Mitochondrial Encephalomyopathies: Defects of Nuclear DNA

The term "mitochondrial diseases" encompasses a heterogeneous group of disorders in which a primary mitochondrial dysfunction is suspected or proven by morphologic, genetic, or biochemical criteria. Clinically, these progressive disorders usually affect muscle, either alone (mitochondrial myopathies) or in combination with other systems, most often brain (encephalomyopathies). Mitochondria are unique among intracellular organelles in that mitochondrial proteins are encoded by two genomes, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). The vast majority of mitochondrial proteins are encoded by the nuclear genome, whereas mtDNA (a circular, double stranded 16.5 kb molecule) encodes only 13 polypeptides, all of them subunits of respiratory chain complexes. In addition to structural genes, mtDNA also codes for 22 transfer RNAs and two ribosomal RNAs. Our understanding of mitochondrial diseases has grown at an impressive rate in the past few years, and most of the progress has been in the area of mtDNA genetics, where several mtDNA mutations have been associated with specific diseases (reviewed in this issue by Zeviani et al.). In comparison, our understanding of mitochondrial disorders due to nDNA lesions has lagged behind and, to date, molecular defects of nuclear genes have been documented in only a few patients. We will review which alterations in the nuclear genome can cause mitochondrial disorders and which criteria are useful in identifying such mutations. While several examples will be provided, this is not intended as a complete review of the subject.     

A collection of 33 novel human mtDNA homoplasmic variants

Mitochondria are involved in cellular energy production via oxidative phosphorylation and this function may be damaged by any mutation in mitochondrial DNA (mtDNA). To identify novel mtDNA mutations, we have developed a program to systematically screen the entire mitochondrial genome in a large number of individuals with clinical and/or morphological features of mitochondrial dysfunction, but still no genetic diagnosis. The sequence-data were obtained with an automated rapid system, which gave us a series of information: in the eleven mitochondrial genomes analyzed we observed the presence of 33 differences from the revised Cambridge Reference Sequence (Andrews et al., 1999), but they were all homoplasmic in the patients' tissues analyzed (skeletal muscle and blood), suggesting that they are unlikely to be primarily pathogenic though they may be co-responsible in the determination of the disease. This work can therefore help complete the already ample mtDNA polymorphism existent database.     

Mitochondrial DNA damage in a mouse model of Alzheimer's disease decreases amyloid beta plaque formation

Mitochondrial DNA (mtDNA) damage and the generation of reactive oxygen species have been associated with and implicated in the development and progression of Alzheimer's disease. To study how mtDNA damage affects reactive oxygen species and amyloid beta (Aβ) pathology in vivo, we generated an Alzheimer's disease mouse model expressing an inducible mitochondrial-targeted endonuclease (Mito-PstI) in the central nervous system. Mito-PstI cleaves mtDNA causing mostly an mtDNA depletion, which leads to a partial oxidative phosphorylation defect when expressed during a short period in adulthood. We found that a mild mitochondrial dysfunction in adult neurons did not exacerbate Aβ accumulation and decreased plaque pathology. Mito-PstI expression altered the cleavage pathway of amyloid precursor protein without increasing oxidative stress in the brain. These data suggest that mtDNA damage is not a primary cause of Aβ accumulation.     

The pathophysiology of mitochondrial disease as modeled in the mouse

It is now clear that mitochondrial defects are associated with a plethora of clinical phenotypes in man and mouse. This is the result of the mitochondria''s central role in energy production, reactive oxygen species (ROS) biology, and apoptosis, and because the mitochondrial genome consists of roughly 1500 genes distributed across the maternal mitochondrial DNA (mtDNA) and the Mendelian nuclear DNA (nDNA). While numerous pathogenic mutations in both mtDNA and nDNA mitochondrial genes have been identified in the past 21 years, the causal role of mitochondrial dysfunction in the common metabolic and degenerative diseases, cancer, and aging is still debated. However, the development of mice harboring mitochondrial gene mutations is permitting demonstration of the direct cause-and-effect relationship between mitochondrial dysfunction and disease. Mutations in nDNA-encoded mitochondrial genes involved in energy metabolism, antioxidant defenses, apoptosis via the mitochondrial permeability transition pore (mtPTP), mitochondrial fusion, and mtDNA biogenesis have already demonstrated the phenotypic importance of mitochondrial defects. These studies are being expanded by the recent development of procedures for introducing mtDNA mutations into the mouse. These studies are providing direct proof that mtDNA mutations are sufficient by themselves to generate major clinical phenotypes. As more different mtDNA types and mtDNA gene mutations are introduced into various mouse nDNA backgrounds, the potential functional role of mtDNA variation in permitting humans and mammals to adapt to different environments and in determining their predisposition to a wide array of diseases should be definitively demonstrated.     

