Mitochondrial respiration controls neoangiogenesis during wound healing and tumour growth.

The vasculature represents a highly plastic compartment, capable of switching from a quiescent to an active proliferative state during angiogenesis. Metabolic reprogramming in endothelial cells (ECs) thereby is crucial to cover the increasing cellular energy demand under growth conditions. Here we assess the impact of mitochondrial bioenergetics on neovascularisation, by deleting cox10 gene encoding an assembly factor of cytochrome c oxidase (COX) specifically in mouse ECs, providing a model for vasculature-restricted respiratory deficiency. We show that EC-specific cox10 ablation results in deficient vascular development causing embryonic lethality. In adult mice induction of EC-specific cox10 gene deletion produces no overt phenotype. However, the angiogenic capacity of COX-deficient ECs is severely compromised under energetically demanding conditions, as revealed by significantly delayed wound-healing and impaired tumour growth. We provide genetic evidence for a requirement of mitochondrial respiration in vascular endothelial cells for neoangiogenesis during development, tissue repair and cancer.

DNA-editing enzymes as potential treatments for heteroplasmic mtDNA diseases.

Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and, in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild-type and mutant mtDNA molecules co-exist within the cell. Disease only manifests once the per cent of mutant mtDNA reaches a high threshold (usually >80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA-editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes and discusses roadblocks of applying these to gene therapy in humans.

Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease.

Mitochondrial diseases are frequently caused by heteroplasmic mitochondrial DNA (mtDNA) mutations. As these mutations express themselves only at high relative ratios, any approach able to manipulate mtDNA heteroplasmy can potentially be curative. In this study, we developed a system to manipulate mtDNA heteroplasmy in all skeletal muscles from neonate mice. We selected muscle because it is one of the most clinically affected tissues in mitochondrial disorders. A mitochondria-targeted restriction endonuclease (mito-ApaLI) expressed from AAV9 particles was delivered either by intraperitoneal or intravenous injection in neonate mice harboring two mtDNA haplotypes, only one of which was susceptible to ApaLI digestion. A single injection was able to elicit a predictable and marked change in mtDNA heteroplasmy in all striated muscles analyzed, including heart. No health problems or reduction in mtDNA levels were observed in treated mice, suggesting that this approach could have clinical applications for mitochondrial myopathies.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease.

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Pathophysiology and fate of hepatocytes in a mouse model of mitochondrial hepatopathies.

BACKGROUND: Although oxidative phosphorylation defects can affect the liver, these conditions are poorly understood, partially because of the lack of animal models. AIMS: To create and characterise the pathophysiology of mitochondrial hepatopathies in a mouse model. METHODS: A mouse model of mitochondrial hepatopathies was created by the conditional liver knockout (KO) of the COX10 gene, which is required for cytochrome c oxidase (COX) function. The onset and progression of biochemical, molecular and clinical phenotypes were analysed in several groups of animals, mostly at postnatal days 23, 56, 78 and 155. RESULTS: Biochemical and histochemical analysis of liver samples from 23-56-day-old KO mice showed liver dysfunction, a severe COX deficiency, marked mitochondrial proliferation and lipid accumulation. Despite these defects, the COX-deficient hepatocytes were not immediately eliminated, and apoptosis followed by liver regeneration could be observed only at age 78 days. Hepatocytes from 56-78-day-old KO mice survived despite very low COX activity but showed a progressive depletion of glycogen stores. In most animals, hepatocytes that escaped COX10 ablation were able to proliferate and completely regenerate the liver between days 78 and 155. CONCLUSIONS: The results showed that when faced with a severe oxidative phosphorylation defect, hepatocytes in vivo can rely on glycolysis/glycogenolysis for their bioenergetic needs for relatively long periods. Ultimately, defective hepatocytes undergo apoptosis and are replaced by COX-positive cells first observed in the perivascular regions.

Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a 'differential multiple cleavage-site' model.

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a 'differential multiple cleavage site' model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.

Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA.

