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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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Targeting of PaMT1-his to mitochondria influences respiration and lifespan in P. anserina.(A) Cu-binding of PaMT1 reduces COX activity and induces AOX. Oxygen-consumption measurements of wild-type, two independent PaMT1 transformants (T4 and T5) and a control, overexpressing PaMT1 in the cytosol (T9-PaMT1 cyto) are shown. The percentage of respiration via COX or AOX, respectively, was determined by inhibition with 10 mM KCN or 4 mM SHAM, respectively. The values represent mean±SE. (B) Western blot analysis of isolated mitochondria of wild-type and the two PaMT1 transformants T4 and T5 probed with an antibody against the AOX of S. guttatum. (C) The mean lifespan of PaMT1 transformants T4 (n = 31) and T5 (n = 30) is doubled compared to the wild-type s (n = 12) (73 d and 75 d vs. 37 d) as determined in race tubes on standard cornmeal medium.
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pone-0004919-g005: Targeting of PaMT1-his to mitochondria influences respiration and lifespan in P. anserina.(A) Cu-binding of PaMT1 reduces COX activity and induces AOX. Oxygen-consumption measurements of wild-type, two independent PaMT1 transformants (T4 and T5) and a control, overexpressing PaMT1 in the cytosol (T9-PaMT1 cyto) are shown. The percentage of respiration via COX or AOX, respectively, was determined by inhibition with 10 mM KCN or 4 mM SHAM, respectively. The values represent mean±SE. (B) Western blot analysis of isolated mitochondria of wild-type and the two PaMT1 transformants T4 and T5 probed with an antibody against the AOX of S. guttatum. (C) The mean lifespan of PaMT1 transformants T4 (n = 31) and T5 (n = 30) is doubled compared to the wild-type s (n = 12) (73 d and 75 d vs. 37 d) as determined in race tubes on standard cornmeal medium.

Mentions: To assess physiological effects of targeting PaMT1 into the mitochondrial matrix of P. anserina, the type of respiration was determined. Oxygen-consumption revealed a reduction by 30% of KCN-sensitive COX-dependent respiration and a concomitant induction of SHAM-sensitive respiration via AOX in the transformants (Fig. 5A), compared to the wild-type which almost exclusively respires via COX. The induction of the AOX was also verified by Western blot analysis (Fig. 5B). Overall, the observed changes in copper-dependent respiration appear not to be due to a reduced amount of copper but rather result from a binding of copper by the mitochondrial targeted metallothionein. Presumably, this binding makes part of the mitochondrial copper pool unavailable for incorporation into COX.


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)

Targeting of PaMT1-his to mitochondria influences respiration and lifespan in P. anserina.(A) Cu-binding of PaMT1 reduces COX activity and induces AOX. Oxygen-consumption measurements of wild-type, two independent PaMT1 transformants (T4 and T5) and a control, overexpressing PaMT1 in the cytosol (T9-PaMT1 cyto) are shown. The percentage of respiration via COX or AOX, respectively, was determined by inhibition with 10 mM KCN or 4 mM SHAM, respectively. The values represent mean±SE. (B) Western blot analysis of isolated mitochondria of wild-type and the two PaMT1 transformants T4 and T5 probed with an antibody against the AOX of S. guttatum. (C) The mean lifespan of PaMT1 transformants T4 (n = 31) and T5 (n = 30) is doubled compared to the wild-type s (n = 12) (73 d and 75 d vs. 37 d) as determined in race tubes on standard cornmeal medium.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004919-g005: Targeting of PaMT1-his to mitochondria influences respiration and lifespan in P. anserina.(A) Cu-binding of PaMT1 reduces COX activity and induces AOX. Oxygen-consumption measurements of wild-type, two independent PaMT1 transformants (T4 and T5) and a control, overexpressing PaMT1 in the cytosol (T9-PaMT1 cyto) are shown. The percentage of respiration via COX or AOX, respectively, was determined by inhibition with 10 mM KCN or 4 mM SHAM, respectively. The values represent mean±SE. (B) Western blot analysis of isolated mitochondria of wild-type and the two PaMT1 transformants T4 and T5 probed with an antibody against the AOX of S. guttatum. (C) The mean lifespan of PaMT1 transformants T4 (n = 31) and T5 (n = 30) is doubled compared to the wild-type s (n = 12) (73 d and 75 d vs. 37 d) as determined in race tubes on standard cornmeal medium.
Mentions: To assess physiological effects of targeting PaMT1 into the mitochondrial matrix of P. anserina, the type of respiration was determined. Oxygen-consumption revealed a reduction by 30% of KCN-sensitive COX-dependent respiration and a concomitant induction of SHAM-sensitive respiration via AOX in the transformants (Fig. 5A), compared to the wild-type which almost exclusively respires via COX. The induction of the AOX was also verified by Western blot analysis (Fig. 5B). Overall, the observed changes in copper-dependent respiration appear not to be due to a reduced amount of copper but rather result from a binding of copper by the mitochondrial targeted metallothionein. Presumably, this binding makes part of the mitochondrial copper pool unavailable for incorporation into COX.

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