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

Mitochondrial dysfunction has been implicated in a variety of human diseases. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases. Therefore, mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve such a strategy, it will be necessary to deliver therapeutic agents into mitochondria in living cells. We report here on an approach to accomplish this via the use of a Dual Function (DF)-MITO-Porter, aimed at the mitochondrial genome, so-called mitochondrial DNA (mtDNA). The DF-MITO-Porter, a nano carrier for mitochondrial delivery, has the ability to penetrate the endosomal and mitochondrial membranes via step-wise membrane fusion. We first constructed a DF-MITO-Porter encapsulating DNase I protein as a bioactive cargo. It was expected that mtDNA would be digested, when the DNase I was delivered to the mitochondria. We observed the intracellular trafficking of the carriers, and then measured mitochondrial activity and mtDNA-levels after the delivery of DNase I by the DF-MITO-Porter. The findings confirm that the DF-MITO-Porter effectively delivered the DNase I into the mitochondria, and provides a demonstration of its potential use in therapies that are selective for the mitochondrial genome.     

The A3243G tRNALeu(UUR) mutation induces mitochondrial dysfunction and variable disease expression without dominant negative acting translational defects in complex IV subunits at UUR codons

Mutations in the mitochondrial tRNA(Leu(UUR)) gene are associated with a large variety of human diseases through a largely undisclosed mechanism. The A3243G tRNA(Leu(UUR)) mutation leads to reduction of mitochondrial DNA (mtDNA)-encoded proteins and oxidative phosphorylation activity even when the cells are competent in mitochondrial translation. These two aspects led to the suggestion that a dominant negative factor may underlie the diversity of disease expression. Here we test the hypothesis that A3243G tRNA(Leu(UUR)) generates such a dominant negative gain-of-function defect through misincorporation of amino acids at UUR codons of mtDNA-encoded proteins. Using an anti-complex IV immunocapture technique and mass spectrometry, we show that the mtDNA-encoded cytochrome c oxidase I (COX I) and COX II exist exclusively with the correct amino acid sequences in A3243G cells in a misassembled complex IV. A dominant negative component therefore cannot account for disease phenotype, leaving tissue-specific accumulation by mtDNA segregation as the most likely cause of variable mitochondrial disease expression.     

线粒体病的基因治疗

线粒体病是一组因线粒体DNA缺失或突变而致氧化磷酸化及能量供应异常的疾病,目前该类疾病的治疗主要是支持治疗。然而由于线粒体结构和功能的特殊性,该疗效并不确切。因此,线粒体病的基因治疗显得越来越迫切和重要。目前,基因治疗的策略包括降低突变型mtDNA/野生型mtDNA的比例、错位表达、输入其他同源性基因以及利用限制性内切酶修复突变型mtDNA等。     

Induction of an unregulated channel by mutations in adenine nucleotide translocase suggests an explanation for human ophthalmoplegia

Adenine nucleotide translocase (Ant) is primarily involved in ATP/ADP exchange across the mitochondrial inner membrane. Recently, the A114P missense mutation in the human Ant1 protein was found to be associated with autosomal dominant progressive external ophthalmoplegia (adPEO). Ant1(A114P) was proposed to cause an imbalance of the mitochondrial deoxynucleotide pool that subsequently affects the accuracy of mtDNA replication, thereby leading to accumulation of mutant mtDNA. In the present study, it has been shown that the A128P mutation of the Saccharomyces cerevisiae Aac2 protein, equivalent to A114P in human Ant1p, does not always affect respiratory growth. However, expression of aac2(A128P) results in depolarization, structural swelling and disintegration of mitochondria, and ultimately an arrest of cell growth in a dominant-negative manner. The aac2(A128P) mutation likely induces an unregulated channel allowing free passage of solutes across the inner membrane. These data raise the possibility that the formation of an unregulated channel, rather than a defect in ATP/ADP exchange, is a direct pathogenic factor in human adPEO. The accumulation of mtDNA mutations might be a consequence of mitochondrial dysfunction.     

