Characterization of human fibroblast lines

We used eight human fibroblast lines—four from young and four from elderly subjects—to examine the mitochondrial theory of aging. First, we examined mitochondrial respiratory function by estimating O 2 consumption rates; we confirmed the presence of age-associated respiration defects in human fibroblast lines (Fig. 1a). However, the lines did not show age-associated increases in the production of superoxide (mitochondrial ROS) (Fig. 1b). Moreover, no decreases in mtDNA copy number (Supplementary Fig. 1a) or abnormalities in mitochondrial morphology (Supplementary Fig. 1b) were observed in the elderly fibroblasts.

Figure 1 Examination of the mitochondrial theory of aging by using human fibroblast lines derived from young and elderly subjects. The ‘young’ group included fibroblast lines TIG3S (fetus), TIG121 (age 8 months [8mo]), TIG120 (6 years [6y]) and TIG118 (12 years [12y]). The ‘elderly’ group included fibroblast lines TIG106 (80 years [80y]), TIG107 (81 years [81y]), TIG101 (86 years [86y]) and TIG102 (97 years [97y]). (a) Comparison of mitochondrial respiratory function between young and elderly groups by estimation of O 2 consumption rates. ‘Average’ indicates the average O 2 consumption rates of each group. (b) Comparison of amounts of mitochondrial ROS (superoxide) between young and elderly groups by estimation of mitochondrial superoxide levels. Relative superoxide levels are expressed as mean fluorescence intensity of MitoSox-Red. ‘Average’ indicates the average fluorescence intensity of each group. Experiments in (a) and (b) were performed in triplicate; error bars, ± SD. *P < 0.05. Black and open bars represent young and elderly groups, respectively. (c) Comparison of mutation frequencies in mtDNA populations from young and elderly groups by using deep sequence analysis. Upper, middle and lower panels represent frequencies of total, rare and frequent mutations, respectively. Rare mutations existing in less than 1% mtDNA correspond to somatic mutations, whereas frequent mutations existing in 1% or more of mtDNA correspond to inherited mutations. Black and open circles represent young and elderly groups, respectively. Full size image

We then quantitatively estimated the mutation frequency at each nucleotide position of mtDNA by deep sequence analysis of whole mtDNA prepared from the eight lines (Supplementary Fig. 2). The results unexpectedly showed that the total frequency of mtDNA mutations did not differ substantially between fibroblast lines from young and elderly subjects (Fig. 1c). Moreover, the frequency of rare mutations (existing in less than 1% of mtDNA), which have been proposed to be somatic mutations12, was not significantly greater in the fibroblast lines from elderly subjects than in those from young ones (Fig. 1c). The frequency of mutations existing in 1% or more of mtDNA, which have been proposed to be inherited mtDNA mutations12, also did not differ substantially between fibroblast lines from the young and elderly (Fig. 1c and Supplementary Table 1). Because we observed no age-associated overproduction of mitochondrial ROS and no age-associated accumulation of somatic mutations in the mtDNA of the human fibroblast lines we used (Fig. 1b and c), neither was associated with the mitochondrial respiration defects found in the fibroblast lines derived from the elderly subjects (Fig. 1a).

Effects of redifferentiation of reprogrammed fibroblasts on respiration defects

Our previous report11 proposed that nuclear-recessive mutations are responsible for age-associated mitochondrial respiration defects in human fibroblast lines, because the respiration defects were restored by the introduction of pure nuclei (uncontaminated by mtDNA) from mtDNA-less HeLa cells into the fibroblasts. However, our previous results could also have been explained by epigenetic regulation of nuclear genes in the absence of nuclear-recessive mutations. In the case of epigenetic regulation, expression of mitochondrial respiration defects would be reversible and restorable with reprogramming.

To examine this possibility, we randomly chose two young fibroblast lines (TIG3S and TIG121) and two elderly fibroblast lines (TIG107 and TIG102) and used them to generate human induced pluripotent stem cells (hiPSCs). These cells were then redifferentiated into fibroblasts and their mitochondrial respiratory function examined. For effective generation of hiPSCs from elderly human fibroblast lines, the conventional reprogramming gene set OCT3/4, SOX2, KLF4 and C-MYC required to isolate hiPSCs13 was replaced by the gene set OCT3/4, SOX2, KLF4, L-MYC, LIN28 and p53shRNA14. Moreover, virus vectors were replaced by episomal plasmids for transient introduction of the gene set into fibroblasts14. Small colonies with flat embryonic-stem-cell-like morphology (Supplementary Fig. 3a) were picked up about 4 weeks after transfection with the gene set. All the colonies expressed pluripotency marker genes of reprogrammed cells15, such as Nanog, TRA-1-60 and SSEA4 (Supplementary Fig. 3b), indicating that these colonies were hiPSCs. The cells were subsequently cultured in the absence of the feeder cells to allow their redifferentiation into fibroblasts16. The resultant growing cells were confirmed to be fibroblasts by their immunostaining with antibody to the beta subunit of prolyl 4-hydroxylase (Fig. 2a), which is a specific marker for fibroblasts16.

