Stem cell aging. A mitochondrial UPR-mediated metabolic checkpoint regulates hematopoietic stem cell aging

Abstract

Deterioration of adult stem cells accounts for much of aging-associated compromised tissue maintenance. How stem cells maintain metabolic homeostasis remains elusive. Here, we identified a regulatory branch of the mitochondrial unfolded protein response (UPR(mt)), which is mediated by the interplay of SIRT7 and NRF1 and is coupled to cellular energy metabolism and proliferation. SIRT7 inactivation caused reduced quiescence, increased mitochondrial protein folding stress (PFS(mt)), and compromised regenerative capacity of hematopoietic stem cells (HSCs). SIRT7 expression was reduced in aged HSCs, and SIRT7 up-regulation improved the regenerative capacity of aged HSCs. These findings define the deregulation of a UPR(mt)-mediated metabolic checkpoint as a reversible contributing factor for HSC aging.

Fig. 1. NRF1 stabilizes SIRT7 at the promoters of mitochondrial translational machinery components

(A and B) Coimmunoprecipitation of transfected Flag-tagged or endogenous SIRT7 with endogenous NRF1 from HEK-293T cells. (C and D) ChIP followed by quantitative real-time polymerase chain reaction (ChIP-qPCR) showing SIRT7 (C) and NRF1 (D) occupancy at gene promoters. Error bars, mean ± SE. **P < 0.01; ***P < 0.001; ns, P > 0.05. Student’s t test.

Fig. 2. SIRT7 limits mitochondrial activity, proliferation, and PFS^mt

(A and B) Increased expression of GFM2 and MRPL24 in SIRT7 KD cells is abrogated by NRF1 siRNA. (C and D) Increased mitochondrial mass in SIRT7 KD cells determined by MTG staining and mitochondrial DNA quantification. n = 3. (E) Increased proliferation in SIRT7 KD cells. n = 3. (F) PFS^mt induces SIRT7 expression. Dox, doxycycline. EB, ethidium bromide. (G) Increased accumulation of misfolded ΔOTC in SIRT7 KD cells is rescued by NRF1 siRNA. OTC is used as a control. (H) Increased apoptosis in SIRT7 KD cells treated with EB. n = 3. (I) NRF1 siRNA abrogates increased PFS^mt in SIRT7 KD cells. (J) A proposed model. Error bars, mean ± SE. *P < 0.05. **P < 0.01. Student’s t test.

Fig. 3. SIRT7 ensures HSC maintenance

(A) qPCR showing increased PFS^mt in SIRT7^−/− HSCs. n = 4. (B and C) MTG staining and mitochondrial DNA quantification showing increased mitochondrial mass in SIRT7^−/− HSCs. n = 4. MP, myeloid progenitor cells. Lin-, lineage-negative cells. (D) In vivo BrdU incorporation showing increased proliferation of SIRT7^−/− HSCs. n = 4. (E) Increased propensity of SIRT7^−/− HSCs to cycle upon ex vivo culture with cytokines, indicated by BrdU pulse. n =4. (F) Mice reconstituted with SIRT7^−/− BMCs have increased sensitivity to 5-fluorouracil. n = 12. Log-rank test. (G) Competitive transplantation using SIRT7^+/+ and SIRT7^−/− BMCs as donors showing reduced reconstitution capacity of SIRT7^−/− HSCs. n = 15. Representative of five transplants. (H) Reduced white blood cell count in SIRT7^−/− mice. (I)Myeloid-biased differentiation in the peripheral blood (PB) of SIRT7^−/− mice. MNCs, mononuclear cells. n = 7. Error bars, mean ± SE. *P < 0.05. ***P < 0.001. ns, P > 0.05. Student’s t test.

Fig. 4. HSC aging is regulated by SIRT7

(A) qPCR showing reduced SIRT7 expression in aged HSCs. n = 3. (B and C) qPCR showing that increased PFS^mt in aged HSCs is rescued by SIRT7 overexpression. n = 3. (D and E) Competitive transplantation using aged HSCs transduced with SIRT7 or control lentivirus as donors. SIRT7 overexpression increases reconstitution capacity and reverses myeloid-biased differentiation of aged HSCs. n =7. Error bars, mean ± SE. *P < 0.05. **P < 0.01. Student’s t test.