The pathophysiology of mitochondrial biogenesis: towards four decades of mitochondrial DNA research

Mitochondria are with very few exceptions ubiquitous organelles in eukaryotic cells where they are essential for cell life and death. Mitochondria play a central role not only in a variety of metabolic pathways including the supply of the bulk of cellular ATP through oxidative phosphorylation (OXPHOS), but also in complex processes such as development, apoptosis, and aging. Mitochondria contain their own genome that is replicated and expressed within the organelle. It encodes 13 polypeptides all of them components of the OXPHOS system, and thus, the integrity of the mitochondrial DNA (mtDNA) is critical for cellular energy supply. In the past 12 years more than 50 point mutations and around 100 rearrangements in the mtDNA have been associated with human diseases. Also in recent years, several mutations in nuclear genes that encode structural or regulatory factors of the OXPHOS system or the mtDNA metabolism have been described. The development of increasingly powerful techniques and the use of cellular and animal models are opening new avenues in the study of mitochondrial medicine. The detailed molecular characterization of the effects produced by different mutations that cause mitochondrial cytopathies will be critical for designing rational therapeutic strategies for this group of devastating diseases.     

Somatic mitochondrial DNA mutations in cortex and substantia nigra in aging and Parkinson's disease

Oxidative damage to mitochondrial DNA (mtDNA) increases with age in the brain and can induce G:C to T:A and T:A to G:C point mutations. Though rare at any particular site, multiple somatic mtDNA mutations induced by oxidative damage or by other mechanisms may accumulate with age in the brain and thus could play a role in aging and neurodegenerative diseases. However, no prior study has quantified the total burden of mtDNA point mutation subtypes in the brain. Using a highly sensitive cloning and sequencing strategy, we find that the aggregate levels of G:C to T:A and T:A to G:C transversions and of all point mutations increase with age in the frontal cortex (FCtx). In the substantia nigra (SN), the aggregate levels of point mutations in young controls are similar to the levels in the SN or FCtx of elderly subjects. Extrapolation from our data suggests an average of 2.7 (FCtx) to 3.2 (SN) somatic point mutations per mitochondrial genome in elderly subjects. There were no significant differences between Parkinson's disease (PD) patients and age-matched controls in somatic mutation levels. These results indicate that individually rare mtDNA point mutations reach a high aggregate burden in FCtx and SN of elderly subjects.     

Approaches to mitochondrial gene therapy

Since their discovery during the end of the 80's the number of diseases found to be associated with defects in the mitochondrial genome has grown significantly. Organs affected by mutations in mitochondrial DNA (mtDNA) include in decreasing order of vulnerability the brain, skeletal muscle, heart, kidney and liver. Hence neuromuscular and neurodegenerative diseases represent the two largest groups of mtDNA diseases. Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is however no satisfactory treatment available to the vast majority of patients. This is largely due to the fact that most of these patients have respiratory chain defects, i.e. defects that involve the final common pathway of oxidative metabolism, making it impossible to bypass the defect by administering alternative metabolic carriers of energy. Conventional biochemical treatment having reached an impasse, the exploration of gene therapeutic approaches for patients with mtDNA defects is warranted. For now mitochondrial gene therapy appears to be only theoretical and speculative. Any possibility for gene replacement is dependent on the development of an efficient mitochondrial transfection vector. In this review we describe the current state of the development of mitochondria-specific DNA delivery systems. We summarize our own efforts in exploring the properties of dequalinium and other similar cationic bolaamphiphiles with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells. Further, we outline some unique hurdles that need to be overcome if the development of such delivery systems is to progress.     

Genetic control of oxidative phosphorylation and experimental models of defects

Energy in the form of ATP is continually produced by all cells for normal growth and function. Anaerobic glycolysis can provide enough ATP for some cells, but energetic cells such as cardiomyocytes and neurons require a more efficient ATP supply, which can only be provided by mitochondrial oxidative phosphorylation. Invented by bacteria that became symbiotically associated with other bacteria to form eukaryotic cells billions of years ago, oxidative phosphorylation carries with it a genetic legacy that is unique. The mitochondrial oxidative phosphorylation complexes are assembled from protein subunits encoded by both the mitochondrial genome (mtDNA) and the nuclear genome (nDNA, located in the chromosomes). The mtDNA is a remnant genome of the bacterial progenitor of mitochondria, and (unlike the biparental diploidy that characterizes the nuclear genome) is present in thousands of copies per cell, is replicated through life, and is inherited (cytoplasmically) only from the female parent. Oxidative phosphorylation comprises five multimeric enzyme complexes that act as a redox pathway, passing electrons from oxidizable intermediates produced by the metabolism of food to molecular oxygen in the mitochondrial matrix, while producing an electrochemical gradient by pumping protons into the intermembranal space. The proton (hydrogen ion) gradient across the inner mitochondrial membrane is used by the H+-transporting ATP synthase to produce ATP from ADP and inorganic phosphate, with the protons released into the mitochondrial matrix then combining with electronated oxygen to form water. Many of the details regarding the control of the synthesis of oxidative phosphorylation enzyme complexes remain to be elucidated. Transmitochondrial cell culture systems have been developed so that defective oxidative phosphorylation can be studied in a controlled nuclear background. Such systems may soon enable the development of mtDNA 'knockout' mice in order to better model mtDNA transmission and mitochondrial disease.     

