Escape from Premature Death Due to Nuclear Mutations inPodospora anserina:Repeal versus Respite

Premature death has been defined as a growth stoppage linked to the accumulation of specific deletions of the mitochondrial genome (mtDNA) inPodospora anserina. This occurs only in strains carrying theAS1-4mutation which lies in a gene encoding a cytosolic ribosomal protein. Here we describe the isolation and genetic characterization of 10 nuclear mutations which either delay the appearance of this syndrome (respite from premature death) or cause a switch to the classical senescence process (repeal of premature death). These mutations lie in at least six genes. Some cause defects at the levels of ascospore germination, growth rates, and/or sensitivity toward inhibitors of protein syntheses. All modify the onset of senescence in wild-type (AS1⁺) strains. The role played by these genes is discussed with respect to the control of diseases due to mtDNA rearrangements in filamentous fungi.     

Targeted enrichment and high‐resolution digital profiling of mitochondrial DNA deletions in human brain

Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed ‘Digital Deletion Detection’ (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10⁻⁸. 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age-related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre-existing mutations is the primary factor contributing to age-related accumulation of mtDNA deletions.     

Escape from Premature Death Due to Nuclear Mutations in Podospora anserina: Repeal versus Respite

Premature death has been defined as a growth stoppage linked to the accumulation of specific deletions of the mitochondrial genome (mtDNA) in Podospora anserina. This occurs only in strains carrying the AS1-4 mutation which lies in a gene encoding a cytosolic ribosomal protein. Here we describe the isolation and genetic characterization of 10 nuclear mutations which either delay the appearance of this syndrome (respite from premature death) or cause a switch to the classical senescence process (repeal of premature death). These mutations lie in at least six genes. Some cause defects at the levels of ascospore germination, growth rates, and/or sensitivity toward inhibitors of protein syntheses. All modify the onset of senescence in wild-type (AS1+) strains. The role played by these genes is discussed with respect to the control of diseases due to mtDNA rearrangements in filamentous fungi. Copyright 1998 Academic Press.     

Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging

A wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) with aging has been observed in animals and humans. These include (i) decline in mitochondrial respiratory function; (ii) increase in mitochondrial production of reactive oxygen species (ROS) and the extent of oxidative damage to DNA, proteins, and lipids; (iii) accumulation of point mutations and large-scale deletions of mtDNA; and (iv) enhanced apoptosis. Recent studies have provided abundant evidence to substantiate the importance of mitochondrial production of ROS in aging. On the other hand, somatic mtDNA mutations can cause premature aging without increasing ROS production. In this review, we focus on the roles that ROS play in the aging-associated decline of mitochondrial respiratory function, accumulation of mtDNA mutations, apoptosis, and alteration of gene expression profiles. Taking these findings together, we suggest that mitochondrial dysfunction, enhanced oxidative stress, subsequent accumulation of mtDNA mutations, altered expression of a few clusters of genes, and apoptosis are important contributors to human aging.     

A Heterozygous Truncating Mutation in RRM2B Causes Autosomal-Dominant Progressive External Ophthalmoplegia with Multiple mtDNA Deletions

Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C→T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.     

Effects of Fcj1-Mos1 and mitochondrial division on aggregation of mitochondrial DNA nucleoids and organelle morphology

Mitochondrial DNA (mtDNA) is packaged into DNA–protein complexes called nucleoids, which are distributed as many small foci in mitochondria. Nucleoids are crucial for the biogenesis and function of mtDNA. Here, using a yeast genetic screen for components that control nucleoid distribution and size, we identify Fcj1 and Mos1, two evolutionarily conserved mitochondrial proteins that maintain the connection between the cristae and boundary membranes. These two proteins are also important for establishing tubular morphology of mitochondria, as mitochondria lacking Fcj1 and Mos1 form lamellar sheets. We find that nucleoids aggregate, increase in size, and decrease in number in fcj1∆ and mos1∆ cells. In addition, Fcj1 form punctate structures and localized adjacent to nucleoids. Moreover, connecting mitochondria by deleting the DNM1 gene required for organelle division enhances aggregation of mtDNA nucleoids in fcj1∆ and mos1∆ cells, whereas single deletion of DNM1 does not affect nucleoids. Conversely, deleting F1Fo-ATP synthase dimerization factors generates concentric ring-like cristae, restores tubular mitochondrial morphology, and suppresses nucleoid aggregation in these mutants. Our findings suggest an unexpected role of Fcj1-Mos1 and organelle division in maintaining the distribution and size of mtDNA nucleoids.     

