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Age-related cellular copper dynamics in the fungal ageing model Podospora anserina and in ageing human fibroblasts.

Scheckhuber CQ, Grief J, Boilan E, Luce K, Debacq-Chainiaux F, Rittmeyer C, Gredilla R, Kolbesen BO, Toussaint O, Osiewacz HD - PLoS ONE (2009)

Bottom Line: Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension.Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs.These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.

ABSTRACT
In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.

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Analysis of senescence biomarkers in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.(A) Senescence-associated β-galactosidase. Control (CTL): cells not exposed to CuSO4 are considered as 100%. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. ***: p<0.001. (B) Estimation of the proliferative potential. At 48 h after the end of incubation, 10,000 cells were incubated for 24 h with 1 µCi [3H]-thymidine. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. *: 0.01<p<0.05.
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pone-0004919-g009: Analysis of senescence biomarkers in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.(A) Senescence-associated β-galactosidase. Control (CTL): cells not exposed to CuSO4 are considered as 100%. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. ***: p<0.001. (B) Estimation of the proliferative potential. At 48 h after the end of incubation, 10,000 cells were incubated for 24 h with 1 µCi [3H]-thymidine. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. *: 0.01<p<0.05.

Mentions: Next we investigated whether biomarkers of senescence are induced by an increase of extracellular copper concentration (Fig. 9). As well known biomarkers of senescence the increase of the proportion of cells positive for senescence-associated β-galactosidase activity and growth arrest were analyzed. The results demonstrate an increase of the proportion of senescence-associated β-galactosidase positive cells (Fig. 9A) and a decrease of proliferative potential since the incorporation of [3H]-thymidine was strongly diminished (Fig. 9B) in WI-38 cells incubated with CuSO4.


Age-related cellular copper dynamics in the fungal ageing model Podospora anserina and in ageing human fibroblasts.

Scheckhuber CQ, Grief J, Boilan E, Luce K, Debacq-Chainiaux F, Rittmeyer C, Gredilla R, Kolbesen BO, Toussaint O, Osiewacz HD - PLoS ONE (2009)

Analysis of senescence biomarkers in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.(A) Senescence-associated β-galactosidase. Control (CTL): cells not exposed to CuSO4 are considered as 100%. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. ***: p<0.001. (B) Estimation of the proliferative potential. At 48 h after the end of incubation, 10,000 cells were incubated for 24 h with 1 µCi [3H]-thymidine. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. *: 0.01<p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654708&req=5

pone-0004919-g009: Analysis of senescence biomarkers in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.(A) Senescence-associated β-galactosidase. Control (CTL): cells not exposed to CuSO4 are considered as 100%. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. ***: p<0.001. (B) Estimation of the proliferative potential. At 48 h after the end of incubation, 10,000 cells were incubated for 24 h with 1 µCi [3H]-thymidine. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. *: 0.01<p<0.05.
Mentions: Next we investigated whether biomarkers of senescence are induced by an increase of extracellular copper concentration (Fig. 9). As well known biomarkers of senescence the increase of the proportion of cells positive for senescence-associated β-galactosidase activity and growth arrest were analyzed. The results demonstrate an increase of the proportion of senescence-associated β-galactosidase positive cells (Fig. 9A) and a decrease of proliferative potential since the incorporation of [3H]-thymidine was strongly diminished (Fig. 9B) in WI-38 cells incubated with CuSO4.

Bottom Line: Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension.Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs.These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.

ABSTRACT
In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.

Show MeSH
Related in: MedlinePlus