A Cytoplasmic Suppressor of a Nuclear Mutation Affecting Mitochondrial Functions in Drosophila

Phenotypes relevant to oxidative phosphorylation (OXPHOS) in eukaryotes are jointly determined by nuclear and mitochondrial DNA (mtDNA). Thus, in humans, the variable clinical presentations of mitochondrial disease patients bearing the same primary mutation, whether in nuclear or mitochondrial DNA, have been attributed to putative genetic determinants carried in the “other” genome, though their identity and the molecular mechanism(s) by which they might act remain elusive. Here we demonstrate cytoplasmic suppression of the mitochondrial disease-like phenotype of the Drosophila melanogaster nuclear mutant tko^25t, which includes developmental delay, seizure sensitivity, and defective male courtship. The tko^25t strain carries a mutation in a mitoribosomal protein gene, causing OXPHOS deficiency due to defective intramitochondrial protein synthesis. Phenotypic suppression was associated with increased mtDNA copy number and increased mitochondrial biogenesis, as measured by the expression levels of porin voltage dependent anion channel and Spargel (PGC1α). Ubiquitous overexpression of Spargel in tko^25t flies phenocopied the suppressor, identifying it as a key mechanistic target thereof. Suppressor-strain mtDNAs differed from related nonsuppressor strain mtDNAs by several coding-region polymorphisms and by length and sequence variation in the noncoding region (NCR), in which the origin of mtDNA replication is located. Cytoplasm from four of five originally Wolbachia-infected strains showed the same suppressor effect, whereas that from neither of two uninfected strains did so, suggesting that the stress of chronic Wolbachia infection may provide evolutionary selection for improved mitochondrial fitness under metabolic stress. Our findings provide a paradigm for understanding the role of mtDNA genotype in human disease.     

Disruption of mitochondrial function in interpopulation hybrids of Tigriopus californicus

Electron transport system (ETS) function in mitochondria is essential for the aerobic production of energy. Because ETS function requires extensive interactions between mitochondrial and nuclear gene products, coadaptation between mitochondrial and nuclear genomes may evolve within populations. Hybridization between allopatric populations may then expose functional incompatibilities between genomes that have not coevolved. The intertidal copepod Tigriopus californicus has high levels of nucleotide divergence among populations at mitochondrial loci and suffers F2 hybrid breakdown in interpopulation hybrids. We hypothesize that hybridization results in incompatibilities among subunits in ETS enzyme complexes and that these incompatibilities result in diminished mitochondrial function and fitness. To test this hypothesis, we measured fitness, mitochondrial function, and ETS enzyme activity in inbred recombinant hybrid lines of Tigriopus californicus. We found that (1) both fitness and mitochondrial function are reduced in hybrid lines, (2) only those ETS enzymes with both nuclear and mitochondrial subunits show a loss of activity in hybrid lines, and (3) positive relationships exist between ETS enzyme activity and mitochondrial function and between mitochondrial function and fitness. We also present evidence that hybrid lines harboring mitochondrial DNA (mtDNA) and mitochondrial RNA polymerase (mtRPOL) from the same parental source population have higher fitness than those with mtDNA and mtRPOL from different populations, suggesting that mitochondrial gene regulation may play a role in disruption of mitochondrial performance and fitness of hybrids. These results suggest that disruption of coadaptation between nuclear and mitochondrial genes contributes to the phenomenon of hybrid breakdown.     

Genetic Architecture of Physiological Phenotypes: Empirical Evidence for Coadapted Gene Complexes

Physiological phenotypes are the result of the coordinated functionof many genes, some of which may be differentiated between conspecificpopulations. Within any one population, natural selection willfavor evolution of a coadapted set of alleles which optimizesphysiological performance and reproductive success. The existenceof such coadapted gene complexes may be assessed by assayingphenotypes of interpopulation hybrids: inferior performanceof hybrids suggests that the allelic combinations present inthe parental populations are coadapted. This approach has beenused to examine the genetic architecture of physiological traitsin the copepod Tigriopus californicus, a species characterizedby sharp genetic differentiation of populations. Developmentaltime and response to osmotic stress both show pronounced F₂hybrid breakdown, a result consistent with genetic coadaptationwithin populations. To better understand the biochemical andmolecular mechanisms underlying hybrid breakdown, we are investigatinga specific biochemical phenotype, the activity of the enzymecytochrome c oxidase (COX). COX (encoded by multiple nuclearand mitochondrial genes) catalyzes the oxidation of cytochromec (encoded by a nuclear gene). Two approaches are being usedto address the extent of coadaptation (both among nuclear genesand between nuclear and mitochondrial genes) underlying COXfunction: (1) studies of the DNA (and inferred amino acid) sequencesof component genes among populations in search of coordinatepatterns of amino acid substitution across loci, and (2) directstudies of COX function in interpopulation hybrids and backcrosses.These approaches provide evidence for the existence of nuclear/nuclearand/or nuclear/mitochondrial coadaptation within natural populationsof T. californicus.     