Suppression of Mitochondrial DNA Instability of Autosomal Dominant Forms of Progressive External Ophthalmoplegia-Associated ANT1 Mutations in Podospora anserina

Maintenance and expression of mitochondrial DNA (mtDNA) are essential for the cell and the organism. In humans, several mutations in the adenine nucleotide translocase gene ANT1 are associated with multiple mtDNA deletions and autosomal dominant forms of progressive external ophthalmoplegia (adPEO). The mechanisms underlying the mtDNA instability are still obscure. A current hypothesis proposes that these pathogenic mutations primarily uncouple the mitochondrial inner membrane, which secondarily causes mtDNA instability. Here we show that the three adPEO-associated mutations equivalent to A114P, L98P, and V289M introduced into the Podospora anserina ANT1 ortholog dominantly cause severe growth defects, decreased reactive oxygen species production (ROS), decreased mitochondrial inner membrane potential (Δψ), and accumulation of large-scale mtDNA deletions leading to premature death. Interestingly, we show that, at least for the adPEO-type M106P and A121P mutant alleles, the associated mtDNA instability cannot be attributed only to a reduced membrane potential or to an increased ROS level since it can be suppressed without restoration of the Δψ or modification of the ROS production. Suppression of mtDNA instability due to the M106P and A121P mutations was obtained by an allele of the rmp1 gene involved in nucleo-mitochondrial cross- talk and also by an allele of the AS1 gene encoding a cytosolic ribosomal protein. In contrast, the mtDNA instability caused by the S296M mutation was not suppressed by these alleles.THE maintenance and expression of mitochondrial DNA (mtDNA) depend on many nuclear-encoded gene products. Recent studies have shown that defects in this maintenance can have devastating consequences for the cell and the organism. In humans, these defects are an important cause of neurological diseases including autosomal dominant (or recessive) progressive external ophthalmoplegia (adPEO) (Chinnery 2003; Copeland 2008). These disorders are characterized by multiple large-scale deletions of mtDNA. Three different genes that can cause PEO with multiple mtDNA deletions have been identified: the mtDNA polymerase (POLG), the heart/muscle isoform of the adenine nucleotide translocator (ANT1), and the mitochondrial DNA helicase, Twinkle.The adenine nucleotide translocator (ANT), also known as the ADP/ATP mitochondrial translocator, is the most abundant protein in the inner mitochondrial membrane (Riccio et al. 1975; Nury et al. 2006; Klingenberg 2008). It exports ATP produced by mitochondrial oxidative phosphorylation toward the cytosol to meet the energy requirements of the cell; in exchange, it transports ADP into the mitochondrial matrix to fuel the conversion of ADP to ATP by the F₁F_O-ATP synthase. In humans, four isoforms of the ANT protein exist, and they are differently expressed in a tissue-specific manner (Stepien et al. 1992; Palmieri 2004; Dolce et al. 2005). The human ANT1 isoform is predominantly expressed in skeletal and cardiac muscle, and specific ANT1 mutations are associated with adPEO characterized by mtDNA instability (Kaukonen et al. 1999, 2000; Napoli et al. 2001; Komaki et al. 2002; Siciliano et al. 2003). In mice, Ant1 knockout induces mitochondrial myopathy (Graham et al. 1997), increased H₂O₂ production, and mtDNA damage and inhibits oxidative phosphorylation (Esposito et al. 1999). Some of these mutations were introduced in the AAC2 gene of Saccharomyces cerevisiae that encodes the major ADP/ATP mitochondrial translocator isoform in this organism. Numerous and sometimes contradictory effects have been reported depending in particular on the yeast laboratory strains examined (Kaukonen et al. 2000; Chen 2002, 2004; Fontanesi et al. 2004; Palmieri et al. 2005; Wang et al. 2008b).In an attempt to better understand how these mutations affect mitochondrial DNA stability and their functional consequences on mitochondrial metabolism, we decided to introduce them in the unique ADP/ATP translocator gene of Podospora anserina, PaAnt. Like S. cerevisiae, the filamentous fungus P. anserina is an excellent system for genetic and molecular analyses. In contrast to S. cerevisiae, it is a strict multicellular aerobe that can display heteroplasmic states in which intact and rearranged mitochondrial genomes coexist. In this organism, life span is a reflection of mtDNA stability, and death is always associated with large mtDNA rearrangements. “Natural death” or aging is accompanied by large-scale reorganizations of the mtDNA whereas a nuclear-controlled premature death syndrome is accompanied by the accumulation of site-specific mtDNA deletions (Belcour et al. 1999; Silar et al. 2001 for reviews). P. anserina therefore occupies an interesting position among model systems for studying the cellular consequences of mutations in the ADP/ATP translocase gene.We show here that the mutations M106P, A121P, and S296M, equivalent to the L98P, A114P (familial), and V289M (sporadic) human mutations, severely impair the vegetative and sexual development of the fungus and are responsible for decreased ROS production and for decreased inner membrane potential (Δψ). The severity of the phenotypes differs according to the mutation. The three mutations show mtDNA instability, which leads to premature death. All these mutated traits are dominant. Interestingly, the mtDNA instability associated with the M106P and A121P mutations depends on the rmp1 gene. This gene exists under two naturally occurring alleles, rmp1-1 and rmp1-2, which control mtDNA integrity in some genetic contexts (Belcour et al. 1991; Contamine et al. 1996, 2004). When associated with the rmp1-1 allele, the M106P and A121P mutations lead to rapid mtDNA instability whereas, in the presence of the rmp1-2 allele, mtDNA instability is suppressed, and life span is considerably increased. Surprisingly, suppression is not accompanied by a restoration of the Δψ or a modification in the ROS level, demonstrating that these parameters are not sufficient to explain the M106P and A121P mtDNA instability. Mitochondrial DNA instability due to the M106P and A121P mutations is also suppressed by a mutation in the AS1 gene encoding a ribosomal protein. The suppressor effects are not observed for the S296M mutation.     