BACKGROUND: The mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder of early childhood characterized by decreased mtDNA copy number in affected tissues. Recently, MDS has been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxy-guanosine kinase (dGK). Mutations in TK2 have been associated with the myopathic form of MDS, and mutations in dGK with the hepatoencephalopathic form. OBJECTIVES: To further characterize the frequency and clinical spectrum of these mutations, the authors screened 20 patients with myopathic MDS. RESULTS: No patient had dGK gene mutations, but four patients from two families had TK2 mutations. Two siblings were compound heterozygous for a previously reported H90N mutation and a novel T77M mutation. The other siblings harbored a homozygous I22M mutation, and one of them had evidence of lower motor neuron disease. The pathogenicity of these mutations was confirmed by reduced TK2 activity in muscle (28% to 37% of controls). CONCLUSIONS: These results show that the clinical expression of TK2 mutations is not limited to myopathy and that the myopathic form of MDS is genetically heterogeneous.

Reactive oxygen species and mitochondrial diseases.

A variety of diseases have been associated with excessive reactive oxygen species (ROS), which are produced mostly in the mitochondria as byproducts of normal cell respiration. The interrelationship between ROS and mitochondria suggests shared pathogenic mechanisms in mitochondrial and ROS-related diseases. Defects in oxidative phosphorylation can increase ROS production, whereas ROS-mediated damage to biomolecules can have direct effects on the components of the electron transport system. Here, we review the molecular mechanisms of ROS production and damage, as well as the existing evidence of mitochondrial ROS involvement in human diseases.

Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease.

Mutations in the mitochondrial DNA (mtDNA) can cause a variety of human diseases. In most cases, such mutations are heteroplasmic (i.e. mutated and wild-type mtDNA coexist) and a small percentage of wild-type sequences can have a strong protective effect against a metabolic defect. Because a genetic approach to correct mtDNA mutations is not currently available, the ability to modulate heteroplasmy would have a major impact in the phenotype of many patients with mitochondrial disorders. We show here that a restriction endonuclease targeted to mitochondria has this ability. A mitochondrially targeted PstI degraded mtDNA harboring PstI sites, in some cases leading to a complete loss of mitochondrial genomes. Recombination between DNA ends released by PstI was not observed. When expressed in a heteroplasmic rodent cell line, containing one mtDNA haplotype with two sites for PstI and another haplotype having none, the mitochondrial PstI caused a significant shift in heteroplasmy, with an accumulation of the mtDNA haplotype lacking PstI sites. These experiments provide proof of the principle that restriction endonucleases are feasible tools for genetic therapy of a sub-group of mitochondrial disorders. Although this approach is limited by the presence of mutation-specific restriction sites, patients with neuropathy, ataxia and retinitis pigmentosa (NARP) could benefit from it, as the T8399G mutation creates a unique restriction site that is not present in wild-type human mitochondrial DNA.

A helicase is born.

One of three loci previously associated with autosomal dominant progressive external ophthalmoplegia (adPEO) encodes ANT1, a mitochondrial nucleotide transporter. Now, mutations in two other genes are found in people with adPEO. One of these encodes a new helicase, Twinkle, which is related to the product of bacteriophage T7 gene 4, and co-localizes with mitochondrial DNA. The identification of Twinkle adds a new star to the expanding constellation of 'helicase diseases'.

What regulates mitochondrial DNA copy number in animal cells?

The study of the control of mitochondrial DNA copy number spans several decades and has identified many factors involved in the replication of the mitochondrial genome. However, the mechanisms involved in the regulation of this process are still obscure, particularly in animal cells. During the past decade, however, the identification of human diseases associated with drastically reduced levels of mtDNA caused renewed interest in this topic. Here, I will discuss recent work that sheds some light on how animal cells might maintain and control mtDNA levels.

Dysfunctional mitochondrial respiration in the wobbler mouse brain.