The A8296G mtDNA mutation associated with several mitochondrial diseases does not cause mitochondrial dysfunction in cybrid cell lines

Transmitochondrial cybrid cell lines homoplasmic for the A8296G mtDNA transition, a mutation associated with several mitochondrial diseases, have a normal oxidative phosphorylation function, as shown by oxygen consumption, lactate production, respiratory enzyme activities, and growth using galactose as the only source of energy. The synthesis of mitochondrial proteins is also similar in mutant and wild-type cybrids. Our results suggest that the A8296G mutation is a polymorphism and reinforce the necessity of performing functional studies to assess the pathogenicity of mtDNA mutations.     

OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution

Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.     

Mitochondrial and nuclear DNA defects in Saccharomyces cerevisiae with mutations in DNA polymerase gamma associated with progressive external ophthalmoplegia

A number of nuclear mutations have been identified in a variety of mitochondrial diseases including progressive external ophthalmoplegia (PEO), Alpers syndrome and other neuromuscular and oxidative phosphorylation defects. More than 50 mutations have been identified in POLG, which encodes the human mitochondrial DNA (mtDNA) polymerase gamma, PEO and Alpers patients. To rapidly characterize the effects of these mutations, we have developed a versatile system that enables the consequences of homologous mutations, introduced in situ into the yeast mtDNA polymerase gene MIP1, to be evaluated in vivo in haploid and diploid cells. Overall, distinct phenotypes for expression of each of the mip1-PEO mutations were observed, including respiration-defective cells with decreased viability, dominant-negative mutant polymerases, elevated levels of mitochondrial and nuclear DNA damage and chromosomal mutations. Mutations in the polymerase domain caused the most severe phenotype accompanied by loss of mtDNA and cell viability, whereas the mutation in the exonuclease domain showed mild dominance with loss of mtDNA. Interestingly, the linker region mutation caused elevated mitochondrial and nuclear DNA damage. The cellular processes contributing to these observations in the mutant yeast cells are potentially relevant to understanding the pathologies observed in human mitochondrial disease patients.     

Infantile-onset spinocerebellar ataxia and mitochondrial recessive ataxia syndrome are associated with neuronal complex I defect and mtDNA depletion

Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.     

Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids

Mitochondrial DNA (mtDNA) mutations cause heterogeneous disorders in humans. MtDNA exists in multiple copies per cell, and mutations need to accumulate beyond a critical threshold to cause disease, because coexisting wild-type mtDNA can complement the genetic defect. A better understanding of the molecular determinants of functional complementation among mtDNA molecules could help us shedding some light on the mechanisms modulating the phenotypic expression of mtDNA mutations in mitochondrial diseases. We studied mtDNA complementation in human cells by fusing two cell lines, one containing a homoplasmic mutation in a subunit of respiratory chain complex IV, COX I, and the other a distinct homoplasmic mutation in a subunit of complex III, cytochrome b. Upon cell fusion, respiration is recovered in hybrids cells, indicating that mitochondria fuse and exchange genetic and protein materials. Mitochondrial functional complementation occurs frequently, but with variable efficiency. We have investigated by native gel electrophoresis the molecular organization of the mitochondrial respiratory chain in complementing hybrid cells. We show that the recovery of mitochondrial respiration correlates with the presence of supramolecular structures (supercomplexes) containing complexes I, III and IV. We suggest that critical amounts of complexes III or IV are required in order for supercomplexes to form and provide mitochondrial functional complementation. From these findings, supercomplex assembly emerges as a necessary step for respiration, and its defect sets the threshold for respiratory impairment in mtDNA mutant cells.