Mitochondrial peptides modulate mitochondrial function during cellular senescence

Su-Jeong Kim, Hemal H Mehta, Junxiang Wan, Chisaka Kuehnemann, Jingcheng Chen, Ji-Fan Hu, Andrew R Hoffman, Pinchas Cohen, Su-Jeong Kim, Hemal H Mehta, Junxiang Wan, Chisaka Kuehnemann, Jingcheng Chen, Ji-Fan Hu, Andrew R Hoffman, Pinchas Cohen

Abstract

Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.

Keywords: SASP (senescence-associated secretory phenotype); mitochondria; mitochondrial energetics; mitochondrial-derived peptides; mtDNA methylation; senescence.

Conflict of interest statement

CONFLICTS OF INTEREST: Pinchas Cohen is a consultant and stockholder of CohBar Inc.

Figures

Figure 1
Figure 1
Mitochondria mass and energetics are altered during doxorubicin-induced senescence. (A) mitochondrial DNA (mtDNA) copy number in non-senescent (quiescent) and senescent cells. (B) Representative images of Tom20 (green; mitochondria) and Hoechst 33258 (blue; nucleus) immunostaining in non-senescent (quiescent) and senescent cells. Scale bar, 20 μm. The area of Tom20 staining per cells were measured using image J. (C) Cellular ATP levels in non-senescent (quiescent) and senescent cells. (D) Cellular oxygen consumption rate (OCR) in non-senescent and senescent cells. The basal respiration, spare respiratory capacity, and ATP production are calculated based on the sequential compound injection according to the manufacture’s instruction. (E) The extracellular acidification rate (ECAR) in non-senescent (quiescent) and senescent cells. Glycolysis, glycolytic capacity, and glycolytic reserve are calculated based on the sequential compound injection according to the manufacture’s instruction. Data are reported as mean ± SEM of three to eight independent experiments. Significant differences were determined by Student’s t-tests. *p<0.05, ***p<0.001. Abbreviations: NS, Non-senescent cells (quiescent); SEN, Senescent cells.
Figure 2
Figure 2
Mitochondrial fuel usage is altered during doxorubicin-induced senescence. (A) Glucose uptake rate was measured by 2-NBDG, a fluorescently labeled deoxyglucose analog. Quantification and representative western blots of (B) carnitine palmitoyltransferase I (CPT1A) and (C) glutaminase (GLS1), both showing beta-actin as a loading control. Data are reported as mean ± SEM of three to six independent experiments. Significant differences were determined by Student’s t-tests. *p<0.05. Abbreviations: NS, Non-senescent cells (quiescent); SEN, Senescent cells.
Figure 3
Figure 3
Mitochondrial respiration was not altered, but glycolysis was enhanced in replicative senescence. (A) Cellular oxygen consumption rate (OCR) in non-senescence (quiescent) and senescent cells. The basal respiration, spare respiratory capacity, and ATP production are calculated based on the sequential compound injection according to the manufacture’s instruction. (B) The extracellular acidification rate (ECAR) in non-senescent (quiescent) and senescent cells. (C) Glucose uptake rate were measured by 2-NBDG, a fluorescently labeled deoxyglucose analog. Data are reported as mean ± SEM of three to eight independent experiments. Significant differences were determined with Student’s t-tests. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: NS, Non-senescent cells (quiescent); SEN, Senescent cells.
Figure 4
Figure 4
Mitochondria DNA methylation changes during doxorubicin-induced senescence. (A) Schematic diagram of mitochondrial genes and CpG sites in the mtDNA. Quantification and representative agarose gel images of mtDNA methylation levels at the site of (B) CpG1 and (C) CpG4. (D) Quantification and representative western blots of COX1 (MT-CO1) in non-senescent and senescent cells. Reduced lamin B1 was used as a senescence marker. Data are reported as mean ± SEM of three to four independent experiments. Significant differences were determined by Student’s t-tests. *p<0.05, ***p<0.001. Abbreviations: NS, Non-senescent cells (quiescent); SEN, Senescent cells.
Figure 5
Figure 5
Expression levels of MDPs are differentially regulated during cellular senescence. Humanin and MOTS-c levels were examined in (A) doxorubicin-induced senescence and (B) replicative senescence. Data are reported as mean ± SEM of three independent experiments. Significant differences were determined by Student’s t-tests. *p<0.05.
Figure 6
Figure 6
Humanin and MOTS-c modulate the mitochondrial respiration. (A) Cellular oxygen consumption rate (OCR; pmole/min/total DNA) in non-senescence (quiescent) and senescent cells in the absence or presence of either HNG (a potent analogue of humanin with a glycine substitution, S14G) or MOTS-c. The basal respiration, spare respiratory capacity, and ATP production are calculated based on the sequential compound injection according to the manufacture’s instruction. (B) Representative western blots of carnitine palmitoyltransferase I (CPT1A) in non-senescence and senescent cells in the absence or presence of either HNG (a potent analogue of humanin with a glycine substitution, S14G) or MOTS-c. Quantification of carnitine palmitoyltransferase I (CPT1A) expression. Data are reported as mean ± SEM of three independent experiments. Significant differences were determined with one-way ANOVA followed by Tukey’s post hoc test. *p<0.05.

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