Figure 2 Effects of reprogramming of fibroblasts on age-associated mitochondrial respiration defects. (a) Immunostaining of original fibroblasts and fibroblasts redifferentiated from hiPSCs (reprogrammed fibroblasts) with antibody to a fibroblast-specific marker enzyme, namely the beta subunit of prolyl 4-hydroxylase. R3S, R121, R107 and R102 represent fibroblasts reprogrammed from the original fibroblasts TIG3S, TIG121, TIG107 and TIG102, respectively. Bars, 100 μm. (b) Estimation of O 2 consumption rates of original and reprogrammed fibroblasts. Black and open bars are original fibroblasts from young and elderly subjects, respectively. Gray bars represent reprogrammed fibroblasts. Experiments were performed in triplicate; error bars indicate ± SD. *P < 0.05. Full size image

Next, we examined the O 2 consumption rates of fibroblast lines redifferentiated from hiPSCs. The O 2 consumption rates of redifferentiated fibroblast lines R107 and R102 were significantly greater than those in the original lines TIG107 and TIG102, respectively. Moreover, all of the fibroblast lines redifferentiated from hiPSCs had O 2 consumption rates comparable to those of the fetal fibroblast line TIG3S, irrespective of whether they were derived from young or elderly subjects (Fig. 2b). Thus, the results in Fig. 2b reflect the reversibility of expression of age-associated mitochondrial respiration defects, indicating that these aging phenotypes are controlled by epigenetic regulation, not by mutations.

Screening for nuclear genes regulating age-associated mitochondrial respiration defects

To identify nuclear-coded genes that were controlled epigenetically and could be responsible for age-associated mitochondrial respiration defects (Fig. 1a), we performed a microarray analysis and compared the gene expression spectra of the two young (TIG3S and TIG121) and two elderly (TIG107 and TIG102) fibroblast lines that had been used to isolate the hiPSCs (all microarray data were deposited at NCBI GEO database and received accession number GSE67000). Because our focus here was age-associated respiration defects, which are considered to be caused by a reduction in mitochondrial translation10,11, genes were selected by using the gene ontology (GO) term ‘mitochondria’ followed by four GO terms related to translation and respiration (Supplementary Fig. 4a). As a result, we selected 371 genes from among the 27,958 nuclear-coded genes used for the microarray analysis. From among these 371 genes, we furthermore selected six genes that showed age-associated regulation in the two sets of fibroblast lines with a log 2 ratio of signal intensities of >0.585 or <–0.585, corresponding respectively to >1.5-fold upregulation or downregulation (Supplementary Fig. 4b).

To confirm the microarray results, we performed real-time quantitative PCR to estimate the mRNA levels of the six genes. Comparison of their mRNA levels between the four lines from young subjects and the four lines from elderly subjects showed that MRPL28 and GCAT were downregulated, whereas EHHADH was upregulated, in elderly fibroblast lines (Fig. 3a, Supplementary Fig. 5). In contrast, the remaining three genes showed no significant differences in age-associated regulation.

Figure 3 Use of real-time quantitative PCR to identify nuclear-coded genes regulating age-associated mitochondrial respiration defects. (a) Comparison of mRNA levels of the candidate six genes in the young and elderly groups. The six gene candidates for regulation of age-associated respiration defects were selected by using gene ontology terms and a microarray heatmap (Supplementary Fig. 4b). Black and open bars are the average gene expression levels of fibroblasts from young and elderly groups, respectively. The young group consisted of fibroblast lines TIG3S (fetus), TIG121 (age 8 months), TIG120 (6 years) and TIG118 (12 years). The elderly group consisted of lines TIG106 (80 years), TIG107 (81 years), TIG101 (86 years) and TIG102 (97 years). Levels of transcripts were normalized against UBC expression. The results of each fibroblast line were shown in the Supplemental Figure 5. Of the six genes examined, age-associate regulation was confirmed to be present in MRPL28, EHHADH and GCAT. (b) Comparison of mRNA levels of MRPL28, EHHADH and GCAT in original and reprogrammed fibroblasts. R3S, R121, R107 and R102 represent fibroblasts reprogrammed from the original fibroblasts TIG3S, TIG121, TIG107 and TIG102, respectively. Levels of transcripts were normalized against UBC expression. Black and open bars are fibroblasts from young and elderly subjects, respectively. Gray bars represent reprogrammed fibroblasts. Experiments were performed in triplicate; error bars indicate ± SD. *P < 0.05. Full size image

