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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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Contents of the mitochondrial matrix are released into the cytosol in senescent P. anserina strains.(A) The localization of eGFP directed into the matrix changes from mitochondrial in middle-aged strains (left) to cytoplasmic distribution in senescent strains (right). As control mitochondria are stained with MitoTracker® red CMXRos. Scale bar: 2 µm. (B) The microscopic results are verified by Western Blot analysis. eGFP (mature form and pre-protein) can be detected in the cytosol (C) of senescent strains (right), but not in middle-aged isolates (left) (M - mitochondria). Antibodies against the MnSOD (mitochondria) and the Cu/Zn-SOD (cytosol) were used as marker proteins for compartments and to show equal loading. The natively tetrameric MnSOD is retained within the mitochondria in senescence showing the size-limit for release should be between 27 kDa and ∼80 kDa.
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pone-0004919-g003: Contents of the mitochondrial matrix are released into the cytosol in senescent P. anserina strains.(A) The localization of eGFP directed into the matrix changes from mitochondrial in middle-aged strains (left) to cytoplasmic distribution in senescent strains (right). As control mitochondria are stained with MitoTracker® red CMXRos. Scale bar: 2 µm. (B) The microscopic results are verified by Western Blot analysis. eGFP (mature form and pre-protein) can be detected in the cytosol (C) of senescent strains (right), but not in middle-aged isolates (left) (M - mitochondria). Antibodies against the MnSOD (mitochondria) and the Cu/Zn-SOD (cytosol) were used as marker proteins for compartments and to show equal loading. The natively tetrameric MnSOD is retained within the mitochondria in senescence showing the size-limit for release should be between 27 kDa and ∼80 kDa.

Mentions: In order to further support the hypothesis of an age-related release of components from mitochondria, we constructed an eGfp expression vector in which the eGfp open reading frame was fused to the mitochondrial target sequence (MTS) of the mitochondrial processing peptidase (MPP) from the closely related ascomycete Neurospora crassa [17]. Middle-aged cultures of the selected transformants expressing the introduced gene display tubular shaped fluorescent structures (Fig. 3A, left panel). Counterstaining with MitoTracker® CMXRos verified that the fluorescing structures represent mitochondria and demonstrate a mitochondrial localization of the eGFP marker protein. Strikingly, in hyphae of senescent cultures of the same transformant a diffuse green fluorescence occurs. MitoTracker® CMXRos staining identified distinct punctuate structures corresponding to fragmented mitochondria as they are characteristic for senescent P. anserina cultures [29] (Fig. 3A, right panel). These data suggest an age-related release of eGFP from mitochondria to the cytoplasm.


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)

Contents of the mitochondrial matrix are released into the cytosol in senescent P. anserina strains.(A) The localization of eGFP directed into the matrix changes from mitochondrial in middle-aged strains (left) to cytoplasmic distribution in senescent strains (right). As control mitochondria are stained with MitoTracker® red CMXRos. Scale bar: 2 µm. (B) The microscopic results are verified by Western Blot analysis. eGFP (mature form and pre-protein) can be detected in the cytosol (C) of senescent strains (right), but not in middle-aged isolates (left) (M - mitochondria). Antibodies against the MnSOD (mitochondria) and the Cu/Zn-SOD (cytosol) were used as marker proteins for compartments and to show equal loading. The natively tetrameric MnSOD is retained within the mitochondria in senescence showing the size-limit for release should be between 27 kDa and ∼80 kDa.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004919-g003: Contents of the mitochondrial matrix are released into the cytosol in senescent P. anserina strains.(A) The localization of eGFP directed into the matrix changes from mitochondrial in middle-aged strains (left) to cytoplasmic distribution in senescent strains (right). As control mitochondria are stained with MitoTracker® red CMXRos. Scale bar: 2 µm. (B) The microscopic results are verified by Western Blot analysis. eGFP (mature form and pre-protein) can be detected in the cytosol (C) of senescent strains (right), but not in middle-aged isolates (left) (M - mitochondria). Antibodies against the MnSOD (mitochondria) and the Cu/Zn-SOD (cytosol) were used as marker proteins for compartments and to show equal loading. The natively tetrameric MnSOD is retained within the mitochondria in senescence showing the size-limit for release should be between 27 kDa and ∼80 kDa.
Mentions: In order to further support the hypothesis of an age-related release of components from mitochondria, we constructed an eGfp expression vector in which the eGfp open reading frame was fused to the mitochondrial target sequence (MTS) of the mitochondrial processing peptidase (MPP) from the closely related ascomycete Neurospora crassa [17]. Middle-aged cultures of the selected transformants expressing the introduced gene display tubular shaped fluorescent structures (Fig. 3A, left panel). Counterstaining with MitoTracker® CMXRos verified that the fluorescing structures represent mitochondria and demonstrate a mitochondrial localization of the eGFP marker protein. Strikingly, in hyphae of senescent cultures of the same transformant a diffuse green fluorescence occurs. MitoTracker® CMXRos staining identified distinct punctuate structures corresponding to fragmented mitochondria as they are characteristic for senescent P. anserina cultures [29] (Fig. 3A, right panel). These data suggest an age-related release of eGFP from mitochondria to the cytoplasm.

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