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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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The abundance of the copper-regulated proteins (Hsp70 and PrP) is increased in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.Proteins were extracted at increasing times after the end of incubation with CuSO4. α-tubulin was used as a loading control. The presented blots are representative of two independent experiments.
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pone-0004919-g008: The abundance of the copper-regulated proteins (Hsp70 and PrP) is increased in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.Proteins were extracted at increasing times after the end of incubation with CuSO4. α-tubulin was used as a loading control. The presented blots are representative of two independent experiments.

Mentions: We subsequently analyzed whether an increase of extracellular copper levels also affects the abundance of Hsp70, PrP and MT2A proteins. Proteins of WI-38 HDFs incubated with CuSO4 or controls cells were analyzed by Western blot method (Fig. 8). The abundance of Hsp70 increased in cells incubated with CuSO4 at 1, 3, 9, 24, 48 and 72 h after the end of incubation (Fig. 8A). For PrP, we observed another band at low molecular weight in cells incubated with CuSO4 at 1, 3, 9 h after the end of incubation (Fig. 8B). This band is likely to represent an unglycosylated PrP form. α-tubulin was used as a reference protein for equal loading on the gels. No antibody against MT2A worked in our experimental conditions. These results show that in WI-38 HDFs, the relative abundance of Hsp70 and PrP is increased in response to elevated copper levels.


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)

The abundance of the copper-regulated proteins (Hsp70 and PrP) is increased in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.Proteins were extracted at increasing times after the end of incubation with CuSO4. α-tubulin was used as a loading control. The presented blots are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004919-g008: The abundance of the copper-regulated proteins (Hsp70 and PrP) is increased in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO4) at 500 µM for 16 h.Proteins were extracted at increasing times after the end of incubation with CuSO4. α-tubulin was used as a loading control. The presented blots are representative of two independent experiments.
Mentions: We subsequently analyzed whether an increase of extracellular copper levels also affects the abundance of Hsp70, PrP and MT2A proteins. Proteins of WI-38 HDFs incubated with CuSO4 or controls cells were analyzed by Western blot method (Fig. 8). The abundance of Hsp70 increased in cells incubated with CuSO4 at 1, 3, 9, 24, 48 and 72 h after the end of incubation (Fig. 8A). For PrP, we observed another band at low molecular weight in cells incubated with CuSO4 at 1, 3, 9 h after the end of incubation (Fig. 8B). This band is likely to represent an unglycosylated PrP form. α-tubulin was used as a reference protein for equal loading on the gels. No antibody against MT2A worked in our experimental conditions. These results show that in WI-38 HDFs, the relative abundance of Hsp70 and PrP is increased in response to elevated copper levels.

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