Because age-associated respiration defects were restored after reprogramming (Fig. 2b), we expected that the reprogramming of elderly fibroblasts would result in the reprogramming of age-associated down- or upregulation of the three genes. We examined this possibility by using redifferentiated fibroblasts from hiPSCs. Reprogramming of gene expression in elderly fibroblasts occurred in GCAT (Fig. 3b), which regulates glycine production in mitochondria17,18. It was therefore likely that reduced glycine production in mitochondria by epigenetic downregulation of GCAT (Fig. 3a) resulted in the age-associated respiration defects (Fig. 1a).

Effects of down- or upregulation of the genes regulating age-associated respiration defects

We then examined whether downregulation of GCAT in TIG3S (from a fetus) would induce respiration defects and whether the gene’s upregulation in TIG102 (from an elderly subject) would restore reduced mitochondrial respiratory function. Downregulation of GCAT in TIG3S by using shRNA led to a reduction in mitochondrial respiratory function (Fig. 4a). Moreover, overexpression of GCAT in TIG102 by infection with lentivirus including the cDNA of GCAT restored the respiration defects (Fig. 4a). These observations suggest that epigenetic downregulation of GCAT with aging is responsible, at least in part, for age-associated respiration defects.

Figure 4 Effects on respiratory function of down- or upregulation of the genes regulating age-associated respiration defects. (a) Down- and upregulation of GCAT and their effects on respiratory function. Control, untreated; scramble, scrambled shRNA treated; shGCAT, downregulation of GCAT in fibroblast line TIG3S by using shRNA; tgGCAT, upregulation of GCAT in line TIG102 by using its cDNA-based transgene. Upper panel, mRNA levels; lower panel, O 2 consumption rates. Black and open bars represent TIG3S and TIG102, respectively. (b) Comparison of mRNA levels of SHMT2 in young and elderly groups (left panel) and in original and reprogrammed fibroblasts (right panel). Black and open bars are young and elderly groups, respectively. ‘Average’ in the left panel indicates the average gene expression levels of each group. R3S, R121, R107 and R102 (gray bars) in the right panel represent fibroblasts reprogrammed from TIG3S, TIG121, TIG107 and TIG102, respectively. (c) Downregulation of SHMT2 or both GCAT and SHMT2 and their effects on respiratory function in TIG3S. Control, untreated; scramble, scrambled-siRNA-treated; siSHMT2, downregulation of SHMT2 by siRNA; shGCAT + siSHMT2, simultaneous downregulation of GCAT and SHMT2 by shRNA and siRNA, respectively. Upper panel, mRNA levels; lower panel, O 2 consumption rates. Black bars represent TIG3S. Experiments were performed in triplicate; error bars indicate ± SD. *P < 0.05. Full size image

Because glycine production in mitochondria is regulated by SHMT217 as well as by GCAT18, downregulation of SHMT2 with aging may also be involved in age-associated respiration defects. To examine this possibility we used real-time quantitative PCR and compared the mRNA levels of SHMT2 in the eight lines from young and elderly subjects. We found age-associated downregulation of SHMT2 (Fig. 4b), even though our microarray results had not revealed its age-associated downregulation (Supplementary Fig. 3b). Moreover, reprogramming of fibroblast lines from aged subjects restored the reduced expression of SHMT2 (Fig. 4b). Furthermore, downregulation of SHMT2 in the TIG3S fibroblast line by using siRNA resulted in a reduction in respiratory function (Fig. 4c) and simultaneous downregulation of GCAT by using shRNA and of SHMT2 by using siRNA had a synergic effect in reducing mitochondrial respiratory function (Fig. 4c). These observations indicated that epigenetic downregulation of both GCAT and SHMT2 with aging (Fig. 3a) was at least partly responsible for the age-associated respiration defects found in elderly fibroblasts (Fig. 1a) by inducing a decrease in glycine production and a resultant decrease in mitochondrial translation. This possibility was supported by our observations that adding glycine to the medium for 10 days restored the reduced respiratory function of TIG102 (Supplementary Fig. 6), suggesting that glycine treatment can effectively prevent elderly fibroblasts from expressing age-associated respiration defects.