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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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Overexpression of copper-regulated genes (MT2A, Hsp70 and PrP) in senescent WI-38 and FS human diploid fibroblasts (HDFs).The results of real-time PCR are expressed as mean fold induction±SD in senescent vs. young cells. The expression of GAPDH was used as reference housekeeping gene. p21Waf-1 (p21) was used as biomarker of senescence. The expression of these genes was investigated in three different strains of cells: FS/RS: replicatively senescent skin HDFs (white columns); WI/RS: replicatively senescent lung WI-38 HDFs (black columns); FS/SIPS: skin HDFs in stress-induced premature senescence (SIPS) via UVB treatment (grey columns).
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pone-0004919-g010: Overexpression of copper-regulated genes (MT2A, Hsp70 and PrP) in senescent WI-38 and FS human diploid fibroblasts (HDFs).The results of real-time PCR are expressed as mean fold induction±SD in senescent vs. young cells. The expression of GAPDH was used as reference housekeeping gene. p21Waf-1 (p21) was used as biomarker of senescence. The expression of these genes was investigated in three different strains of cells: FS/RS: replicatively senescent skin HDFs (white columns); WI/RS: replicatively senescent lung WI-38 HDFs (black columns); FS/SIPS: skin HDFs in stress-induced premature senescence (SIPS) via UVB treatment (grey columns).

Mentions: Finally we investigated the age-related expression of the copper induced genes encoding Hsp70, PrP and MT2A. RNA of young and replicatively senescent cultures of different HDFs cell lines (BJ HDFs: skin fibroblasts; WI-38 HDFs: lung fibroblasts) grown in standard media. In addition, one cell line (BJ HDFs) in which senescence was induced prematurely by treatment with UV-B radiation [11] was analyzed. Transcription of p21Waf-1 was used as a biomarker of senescence. In fact p21Waf-1 is a cyclin-dependent kinase inhibitor overexpressed in senescent cells [38]–[40]. The data were normalized to the transcription of the housekeeping gene coding for GAPDH. Transcript levels of these three copper-regulated genes were found to increase during senescence (Fig. 10). Depending on the cell lines and type of senescence mRNA abundance of Hsp70 increased by 1.66±0.31 fold and 1.92±0.40 fold. mRNA abundance of MT2A increased 1.83±0.31 fold and 4.18±0.62 fold, and mRNA abundance of PrP between 2.22±0.87 fold and 3.94±0.008 fold.


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)

Overexpression of copper-regulated genes (MT2A, Hsp70 and PrP) in senescent WI-38 and FS human diploid fibroblasts (HDFs).The results of real-time PCR are expressed as mean fold induction±SD in senescent vs. young cells. The expression of GAPDH was used as reference housekeeping gene. p21Waf-1 (p21) was used as biomarker of senescence. The expression of these genes was investigated in three different strains of cells: FS/RS: replicatively senescent skin HDFs (white columns); WI/RS: replicatively senescent lung WI-38 HDFs (black columns); FS/SIPS: skin HDFs in stress-induced premature senescence (SIPS) via UVB treatment (grey columns).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654708&req=5

pone-0004919-g010: Overexpression of copper-regulated genes (MT2A, Hsp70 and PrP) in senescent WI-38 and FS human diploid fibroblasts (HDFs).The results of real-time PCR are expressed as mean fold induction±SD in senescent vs. young cells. The expression of GAPDH was used as reference housekeeping gene. p21Waf-1 (p21) was used as biomarker of senescence. The expression of these genes was investigated in three different strains of cells: FS/RS: replicatively senescent skin HDFs (white columns); WI/RS: replicatively senescent lung WI-38 HDFs (black columns); FS/SIPS: skin HDFs in stress-induced premature senescence (SIPS) via UVB treatment (grey columns).
Mentions: Finally we investigated the age-related expression of the copper induced genes encoding Hsp70, PrP and MT2A. RNA of young and replicatively senescent cultures of different HDFs cell lines (BJ HDFs: skin fibroblasts; WI-38 HDFs: lung fibroblasts) grown in standard media. In addition, one cell line (BJ HDFs) in which senescence was induced prematurely by treatment with UV-B radiation [11] was analyzed. Transcription of p21Waf-1 was used as a biomarker of senescence. In fact p21Waf-1 is a cyclin-dependent kinase inhibitor overexpressed in senescent cells [38]–[40]. The data were normalized to the transcription of the housekeeping gene coding for GAPDH. Transcript levels of these three copper-regulated genes were found to increase during senescence (Fig. 10). Depending on the cell lines and type of senescence mRNA abundance of Hsp70 increased by 1.66±0.31 fold and 1.92±0.40 fold. mRNA abundance of MT2A increased 1.83±0.31 fold and 4.18±0.62 fold, and mRNA abundance of PrP between 2.22±0.87 fold and 3.94±0.008 fold.

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