conditions. b, Apoptosis levels in cultured control (Cnt) and Atg12cKO HSCs and GMPs (n = 3). Results are expressed as percent caspase activation compared to +cytokines Cnt HSCs (100%). c, Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice (n = 4–8 mice per group). Results are expressed as percent of HSCs in fed mice. Data are means ± s.d. Hatching indicates starved conditions. *P ≤ 0.05, **P ≤ 0.01.

Figure 3

Figure 3. FoxO3a poises HSCs for rapid autophagy induction

a, Status of the autophagy machinery in WT HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (set to 0). b, c, qRT-PCR analyses of pro-autophagic genes in WT (b) and FoxO3a−/− (c) HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (b) or FoxO3a+/+ HSCs (c) (set to 0). d, Autophagy flux in cultured FoxO3a−/−::Gfp-Lc3 HSCs (n = 3). Results are expressed as percent GFP-LC3 MFI in -BafA vs. +BafA conditions, and normalized to FoxO3a+/+−::Gfp-Lc3 HSCs. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4

Figure 4. Ongoing autophagy in old HSCs

Electron microscopy images of young and old HSCs. Arrowheads indicate autophagic vesicles.

Figure 5

Figure 5. Ongoing autophagy is essential for the continued survival of old HSCs

a, qRT-PCR analyses of pro-autophagic genes in old HSCs (n = 3–5). Results are expressed as log2 fold expression compared to young HSCs (set to 0). b, Apoptosis levels in cultured young and old HSCs (n = 3). Results are expressed as percent caspase activation compared to +cytokines young HSCs. Hatching indicates –cytokines conditions. c, 2-NBD glucose uptake in cultured young and old HSCs (n = 3). d, Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate (M-Pyr) (n = 3). Colonies were counted at day 10 and normalized to -BafA conditions. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Comment in

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