Somatic mutations of mitochondrial genome in hepatocellular carcinoma

Somatic mutations have been identified in mitochondrial DNA (mtDNA) of various human primary cancers. However, their roles in the pathophysiology of cancers are still unclear. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of hepatocellular carcinomas (HCCs). In the present study, we examined 44 HCCs and corresponding non-cancerous liver tissues, and identified 13 somatic mutations in the coding region of mtDNAs from 11 HCC samples (11/44, 25%). Among the 13 mtDNA mutations, six mutations (T6787C, G7976A, A9263G, G9267A, A9545G and A11708G) were homoplasmic while seven mutations (956delC, T1659C, G3842A, G5650A, 11032delA, 12418insA and a 66 bp deletion) were heteroplasmic. Moreover, the G3842A transition created a premature stop codon and the 66 bp deletion could omit 22 amino acid residues in the NADH dehydrogenase (ND) subunit 1 (ND1) gene. The 11032delA and 12418insA could result in frame-shift mutation in the ND4 and ND5 genes, respectively. The T1659C transition in tRNA^Val gene and G5650A in tRNA^Ala gene were reported to be clinically associated with some mitochondrial disorders. In addition, the T6787C (cytochrome c oxidase subunit I, COI), G7976A (COII), G9267A (COIII) and A11708G (ND4) mutations could result in amino acid substitutions in the highly conserved regions of the affected mitochondrial genes. These mtDNA mutations (10/13, 76.9%) have the potential to cause mitochondrial dysfunction in HCCs. Taken these results together, we suggest that there may be a higher frequency of mtDNA mutations in HCC than in normal liver tissues from the same individuals.     

