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.
Highlights
During the course of investigations to unravel the molecular mechanisms leading to an increased lifespan in the nuclear longlived grisea mutant of the fungal ageing model Podospora anserina, an impact of a tight regulation of cellular copper levels was identified [1,2,3,4,5]
Age-related changes of copper levels in the cytoplasm Previous studies suggested an increase in cytosolic copper levels during ageing of P. anserina cultures
In the current study we investigated the age-related changes in copper abundance more directly by measuring copper concentrations in the post-mitochondrial supernatant and in mitochondria purified by sucrosegradient density centrifugation
Summary
During the course of investigations to unravel the molecular mechanisms leading to an increased lifespan in the nuclear longlived grisea mutant of the fungal ageing model Podospora anserina, an impact of a tight regulation of cellular copper levels was identified [1,2,3,4,5]. During the course of investigations, data were obtained suggesting age-related increases in cytosolic copper levels during ageing of P. anserina cultures. This conclusion was drawn from transcript levels of the copper regulated genes PaMt1, PaCtr, PaSod which were found to be either increased or reduced in senescent fungal cultures [2,5]. In addition PaSOD1 activity, depending on copper as a cofactor, was found to be higher in senescent cultures, while PaSOD2 activity, as a result of reduced transcript levels, is decreased
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