A causal link between respiration and senescence in Podospora anserina

Abstract

Senescence, a progressive degenerative process leading to age-related increase in mortality, is found in most eukaryotes. However, the molecular events underlying aging remain largely unknown. Understanding how longevity is regulated is a fundamental problem. Here we demonstrate that the respiratory function is a key factor that contributes to shortening lifespan of the filamentous fungus Podospora anserina. In this organism, senescence is systematically associated with mitochondrial DNA instabilities. We show that inactivation of the nuclear COX5 gene encoding subunit V of the cytochrome c oxidase complex leads to the exclusive use of the alternative respiratory pathway and to a decrease in production of reactive oxygen species. This inactivation results in a striking increase of longevity associated with stabilization of the mitochondrial chromosome. Moreover, accumulation of several senescence-specific mitochondrial DNA molecules is prevented in this nuclear mutant. These findings provide direct evidence of a causal link between mitochondrial metabolism and longevity in Podospora anserina.

Figure 1

(A) Construction of the plasmid pPH52 designed for gene disruption. The plasmid pPH51 containing the HPH gene and a 2.6-kb fragment including the COX5 gene and its flanking sequences (filled bar) was amplified in a PCR experiment using two divergent primers (*). The amplification product (thick line) is deleted for 139 bp, including the start codon and the complete mitochondrial targeting sequence. It was ligated to the BLE cassette, giving plasmid pPH52. The disrupted COX5 gene is designed cox5∷BLE. (B) Diagram of integration by homologous recombination. (C) Southern analysis of transformants. Genomic DNA was digested by NheI (position shown in B) and hybridized with the probe located in the COX5 gene and indicated by a square outside pPH52 in A. Lane 1 is a primary transformant; lane 5 the recipient wild-type strain, lanes 2 to 4 are secondary monokaryotic progenies derived from the cross between the primary transformant and the wild-type strain. The 2.1- and 2.6-kb fragments respectively correspond to the wild-type and disrupted COX5 locus.

Figure 2

Effects of cyanide and SHAM on the respiration of COX5 and cox5∷BLE strains. Green arrow indicates the addition of the cells. Addition of KCN and SHAM (1 mM and 2.5 mM, respectively) is respectively indicated as red and blue arrows. The initial amount of oxygen present in the chamber corresponds to 100%; KCN and SHAM (when KCN did not completely block respiration) are respectively added when 60% and 30% of oxygen is still present. Without addition of inhibitors, 100% of the oxygen is consumed.

Figure 3

Measurement of ROS elimination in the different strains. COX5 (■), mex16 (♦), and cox5∷BLE (●) protoplasts from each strain were incubated with H₂DCF diacetate, and the DCF fluorescence was measured every 15 min by flow cytometry. Cells with altered membranes (BET positives) were excluded from the analysis. Values of DCF fluorescence, expressed in arbitrary units, are average values calculated with at least 10,000 protoplasts. (A) ROS elimination in standard conditions. (B) ROS elimination in 0.018% H₂O₂ medium. (C) ROS elimination by COX5 protoplasts in the absence (blue curve) or in the presence (violet curve) of 1 mM KCN compared with the ROS elimination by cox5∷BLE protoplasts in the absence of KCN.

Figure 4

Southern blot analysis of mtDNA extracted from young and senescent wild-type, cox5∷BLE, and cox5∷BLE[COX5] subcultures. DNA is digested by HaeIII. Lanes 1 and 2 respectively correspond to young cultures of wild-type and cox5∷BLE; lane 3, to a senescent wild-type culture; lanes 4, 6, and 7, to a cox5∷BLE culture that had stopped growing; and lane 5, to a senescent cox5∷BLE[COX5] culture carrying both the cox5∷BLE deletion and an ectopic integration of the COX5 gene. Hybridization was performed with probes specific for the regions of the mitochondrial chromosome from which senDNA α (lanes 1 to 5), senDNAs β (lane 6), and senDNAs γ (lane 7), respectively, originate. The black arrow indicates the expected size for senDNA α; ● corresponds to the encompassed HaeIII mtDNA fragments expected to hybridize with the α probe; ♦ corresponds to the encompassed HaeIII fragments expected to hybridize with the β and γ probes; and white arrow indicates an additional fragment generated by circularization of γ senDNA.