HETEROSIS AND OUTBREEDING DEPRESSION IN INTERPOPULATION CROSSES SPANNING A WIDE RANGE OF DIVERGENCE

The intertidal copepod Tigriopus californicus was used as a model organism to look at effects of crossing distance on fitness and to investigate the genetic mechanisms responsible. Crosses were conducted between 12 pairs of populations spanning a broad range of both geographic distance (5 m to 2007 km) and genetic distance (0.2% to 22.3% sequence divergence for a 606-bp segment of the mitochondrial COI gene). For each pair of populations, three fitness components (hatching number, survivorship number, and metamorphosis number) were measured in up to 16 cohorts including parentals, reciprocal F₁, F₂, F₃, and first-generation backcross hybrids. Comparisons of each set of cohorts allowed estimation of within- and between-locus gene interaction. Relative to parentals, F₁ hybrids showed a trend toward increased fitness, with no correspondence with population divergence, and a decrease in variance, which in some cases correlated with population divergence. In sharp contrast, F₂ hybrids had a decrease in fitness and an increase in variance that both corresponded to population divergence. Genetic interpretation of these patterns suggests that both the beneficial effects of dominance and the detrimental effects of breaking up coadaptation are magnified by increasing evolutionary distance between populations. Because there is no recombination in T. californicus females, effects of recombination can be assessed by comparing F₁ hybrid males and females backcrossed to parentals. Both recombinant and nonrecombinant backcross hybrids showed a decline in fitness correlated with population divergence, indicating that segregation among chromosomes contributes to the breakup of coadaptation. Although there was no difference in mean fitness between the two backcross types, recombinational backcrosses showed greater variance for fitness than nonrecombinational backcrosses, suggesting that the breakup of parental gene ombinations within chromosomes has both beneficial and detrimental effects.     

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.     

Mitochondrial DNA mutations and human disease

Mitochondrial disorders are a group of clinically heterogeneous diseases, commonly defined by a lack of cellular energy due to oxidative phosphorylation (OXPHOS) defects. Since the identification of the first human pathological mitochondrial DNA (mtDNA) mutations in 1988, significant efforts have been spent in cataloguing the vast array of causative genetic defects of these disorders. Currently, more than 250 pathogenic mtDNA mutations have been identified. An ever-increasing number of nuclear DNA mutations are also being reported as the majority of proteins involved in mitochondrial metabolism and maintenance are nuclear-encoded. Understanding the phenotypic diversity and elucidating the molecular mechanisms at the basis of these diseases has however proved challenging. Progress has been hampered by the peculiar features of mitochondrial genetics, an inability to manipulate the mitochondrial genome, and difficulties in obtaining suitable models of disease. In this review, we will first outline the unique features of mitochondrial genetics before detailing the diseases and their genetic causes, focusing specifically on primary mtDNA genetic defects. The functional consequences of mtDNA mutations that have been characterised to date will also be discussed, along with current and potential future diagnostic and therapeutic advances.     

Hybrid breakdown and mitochondrial dysfunction in hybrids of Nasonia parasitoid wasps

Male F(2) hybrids of the wasps Nasonia giraulti and Nasonia vitripennis suffer increased mortality during development. Previous studies suggested that the mitochondria may play an important role in this pattern of hybrid breakdown. The mitochondrial genome encodes 13 polypeptides, which are integral subunits of the oxidative phosphorylation enzyme complexes I, III, IV and V. We show that the mitochondrial ATP production rate and the efficacy of the enzyme complexes I, III and IV, but not that of the completely nuclear-encoded complex II, are reduced in F(2) hybrid males of N. giraulti and N. vitripennis. We hypothesize that nuclear-mitochondrial protein interactions in the oxidative phosphorylation pathway are disrupted in these hybrids, reducing energy generation capacity and potentially reducing hybrid fitness. Our results suggest that dysfunctional cytonuclear interactions could represent an under-appreciated post-zygotic isolation mechanism that, due to elevated evolutionary rates of mitochondrial genes, evolves very early in the speciation process.     

