Age-related cellular copper dynamics in the fungal ageing model Podospora anserina and in ageing human fibroblasts.

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.

Mitochondrial free radical generation and lifespan control in the fungal aging model Podospora anserina.

In the filamentous fungus Podospora anserina a central role of mitochondria in the control of aging has been repeatedly demonstrated. Interestingly, impairments in cytochrome c oxidase (COX) activity induce an enhancement in the expression of the quinol-oxygen alternative oxidoreductase (AOX) correlating with an extension of lifespan. This effect is thought to be determined by a reduction of the free radical generation in mitochondria. In the current investigation we have analyzed the electron transport chain composition of P. anserina and the superoxide generation rate in wild type s and in mutant grisea, a long-lived mutant with complex IV deficiency. Here we report that, similarly to other fungi, mitochondrial respiration in P. anserina is a combination of standard and alternative routes. A switch in the COX/AOX respiration balance affects the mitochondrial free radical generation. Lower mitochondrial rates of superoxide generation were found in the long-lived mutant, supporting the central role of mitochondrial free radical generation in the lifespan control of P. anserina. The question of how the activity of the alternative respiratory pathway influences the rate of free radical generation in P. anserina mitochondria is discussed.

Mode of translational activation of the catalase (cat1) mRNA of rye leaves (Secale cereale L.) and its control through blue light and reactive oxygen.

The enzyme catalase (EC 1.11.1.6) is inactivated by light and must be continuously replaced by new synthesis in order to maintain a constant enzyme activity in leaves. In winter rye leaves (Secale cereale L.) posttranscriptional mechanisms determine the rate of new catalase synthesis, including a light-controlled reversible modification of the catalase cat1 mRNA by methylation which greatly enhanced its translation efficiency. The specificity and regulation of this mRNA activation were further investigated. The translation efficiency of the rye cat1 mRNA was much more enhanced by N-7 methylation of the cap than that of an lhcb transcript. Investigations with truncated rye cat1 mRNAs indicated that the translational enhancement resulting from N-7 cap methylation did not require the presence of specific sequences of cat1 5'- and 3'-untranslated regions. Translational activation of the cat1 mRNA in rye leaves was independent of photosynthesis and most effectively induced by blue light. Peroxides (H(2)O(2), tertiary butyl hydroperoxide) and conditions enforcing an H(2)O(2) accumulation in the leaves (aminotriazole, paraquat) also caused an activation of the cat1 mRNA. A search for further signalling systems controlling the replenishment of inactivated catalase in light suggested that an inositol-1,4,5-triphosphate-mediated liberation of Ca(2+) from internal stores and a protein phosphatase played some role. However, these signalling systems did not affect the activation of the cat1 mRNA. After removal of Ca(2+) by EGTA the cat1 mRNA was rapidly degraded.