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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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Increase in cytoplasmic copper concentration during ageing of P. anserina wild-type s.(A) Cellular extracts were fractionated by differential centrifugation and subsequent sucrose-gradient ultra-centrifugation and the copper content of cytosol and mitochondria was determined by TXRF. The values represent mean±SE. Statistical analysis: Wilcoxon test, n.s.: nonsignificant (p>0.05). (B) DNA preparations were hybridized to a plDNA-specific probe after digestion with BglII and transferred onto a nylon membrane. In juvenile strains signals of 4.5 kbp (BglII-4) and 1.9 kbp (BglII-17), indicative for intact mtDNA, are detected which are replaced by the signal of amplified plDNA (2.5 kbp) in senescent preparations. In the mtDNAs isolated from mitochondria of senescent strains, a greater amount of 4.5 kbp and 1.9 kbp fragments is observed (arrows) compared to the analysis which was performed using a total DNA preparation from a senescent strain including nuclear DNA and mtDNA. M: DIG-labelled λ DNA [HindIII].
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pone-0004919-g001: Increase in cytoplasmic copper concentration during ageing of P. anserina wild-type s.(A) Cellular extracts were fractionated by differential centrifugation and subsequent sucrose-gradient ultra-centrifugation and the copper content of cytosol and mitochondria was determined by TXRF. The values represent mean±SE. Statistical analysis: Wilcoxon test, n.s.: nonsignificant (p>0.05). (B) DNA preparations were hybridized to a plDNA-specific probe after digestion with BglII and transferred onto a nylon membrane. In juvenile strains signals of 4.5 kbp (BglII-4) and 1.9 kbp (BglII-17), indicative for intact mtDNA, are detected which are replaced by the signal of amplified plDNA (2.5 kbp) in senescent preparations. In the mtDNAs isolated from mitochondria of senescent strains, a greater amount of 4.5 kbp and 1.9 kbp fragments is observed (arrows) compared to the analysis which was performed using a total DNA preparation from a senescent strain including nuclear DNA and mtDNA. M: DIG-labelled λ DNA [HindIII].

Mentions: As expected, a clear increase in copper levels was observed in the post-mitochondrial fraction isolated from senescent cultures (juv: 1.3±0.3 nmol Cu/mg protein; sen: 5.3±2.1 nmol Cu/mg protein; p<0.03, Wilcoxon test, two-tailed) (Fig. 1A). However, in contrast to our expectation, copper concentrations in the mitochondrial fraction did not change significantly. We therefore verified the ‘age’ of the isolated mitochondria investigating the reorganization of mitochondrial DNA (mtDNA) in the cytochrome-c oxidase subunit I (CoxI) gene region. Rearrangements occurring in this region are a robust biomarker of ageing in P. anserina [23], [28]. The analysis revealed that in mitochondrial fractions purified by sucrose gradients a significantly higher fraction of two mtDNA BglII fragments, 1.9 kbp and 4.5 kbp in size, are present in senescent cultures than in total DNA preparations. In addition, plDNA, a 2.5 kbp autonomous DNA molecule liberated from the corresponding mtDNA region is more abundant in total DNA preparations than in purified mitochondria (Fig. 1B). These data indicate that purification of mitochondria by sucrose gradient centrifugation appears to enrich structural intact mitochondria while damaged, ‘aged’ mitochondria which may have released copper to the cytoplasm are lost.


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)

Increase in cytoplasmic copper concentration during ageing of P. anserina wild-type s.(A) Cellular extracts were fractionated by differential centrifugation and subsequent sucrose-gradient ultra-centrifugation and the copper content of cytosol and mitochondria was determined by TXRF. The values represent mean±SE. Statistical analysis: Wilcoxon test, n.s.: nonsignificant (p>0.05). (B) DNA preparations were hybridized to a plDNA-specific probe after digestion with BglII and transferred onto a nylon membrane. In juvenile strains signals of 4.5 kbp (BglII-4) and 1.9 kbp (BglII-17), indicative for intact mtDNA, are detected which are replaced by the signal of amplified plDNA (2.5 kbp) in senescent preparations. In the mtDNAs isolated from mitochondria of senescent strains, a greater amount of 4.5 kbp and 1.9 kbp fragments is observed (arrows) compared to the analysis which was performed using a total DNA preparation from a senescent strain including nuclear DNA and mtDNA. M: DIG-labelled λ DNA [HindIII].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004919-g001: Increase in cytoplasmic copper concentration during ageing of P. anserina wild-type s.(A) Cellular extracts were fractionated by differential centrifugation and subsequent sucrose-gradient ultra-centrifugation and the copper content of cytosol and mitochondria was determined by TXRF. The values represent mean±SE. Statistical analysis: Wilcoxon test, n.s.: nonsignificant (p>0.05). (B) DNA preparations were hybridized to a plDNA-specific probe after digestion with BglII and transferred onto a nylon membrane. In juvenile strains signals of 4.5 kbp (BglII-4) and 1.9 kbp (BglII-17), indicative for intact mtDNA, are detected which are replaced by the signal of amplified plDNA (2.5 kbp) in senescent preparations. In the mtDNAs isolated from mitochondria of senescent strains, a greater amount of 4.5 kbp and 1.9 kbp fragments is observed (arrows) compared to the analysis which was performed using a total DNA preparation from a senescent strain including nuclear DNA and mtDNA. M: DIG-labelled λ DNA [HindIII].
Mentions: As expected, a clear increase in copper levels was observed in the post-mitochondrial fraction isolated from senescent cultures (juv: 1.3±0.3 nmol Cu/mg protein; sen: 5.3±2.1 nmol Cu/mg protein; p<0.03, Wilcoxon test, two-tailed) (Fig. 1A). However, in contrast to our expectation, copper concentrations in the mitochondrial fraction did not change significantly. We therefore verified the ‘age’ of the isolated mitochondria investigating the reorganization of mitochondrial DNA (mtDNA) in the cytochrome-c oxidase subunit I (CoxI) gene region. Rearrangements occurring in this region are a robust biomarker of ageing in P. anserina [23], [28]. The analysis revealed that in mitochondrial fractions purified by sucrose gradients a significantly higher fraction of two mtDNA BglII fragments, 1.9 kbp and 4.5 kbp in size, are present in senescent cultures than in total DNA preparations. In addition, plDNA, a 2.5 kbp autonomous DNA molecule liberated from the corresponding mtDNA region is more abundant in total DNA preparations than in purified mitochondria (Fig. 1B). These data indicate that purification of mitochondria by sucrose gradient centrifugation appears to enrich structural intact mitochondria while damaged, ‘aged’ mitochondria which may have released copper to the cytoplasm are lost.

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