Mitochondrial haplotype does not affect sperm velocity in the zebra finch Taeniopygia guttata

Mitochondrial DNA (mtDNA) variation has been suggested as a possible cause of variation in male fertility because sperm activity is tightly coupled to mitochondrial oxidative phosphorylation and ATP production, both of which are sensitive to mtDNA mutations. Since male‐specific phenotypes such as sperm have no fitness consequences for mitochondria due to maternal mitochondrial (and mtDNA) inheritance, mtDNA mutations that are deleterious in males but which have negligible or no fitness effect in females can persist in populations. How often such mutations arise and persist is virtually unknown. To test whether there were associations between mtDNA variation and sperm performance, we haplotyped 250 zebra finches Taeniopygia guttata from a large pedigreed‐population and measured sperm velocity using computer‐assisted sperm analysis. Using quantitative genetic ‘animal’ models, we found no effect of mtDNA haplotype on sperm velocity. Therefore, there is no evidence that in this system mitochondrial mutations have asymmetric fitness effects on males and females, leading to genetic variation in male fertility that is blind to natural selection.     

Tracing hybrid incompatibilities to single amino acid substitutions

Deleterious interactions among genes cause reductions in fitness of interpopulation hybrids (hybrid breakdown). Identifying genes involved in hybrid breakdown has proven difficult, and few studies have addressed the molecular basis of this widespread phenomenon. Because proper function of the mitochondrial electron transport system (ETS) requires a coadapted set of nuclear and mitochondrial gene products, ETS genes present an attractive system for studying the evolution of coadapted gene complexes within isolated populations and the loss of fitness in interpopulation hybrids. Here we show the effects of single amino acid substitutions in cytochrome c (CYC) on its functional interaction with another ETS protein, cytochrome c oxidase (COX) in the intertidal copepod Tigriopus californicus. The individual and pairwise consequences of three naturally occurring amino acid substitutions in CYC are examined by site-directed mutagenesis and found to differentially effect the rates of CYC oxidation by COX variants from different source populations. In one case, we show that interpopulation hybrid breakdown in COX activity can be attributed to a single naturally occurring amino acid substitution in CYC.     

Seed bank dynamics govern persistence of Brassica hybrids in crop and natural habitats

Background and Aims Gene flow from crops to their wild relatives has the potential to alter population growth rates and demography of hybrid populations, especially when a new crop has been genetically modified (GM). This study introduces a comprehensive approach to assess this potential for altered population fitness, and uses a combination of demographic data in two habitat types and mathematical (matrix) models that include crop rotations and outcrossing between parental species.Methods Full life-cycle demographic rates, including seed bank survival, of non-GM Brassica rapa × B. napus F₁ hybrids and their parent species were estimated from experiments in both agricultural and semi-natural habitats. Altered fitness potential was modelled using periodic matrices including crop rotations and outcrossing between parent species.Key Results The demographic vital rates (i.e. for major stage transitions) of the hybrid population were intermediate between or lower than both parental species. The population growth rate (λ) of hybrids indicated decreases in both habitat types, and in a semi-natural habitat hybrids became extinct at two sites. Elasticity analyses indicated that seed bank survival was the greatest contributor to λ. In agricultural habitats, hybrid populations were projected to decline, but with persistence times up to 20 years. The seed bank survival rate was the main driver determining persistence. It was found that λ of the hybrids was largely determined by parental seed bank survival and subsequent replenishment of the hybrid population through outcrossing of B. rapa with B. napus.Conclusions Hybrid persistence was found to be highly dependent on the seed bank, suggesting that targeting hybrid seed survival could be an important management option in controlling hybrid persistence. For local risk mitigation, an increased focus on the wild parent is suggested. Management actions, such as control of B. rapa, could indirectly reduce hybrid populations by blocking hybrid replenishment.     

Impaired mitochondrial respiration promotes dendritic branching via the AMPK signaling pathway

Functional neuronal circuits require a constant remodeling of their network composed of highly interconnected neurons. The plasticity of synapses and the shaping of elaborated dendritic branches are energy demanding and therefore depend on an efficient mitochondrial oxidative phosphorylation (OXPHOS). The spatial and functional regulations of dendritic patterning occur also after cell fate specification; however, the molecular mechanisms underlying this complex process remain elusive. Here, we exploit the changes in dendritic architecture in highly branched neurons as a result of aberrant mitochondrial activity. In sensory neurons of Caenorhabditis elegans, genetic manipulations of mitochondrial complex I subunits cause an unexpected outgrowth of dendritic arbors and ectopic structures. The increased number of dendritic branches is coordinated through a specific signaling cascade rather than as a simple consequence of oxidative stress. On the basis of genetic and pharmacological evidence, we show that OXPHOS deficiency promotes branching through the activation of the AMP-activated protein kinase AMPK and the downstream target phosphoinositide 3-kinase PI3K. Taken together, our findings describe a well-defined signaling pathway that regulates dendritic outgrowth in conditions of compromised OXPHOS and the resulting AMPK activation.     