How mitochondrial damage affects cell function

The pathophysiology of mitochondrial DNA (mtDNA) diseases is caused by increased cell death and dysfunction due to the accumulation of mutations to mtDNA. While the disruption of oxidative phosphorylation is central to mtDNA diseases, many other factors, such as Ca²⁺ dyshomeostasis, increased oxidative stress and defective turnover of mitochondrial proteins, may also contribute. The relative importance of these processes in causing cell dysfunction and death is uncertain. It is also unclear whether these damaging processes lead to the disease phenotype through affecting cell function, increasing cell death or a combination of both. These uncertainties limit our understanding of mtDNA disease pathophysiology and our ability to develop rational therapies. Here, we outline how the accumulation of mtDNA mutations can lead to cell dysfunction by altering oxidative phosphorylation, Ca²⁺ homeostasis, oxidative stress and protein turnover and discuss how these processes affect cell function and susceptibility to cell death. A better understanding of these processes will eventually clarify why particular mtDNA mutations cause defined syndromes in some cases but not in others and why the same mutation can lead to different phenotypes.     

Association of mitochondrial DNA damage with aging and coronary atherosclerotic heart disease.

The role of somatic mitochondrial DNA (mtDNA) damage in human aging and progressive diseases of oxidative phosphorylation (OXPHOS) was examined by quantitating the accumulation of mtDNA deletions in normal hearts and hearts with coronary atherosclerotic disease. In normal hearts, mtDNA deletions appeared after 40 and subsequently accumulated with age. The common 4977 nucleotide pair (np) deletion (mtDNA4977) reached a maximum of 0.007%, with the mtDNA7436 and mtDNA10,422 deletions appearing at the same time. In hearts deprived of mitochondrial substrates due to coronary artery disease, the level of the mtDNA4977 deletion was elevated 7-220-fold over age-matched controls, with the mtDNA7436 and mtDNA10,422 deletions increasing in parallel. This cumulative mtDNA damage was associated with a compensatory 3.5-fold induction of nuclear OXPHOS gene mRNA and regions of ischemic hearts subjected to the greatest work load (left ventricle) showed the greatest accumulation of mtDNA damage and OXPHOS gene induction. These observations support the hypothesis that mtDNA damage does accumulate with age and indicates that respiratory stress greatly elevates mitochondrial damage.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long‐lived animals

Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (López‐Otín et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematode Caenorhabditis elegans also exhibits age‐dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role in C. elegans aging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age‐dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age‐dependent functional decline commonly observed in C. elegans. Computational modeling of mtDNA dynamics in C. elegans indicates that the lifespan of short‐lived animals such as C. elegans is likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long‐lived animals such as humans and rhesus monkey and possibly in rodents.     

Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein

Biochemical studies of the mitochondrial DNA (mtDNA) replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence) are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold). Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.     

Transcriptional response to mitochondrial NADH kinase deficiency in Saccharomyces cerevisiae

Yeast cells lacking the mitochondrial NADH kinase encoded by POS5 display increased sensitivity to hydrogen peroxide, a slow-growth phenotype, reduced mitochondrial function and increased levels of mitochondrial protein oxidation and mtDNA mutations. Here we examined gene expression in pos5Δ cells, comparing these data to those from cells containing deletions of superoxide dismutase-encoding genes SOD1 or SOD2. Surprisingly, stress–response genes were down-regulated in pos5Δ, sod1Δ and sod2Δ cells, implying that cells infer stress levels from mitochondrial activity rather than sensing reactive oxygen species directly. Additionally, pos5Δ, but not sod1 or sod2, cells displayed an anaerobic expression profile, indicating a defect in oxygen sensing that is specific to pos5, and is not a general stress–response. Finally, the pos5Δ expression profile is quite similar to the hap1Δ expression profile previously reported, which may indicate a shared mechanism.     

