Annie Sainsard-Chanet

Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long‐lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.

Gene deletion and allelic replacement in the filamentous fungus Podospora anserina

Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the ΔPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.

Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence in Podospora anserina

ABSTRACT Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (α) has been thought to play a prominent role in this syndrome. Intron α is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of theCOX1 gene. We describe here the first mutant of P. anserina that has the α sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron α. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron α is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron α plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, “immortality” can be acquired not by the absence of intron α but rather by the lack of active cytochromec oxidase.

Respiratory Complexes III and IV Are Not Essential for the Assembly/Stability of Complex I in Fungi

The functional relevance of respiratory supercomplexes in various eukaryotes including mammals, plants, and fungi is hitherto poorly elucidated. However, substantial evidence indicates as a major role the assembly and/or stabilization of mammalian complex I by supercomplex formation with complexes III and IV. Here, we demonstrate by using native electrophoresis that the long-lived Podospora anserina mutant Cyc1-1, respiring exclusively via the alternative oxidase (AOX), lacks an assembled complex III and possesses complex I partially assembled with complex IV into a supercomplex. This resembles the situation in complex-IV-deficient mutants displaying a corresponding phenotype but possessing I-III supercomplexes instead, suggesting that either complex III or complex IV is in a redundant manner necessary for assembly/stabilization of complex I as previously shown in mammals. To corroborate this notion, we constructed the double mutant Cyc1-1,Cox5::ble. Surprisingly, this mutant lacking both complexes III and IV is viable and essentially a phenocopy of mutant Cyc1-1 including the reversal of the phenotype towards wild-type-like characteristics by the several-fold overexpression of the AOX in mutant Cyc1-1,Cox5::ble(Gpd-Aox). Fungal specific features (not found in mammals) that must be responsible for assembly/stabilization of fungal complex I when complexes III and IV are absent, such as the presence of the AOX and complex I dimerization, are addressed and discussed. These intriguing results unequivocally prove that complexes III and IV are dispensable for assembly/stability of complex I in fungi contrary to the situation in mammals, thus highlighting the imperative to unravel the biogenesis of complex I as well as the true supramolecular organization of the respiratory chain and its functional significance in a variety of model eukaryotes. In summary, we present the first obligatorily aerobic eukaryote with an artificial, simultaneous lack of the respiratory complexes III and IV.

Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing.

A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.

Genetic background influences mitochondrial function: modeling mitochondrial disease for therapeutic development

Genetic background strongly influences the phenotype of human mitochondrial diseases. Mitochondrial biogenesis and function require up to 1500 nuclear genes, providing myriad opportunities for effects on disease expression. Phenotypic variability, combined with relative rarity, constitutes a major obstacle to establish cohorts for clinical trials. Animal models are, therefore, potentially valuable. However, several of these show no or very mild disease phenotypes compared with patients and can not be used for therapeutic studies. One reason might be the insufficient attention paid to the need for genetic diversity in order to capture the effects of genetic background on disease expression. Here, we use data from various models to emphasize the need to preserve genetic diversity when studying mitochondrial disease phenotypes or drug effects.

Two co-existing mechanisms account for the large-scale deletions of mitochondrial DNA in Podospora anserina that involve the 5′ border of a group-II intron

Abstract A degenerative syndrome associated with the accumulation of site-specific deletions within mitochondrial chromosomes occurs in strains of Podospora anserina carrying the AS1-4 nuclear mutation. The site-specific deletion event has been assumed to result from the transposition of a group-II intron (intron α) behind an IBS motif, followed by recombination between the two intron repeats. We show here that a number of distinct deletions can accumulate in AS1-4 strains. Most of them are present in low amounts in wild-type cells where they are only detectable in PCR experiments. The deletions can be divided into two classes. In class I, intron α is joined to an IBS motif. In class II, the intron is not joined to an IBS site, it can be truncated or contain a few upstream exonic nucleotides; some junctions carry non-templated nucleotides. These results indicate that at least two mechanisms are involved in the generation of large-scale mitochondrial deletions in Podospora. One of them seems to be based on the transposition properties of the group-II α intron, the other one on illegitimate recombination. We propose that these two mechanisms use DNA double-strand breaks occurring within the 5′ region of intron α.

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 II. Cyanide-insensitive respiration. Function and regulation

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.

Mobile group II introns, DNA circles, reverse transcriptase and senescence (group II introns, transposition, aging, mitochondria, fungi).

Group II introns are widely distributed as attested by their recent discovery in several bacterial species (Ferat & Michel, 1993; Ferat, Le Gouar & Michel, 1994; Knoop & Brennicke, 1994). They are supposed to be relics of the RNA world and ancestors of the nuclear spliceosomal machinery (Cavalier-Smith, 1991; Sharp, 1991). Recent progress on their molecular biology has shed light on some of their interesting properties as mobile genetic sequences. They display ribozymic properties. Self-splicing, reverse splicing and erroneous reverse splicing have been demonstrated at the RNA level by in vitro experiments (Peebles et al., 1986; Augustin, MUller & Schweyen, 1990; M6rl & Schmelzer, 1990). They often contain an open reading frame related to reverse transcriptase (Michel & Lang, 1985) and recent results have shown that the Saccharomyces cerevisiae mitochondrial coxl-i2 intronic ORF indeed encodes a protein endowed with reverse transcriptase activity (Kennell et al., 1993). Like group I introns, they are able of homing, i.e. of integration into a homologous gene devoid of them. This has been demonstrated for the Saccharomyces cerevisiae coxl-il and coxl-i2 (Meunier et al., 1990), Kluyveromyces lactis coxl=il (Skelly, Hardy & ClarkWalker, 1991) and Podospora anserina coxl-i4 (our unpublished results) mitochondrial introns. Homing of group II introns is dependent upon the expression of their internal ORF (Meunier et al., 1990). More recently, their ability to transpose, i.e. to move to a novel location, has been demonstrated in yeast and in Podospora anserina (Mueller et al., 1993; Sellem, Lecellier & Belcour, 1993). These recurrent events of transposition would account for site-specific deletions of the mitochondrial chromosome leading to respiration deficient mutants in yeast and to the premature death syndrome in Podospora anserina (Mueller et al., 1993; Sellem, Lecellier & Belcour, 1993). Transposition of group II introns could explain their spreading in the course of evolution.

Molecular Gene Therapy: Overexpression of the Alternative NADH Dehydrogenase NDI1 Restores Overall Physiology in a Fungal Model of Respiratory Complex I Deficiency

Defects in oxidative phosphorylation lie at the heart of a wide variety of degenerative disorders, cancer, and aging. Here, we show, using the fungal model Podospora anserina, that the overexpression of the native mitochondrial matrix-faced type II NADH dehydrogenase NDI1, paralogue of the human apoptosis inducing factor AIF1, can fully restore all physiological consequences of respiratory complex I deficiency. We disrupted the 19.3-kDa subunit of the complex I catalytic core, orthologue of the human PSST subunit, leading to a complete absence of the complex without affecting the assembly and/or stability of the rest of the respiratory chain. This disruption caused a several-fold life span extension at the expense of both male and female fertility. The effect was generally similar but markedly milder than that caused by defects in the complex III/IV-dependent pathway and not associated with a clear reduction in the steady-state level of mitochondrial reactive oxygen species. Whereas the native expression of NDI1 was sufficient to overcome lethality, only the artificial, constitutive overexpression of NDI1 could fully remedy this deficiency: The latter strikingly restored both life span and fertility to levels indistinguishable from wild type, thus demonstrating its unique potential in molecular gene therapy.