SIRT3 reverses aging-associated degeneration

Abstract

Despite recent controversy about their function in some organisms, sirtuins are thought to play evolutionarily conserved roles in lifespan extension. Whether sirtuins can reverse aging-associated degeneration is unknown. Tissue-specific stem cells persist throughout the entire lifespan to repair and maintain tissues, but their self-renewal and differentiation potential become dysregulated with aging. We show that SIRT3, a mammalian sirtuin that regulates the global acetylation landscape of mitochondrial proteins and reduces oxidative stress, is highly enriched in hematopoietic stem cells (HSCs) where it regulates a stress response. SIRT3 is dispensable for HSC maintenance and tissue homeostasis at a young age under homeostatic conditions but is essential under stress or at an old age. Importantly, SIRT3 is suppressed with aging, and SIRT3 upregulation in aged HSCs improves their regenerative capacity. Our study illuminates the plasticity of mitochondrial homeostasis controlling stem cell and tissue maintenance during the aging process and shows that aging-associated degeneration can be reversed by a sirtuin.

Figure 1. SIRT3 is highly enriched in HSCs and SIRT3 deficiency does not affect the HSC pool at a young age

A, B, C. BM subpopulations were isolated based on cell surface markers. SIRT3 expression levels were quantified by real time PCR (A, B) or western blot (C). n=5. D, E, F. The frequency of HSPCs and HSCs in the BMs of young mice was determined via flow cytometry (n=3). Flow cytometry plots are gated on Lin⁻ BM cells. Data presented are the numbers of specified cell populations per leg. G. The numbers of total BM cellularity per leg of young WT and SIRT3 KO mice (n=3). H, I, J. BM transplantation. Schematic representation of competitive transplantation assays using BM cells from young WT and SIRT3 KO mice as donors (H). Data shown are the percentage of total donor-derived cells (I) and donor-derived individual lineages (J) in the peripheral blood of the recipients. Donors: n=3. Recipients: n=15. K. The number of colonies formed in a colony-forming assay using BM cells of young WT and SIRT3 KO mice (n=6). Error bars represent standard errors. ***: p<0.001.

Figure 2. SIRT3 regulates HSC self-renewal at an old age or under transplantation stress

A, B, C. The frequency of HSPCs and HSCs in the BM of aged mice determined via flow cytometry (n=4). Data presented are the numbers of specified cell populations per leg. D. The numbers of total BM cellularity per leg of aged WT and SIRT3 KO mice (n=4). E, F. Competitive transplantation using aged mice as donors. The percentage of total donor-derived cells (E) and donor-derived individual lineages (F) in the peripheral blood of the recipients are shown. Donors: n=3. Recipients: n=15. G. The number of colonies formed in a colony-forming assay using BM cells of aged WT and SIRT3 KO mice (n=6). H–K. Schematic representation of competitive serial transplantation assays. BM cells from the competitive transplant recipients were used as donors for the next round of transplantation (H). Data shown are the percentage of donor-derived HSCs (LKSCD150⁺) in the BM (I), total donor-derived cells (J) and donor-derived individual lineages (K) in the peripheral blood using BM cells from young WT and SIRT3 KO mice as donors. Donors: n=3. Recipients: n=15. Error bars represent standard errors. *: p<0.05. ***:p<0.001. See also Figure S1.

Figure 3. SIRT3 regulates mitochondrial metabolic homeostasis in HSCs and SIRT3 reduces with age

A. Intracellular ROS levels were determined by H₂DCFDA staining in various subpopulations of BMs of old WT and SIRT3 KO mice in a transplant setting. MFI: mean fluorescence intensity. n=4. B. BM cells isolated from WT and SIRT3 KO mice were treated with paraquat and cell survival in various cell populations were scored by flow cytometry (HSC: LKSCD150⁺). n=3. C. Dead cells were quantified in various subpopulations of BM cells of old WT and SIRT3 KO mice by propidium iodide staining (HSC: LKSCD150⁺). n=4. D. Cycling of BM cells derived from old mice was assessed in transplant recipients using PY staining. n=4. E. Competitive transplantation assays using BM cells from old WT or SIRT3 KO mice as donors. Recipient mice were either untreated or supplemented with NAC throughout the entire experiment. Data shown are the percentage of donor-derived cells in the peripheral blood. Donors: n=3. Recipients: n=15. F, G. SOD2 mRNA levels (F) and the enzymatic activity (G) in HSPCs of old WT and SIRT3 KO mice were determined. n=4. H. Dysfunctional non-respiring mitochondria in HSCs of old WT and SIRT3 KO mice were determined by MitoTracker Green (MTG) and MitoTracker Red (MTR) staining. I, J. HSPCs were isolated from the BM of young or old mice. SIRT3 expression levels were quantified by real time PCR (I) and western blotting (J) (n=3). K, L. SOD2 mRNA levels (K) and the enzymatic activity (L) in the HSPCs of young and old mice were determined. n=4. Error bars represent standard errors. *: p<0.05. **: p<0.005. ***: p<0.001. See also Figure S2.

Figure 4. SIRT3 overexpression rescues functional defects of aged HSCs

A–F. SIRT3 was overexpressed in Lin⁻ cells isolated from old mice via lentiviral transduction. The cellular ROS levels in the HSC population (LKSCD150⁺) were determined by H₂DCFDA staining (n=3) (A). MFI: mean fluorescence intensity. The expression of p19 and BAX was compared in LKS cells of aged WT and SIRT3 KO mice, and aged LKS cells transduced with control or SIRT3 lentivirus by RT-PCR (B). Colony-forming activity was determined in a colony-forming assay (n=6) (C). Schematic representation of a competitive transplantation assay to compare in vivo reconstitution activity of HSCs transduced with control lentivirus or SIRT3 lentivirus (D). Data shown are the percentage of total donor-derived cells (E) and donor-derived individual lineages (F) in the peripheral blood at 4, 6,10, 12, 16 weeks posttransplant. Donor: n=3. Recipients: n=15. G, H. Lin⁻ cells isolated from old SIRT3 KO mice were infected with a control virus, WT SOD2 virus, or SOD2 K53/89R virus. Cells were treated with paraquat and HSC survival was scored by flow cytometry (G). n=3. Colony-forming activity was determined in a colony-forming assay (H). n=6. I. A proposed model on stem cell aging and rejuvenation regulated by SIRT3-mediated mitochondrial homeostasis. Error bars represent standard errors. *: p<0.05. **: p<0.005. ***: p<0.001. See also Figure S3.