Regular bottlenecks and restrictions to somatic fusion prevent the accumulation of mitochondrial defects in Neurospora

The replication and segregation of multi-copy mitochondrial DNA (mtDNA) are not under strict control of the nuclear DNA. Within-cell selection may thus favour variants with an intracellular selective advantage but a detrimental effect on cell fitness. High relatedness among the mtDNA variants of an individual is predicted to disfavour such deleterious selfish genetic elements, but experimental evidence for this hypothesis is scarce. We studied the effect of mtDNA relatedness on the opportunities for suppressive mtDNA variants in the fungus Neurospora carrying the mitochondrial mutator plasmid pKALILO. During growth, this plasmid integrates into the mitochondrial genome, generating suppressive mtDNA variants. These mtDNA variants gradually replace the wild-type mtDNA, ultimately culminating in growth arrest and death. We show that regular sequestration of mtDNA variation is required for effective selection against suppressive mtDNA variants. First, bottlenecks in the number of mtDNA copies from which a ‘Kalilo’ culture started significantly increased the maximum lifespan and variation in lifespan among cultures. Second, restrictions to somatic fusion among fungal individuals, either by using anastomosis-deficient mutants or by generating allotype diversity, prevented the accumulation of suppressive mtDNA variants. We discuss the implications of these results for the somatic accumulation of mitochondrial defects during ageing.     

Somatic mtDNA Mutation Spectra in the Aging Human Putamen

The accumulation of heteroplasmic mitochondrial DNA (mtDNA) deletions and single nucleotide variants (SNVs) is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq) to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the “common” deletion and other “major arc” deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ⁻ mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states.     

Retinal ganglion cell neurodegeneration in mitochondrial inherited disorders

Since the early days of mitochondrial medicine, it has been clear that optic atrophy is a very common and sometimes the singular pathological feature in mitochondrial disorders. The first point mutation of mitochondrial DNA (mtDNA) associated with the maternally inherited blinding disorder, Leber's hereditary optic neuropathy (LHON), was recognized in 1988. In 2000, the other blinding disorder, dominant optic atrophy (DOA) Kjer type, was found associated with mutations in the nuclear gene OPA1 that encodes a mitochondrial protein. Besides these two non-syndromic optic neuropathies, optic atrophy is a prominent feature in many other neurodegenerative diseases that are now recognized as due to primary mitochondrial dysfunction.We will consider mtDNA based syndromes such as LHON/dystonia/Mitochondrial Encephalomyopahty Lactic Acidosis Stroke-like (MELAS)/Leigh overlapping syndrome, or nuclear based diseases such as Friedreich ataxia (mutations in FXN gene), deafness-dystonia-optic atrophy (Mohr-Tranebjerg) syndrome (mutations in TIMM8A), complicated hereditary spastic paraplegia (mutations in SPG7), DOA “plus” syndromes (mutations in OPA1), Charcot-Marie-Tooth type 2A (CMT2A) with optic atrophy or hereditary motor and sensory neuropathy type VI (HMSN VI) (mutations in MFN2), and Costeff syndrome and DOA with cataract (mutations in OPA3). Thus, genetic errors in both nuclear and mitochondrial genomes often lead to retinal ganglion cell death, a specific target for mitochondrial mediated neurodegeneration. Many mechanisms have been studied and proposed as the bases for the pathogenesis of mitochondrial optic neuropathies including bioenergetic failure, oxidative stress, glutamate toxicity, abnormal mitochondrial dynamics and axonal transport, and susceptibility to apoptosis.     

Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae

The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli^r mutants in an ogg1Δ strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli^r mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8^m recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial genome.