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A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging.

Schauer M, Kottek T, Schönherr M, Bhattacharya A, Ibrahim SM, Hirose M, Köhling R, Fuellen G, Schmitz U, Kunz M - Oncotarget (2015)

Bottom Line: Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers.In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts.In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, University of Leipzig, Leipzig 04103, Germany.

ABSTRACT
Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

No MeSH data available.


Related in: MedlinePlus

Characteristic features of cellular senescence in skin fibroblasts of Nd2-mutant mouse strainA. For primary skin fibroblasts of 3-month-old and 12-month-old mice, cell proliferation of Nd2-mutant or control mouse fibroblasts was analysed by BrdU incorporation assay. n = 3–13, *p < 0.05, **p < 0.01. B. Fibroblasts from 12-month-old mice were fixed and stained for SA-ß-gal 24 h after seeding. Representative picture of SA-β-gal-staining is shown (left panel). The number of positive blue cells was divided by the total number of counted cells resulting in the percentage of ß-gal-positive cells (right panel). n = 3, *p < 0.05. Data are expressed as mean ± SEM. C. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and supernatants were collected and analysed for IL-6 concentrations by ELISA at indicated time points. n = 6–7, *p < 0.05 ***p < 0.001. Data expressed as mean ± SEM. D. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and whole cell lysates were collected at the indicated days thereafter. Protein expression of γH2A.X and p-p53 was analysed by immunoblotting. Lysates were pooled from fibroblasts of three different mice. RPL26 was used as loading control.
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Figure 3: Characteristic features of cellular senescence in skin fibroblasts of Nd2-mutant mouse strainA. For primary skin fibroblasts of 3-month-old and 12-month-old mice, cell proliferation of Nd2-mutant or control mouse fibroblasts was analysed by BrdU incorporation assay. n = 3–13, *p < 0.05, **p < 0.01. B. Fibroblasts from 12-month-old mice were fixed and stained for SA-ß-gal 24 h after seeding. Representative picture of SA-β-gal-staining is shown (left panel). The number of positive blue cells was divided by the total number of counted cells resulting in the percentage of ß-gal-positive cells (right panel). n = 3, *p < 0.05. Data are expressed as mean ± SEM. C. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and supernatants were collected and analysed for IL-6 concentrations by ELISA at indicated time points. n = 6–7, *p < 0.05 ***p < 0.001. Data expressed as mean ± SEM. D. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and whole cell lysates were collected at the indicated days thereafter. Protein expression of γH2A.X and p-p53 was analysed by immunoblotting. Lysates were pooled from fibroblasts of three different mice. RPL26 was used as loading control.

Mentions: Therefore, primary skin fibroblasts isolated from 3- and 12-month-old Nd2-mutant mice and control mice were analyzed for the expression of markers of cellular senescence. At a cellular level, senescence in primary fibroblasts might be detrimental for skin function in general. In a first series of experiments, the impact of the Nd2 mutation on cellular proliferation of primary skin fibroblasts in both strains was analyzed. For this purpose, BrdU incorporation assays were performed. Nd2-mutant fibroblasts showed a more than 50% higher proliferation rate than control fibroblasts in both 3- and in 12-month-old mice (Figure 3A).


A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging.

Schauer M, Kottek T, Schönherr M, Bhattacharya A, Ibrahim SM, Hirose M, Köhling R, Fuellen G, Schmitz U, Kunz M - Oncotarget (2015)

Characteristic features of cellular senescence in skin fibroblasts of Nd2-mutant mouse strainA. For primary skin fibroblasts of 3-month-old and 12-month-old mice, cell proliferation of Nd2-mutant or control mouse fibroblasts was analysed by BrdU incorporation assay. n = 3–13, *p < 0.05, **p < 0.01. B. Fibroblasts from 12-month-old mice were fixed and stained for SA-ß-gal 24 h after seeding. Representative picture of SA-β-gal-staining is shown (left panel). The number of positive blue cells was divided by the total number of counted cells resulting in the percentage of ß-gal-positive cells (right panel). n = 3, *p < 0.05. Data are expressed as mean ± SEM. C. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and supernatants were collected and analysed for IL-6 concentrations by ELISA at indicated time points. n = 6–7, *p < 0.05 ***p < 0.001. Data expressed as mean ± SEM. D. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and whole cell lysates were collected at the indicated days thereafter. Protein expression of γH2A.X and p-p53 was analysed by immunoblotting. Lysates were pooled from fibroblasts of three different mice. RPL26 was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496166&req=5

Figure 3: Characteristic features of cellular senescence in skin fibroblasts of Nd2-mutant mouse strainA. For primary skin fibroblasts of 3-month-old and 12-month-old mice, cell proliferation of Nd2-mutant or control mouse fibroblasts was analysed by BrdU incorporation assay. n = 3–13, *p < 0.05, **p < 0.01. B. Fibroblasts from 12-month-old mice were fixed and stained for SA-ß-gal 24 h after seeding. Representative picture of SA-β-gal-staining is shown (left panel). The number of positive blue cells was divided by the total number of counted cells resulting in the percentage of ß-gal-positive cells (right panel). n = 3, *p < 0.05. Data are expressed as mean ± SEM. C. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and supernatants were collected and analysed for IL-6 concentrations by ELISA at indicated time points. n = 6–7, *p < 0.05 ***p < 0.001. Data expressed as mean ± SEM. D. Fibroblasts of 12-month-old Nd2-mutant and control mice were treated for 1 h with 250 nM doxorubicin and whole cell lysates were collected at the indicated days thereafter. Protein expression of γH2A.X and p-p53 was analysed by immunoblotting. Lysates were pooled from fibroblasts of three different mice. RPL26 was used as loading control.
Mentions: Therefore, primary skin fibroblasts isolated from 3- and 12-month-old Nd2-mutant mice and control mice were analyzed for the expression of markers of cellular senescence. At a cellular level, senescence in primary fibroblasts might be detrimental for skin function in general. In a first series of experiments, the impact of the Nd2 mutation on cellular proliferation of primary skin fibroblasts in both strains was analyzed. For this purpose, BrdU incorporation assays were performed. Nd2-mutant fibroblasts showed a more than 50% higher proliferation rate than control fibroblasts in both 3- and in 12-month-old mice (Figure 3A).

Bottom Line: Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers.In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts.In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, University of Leipzig, Leipzig 04103, Germany.

ABSTRACT
Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

No MeSH data available.


Related in: MedlinePlus