The control of mtDNA replication during differentiation and development

Background

Mitochondrial DNA (mtDNA) is important for energy production as it encodes some of the key genes of electron transfer chain, where the majority of cellular energy is generated through oxidative phosphorylation (OXPHOS). MtDNA replication is mediated by nuclear DNA-encoded proteins or enzymes, which translocate to the mitochondria, and is strictly regulated throughout development. It starts with approximately 200 copies in each primordial germ cell and these copies undergo expansion and restriction events at various stages of development.

Scope of review

I describe the patterns of mtDNA replication at key stages of development. I explain that it is essential to regulate mtDNA copy number and to establish the mtDNA set point in order that the mature, specialised cell acquires the appropriate numbers of mtDNA copy to generate sufficient adenosine triphosphate (ATP) through OXPHOS to undertake its specialised function. I discuss how these processes are dependent on the controlled expression of the nuclear-encoded mtDNA-specific replication factors and that this can be modulated by mtDNA haplotypes. I discuss how these events are altered by certain assisted reproductive technologies, some of which have been proposed to prevent the transmission of mutant mtDNA and others to overcome infertility. Furthermore, some of these technologies are predisposed to transmitting two or more populations of mtDNA, which can be extremely harmful.

Major conclusions

The failure to regulate mtDNA replication and mtDNA transmission during development is disadvantageous.

General significance

Manipulation of oocytes and embryos can lead to significant implications for the maternal-only transmission of mtDNA.This article is part of a Special Issue entitled Frontiers of mitochondrial research.     

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.     

Mitochondrial DNA mutations and breast tumorigenesis

Breast cancer is a heterogeneous disease and genetic factors play an important role in its genesis. Although mutations in tumor suppressors and oncogenes encoded by the nuclear genome are known to play a critical role in breast tumorigenesis, the contribution of the mitochondrial genome to this process is unclear. Like the nuclear genome, the mitochondrial genome also encodes proteins critical for mitochondrion functions such as oxidative phosphorylation (OXPHOS), which is known to be defective in cancer including breast cancer. Mitochondrial DNA (mtDNA) is more susceptible to mutations due to limited repair mechanisms compared to nuclear DNA (nDNA). Thus changes in mitochondrial genes could also contribute to the development of breast cancer. In this review we discuss mtDNA mutations that affect OXPHOS. Continuous acquisition of mtDNA mutations and selection of advantageous mutations ultimately leads to generation of cells that propagate uncontrollably to form tumors. Since irreversible damage to OXPHOS leads to a shift in energy metabolism towards enhanced aerobic glycolysis in most cancers, mutations in mtDNA represent an early event during breast tumorigenesis, and thus may serve as potential biomarkers for early detection and prognosis of breast cancer. Because mtDNA mutations lead to defective OXPHOS, development of agents that target OXPHOS will provide specificity for preventative and therapeutic agents against breast cancer with minimal toxicity.     

Mitochondrial genome changes and neurodegenerative diseases

Mitochondria are essential organelles within the cell where most of the energy production occurs by the oxidative phosphorylation system (OXPHOS). Critical components of the OXPHOS are encoded by the mitochondrial DNA (mtDNA) and therefore, mutations involving this genome can be deleterious to the cell. Post-mitotic tissues, such as muscle and brain, are most sensitive to mtDNA changes, due to their high energy requirements and non-proliferative status. It has been proposed that mtDNA biological features and location make it vulnerable to mutations, which accumulate over time. However, although the role of mtDNA damage has been conclusively connected to neuronal impairment in mitochondrial diseases, its role in age-related neurodegenerative diseases remains speculative. Here we review the pathophysiology of mtDNA mutations leading to neurodegeneration and discuss the insights obtained by studying mouse models of mtDNA dysfunction. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Interpopulation hybrid breakdown maps to the mitochondrial genome

Hybrid breakdown, or outbreeding depression, is the loss of fitness observed in crosses between genetically divergent populations. The role of maternally inherited mitochondrial genomes in hybrid breakdown has not been widely examined. Using laboratory crosses of the marine copepod Tigriopus californicus, we report that the low fitness of F(3) hybrids is completely restored in the offspring of maternal backcrosses, where parental mitochondrial and nuclear genomic combinations are reassembled. Paternal backcrosses, which result in mismatched mitochondrial and nuclear genomes, fail to restore hybrid fitness. These results suggest that fitness loss in T. californicus hybrids is completely attributable to nuclear-mitochondrial genomic interactions. Analyses of ATP synthetic capacity in isolated mitochondria from hybrid and backcross animals found that reduced ATP synthesis in hybrids was also largely restored in backcrosses, again with maternal backcrosses outperforming paternal backcrosses. The strong fitness consequences of nuclear-mitochondrial interactions have important, and often overlooked, implications for evolutionary and conservation biology.