The involvement of mitochondrial dysfunction promoting neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), has been suggested. Histopathological and biochemical mitochondrial abnormalities have been reported in both sporadic and familial patients and suggest the contention that mitochondria may play a key role promoting ALS. Animal models of ALS provide a unique opportunity to study this incurable and fatal human disease. In the present study we tested the hypothesis that alterations in mitochondrial physiology occur in the brain of wobbler mice. No significant difference was found in the respiratory control index or adenosine diphosphate/oxygen ratio values between isolated mitochondria of wobbler and control mice. When pyruvate and malate were used as substrates, oxygen consumption was decreased significantly by approximately 33% in mitochondria isolated from wobbler mouse brain compared to controls. Oxygen consumption in the presence of ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was decreased significantly by approximately 21% in wobbler brain mitochondria compared to controls, which suggests impairment in the function of complex IV. These findings are the first demonstration of mitochondrial respiratory chain dysfunction in the brain of the wobbler mouse.

Rho(0) tumor cells: a model for studying whether mitochondria are targets for rhodamine 123, doxorubicin, and other drugs.

A human osteosarcoma cell line devoid of mitochondrial DNA (rho(0)) and its wild-type parental cell counterpart (wt) are presented as a model to investigate drug targeting. By virtue of the absence of mitochondrial DNA, rho(0) cells cannot perform electron transport or oxidative phosphorylation. Since most of the drugs studied are transported by the efflux pumping systems controlled by the MDR1 and MRP1 genes, both cell lines were examined for the expression of these genes, and it was found that no MDR1 and only low amounts of MRP1 were expressed. Growth inhibition experiments indicated that doxorubicin (Dox), vinblastine, and paclitaxel were equitoxic in these cell lines. On the other hand, the IC(50) for rhodamine 123 (Rho 123) in rho(0) cells was 50 times higher than in wt cells. This result correlates with a lower accumulation of Rho 123 in rho(0) cells as measured by fluorescence microscopy and flow cytometry (3 times less than in wt cells). In contrast, when stained with Dox, both cell types accumulated similar amounts. Surprisingly, in these non-P-glycoprotein expressing cells, verapamil increased both Dox and Rho 123 retention. Overall, these data suggest that: (i) functional mitochondria do not appear to be targets for the growth inhibitory activities of Dox, paclitaxel, or vinblastine; (ii) for lipophilic cations like Rho 123, however, normal functioning mitochondria and maintenance of a normal mitochondrial membrane potential (Deltapsi(mt)) appear to play a critical role in the intracellular accumulation and subsequent cytotoxicities of these compounds; and (iii) verapamil increases drug accumulation in non-P-glycoprotein expressing cell lines, most likely by direct action on Deltapsi(mt) for Rho 123 and safranin O, and on heretofore unidentified plasma membrane transporters, as well as via interaction with low levels of MRP1, for Dox. These results should be considered when Rho 123 and verapamil are used to detect P-glycoprotein.

An out-of-frame cytochrome b gene deletion from a patient with parkinsonism is associated with impaired complex III assembly and an increase in free radical production.

We have isolated transmitochondrial cybrids containing a mitochondrial DNA cytochrome b 4-base pair deletion previously identified in a patient with parkinsonism. This presentation is in contrast to that of most patients with cytochrome b mutations, who present with exercise intolerance. Clones containing different levels of the cytochrome b 4-base pair deletion showed that high levels of the mutation were associated with a respiratory deficiency and a specific complex III defect. Newly synthesized full-length cytochrome b was undetectable by metabolic labeling of mutant cells, and these cells were unable to grow in media that restricts proliferation of cells with defective oxidative phosphorylation. Steady state levels of some subunits previously found to be in close association with cytochrome b by crystallography and biochemical analysis (ie, Rieske [2Fe-2S] protein and subunit VI) were drastically reduced in clones containing high levels of the mutation, whereas the reduction in the core-1 subunit was milder. The absence of cytochrome b and complex III activity was also associated with increased hydrogen peroxide production. These findings, together with the variable tissue distribution of pathogenic mitochondrial DNA molecules, provide clues to the heterogeneous phenotypes associated with mitochondrial DNA mutations and establish a link between different forms of parkinsonism and oxidative phosphorylation defects.

Cytochrome c oxidase assembly in primates is sensitive to small evolutionary variations in amino acid sequence.

Respiring mitochondria require many interactions between nuclear and mitochondrial genomes. Although mitochondrial DNA (mtDNA) from the gorilla and the chimpanzee are able to restore oxidative phosphorylation in a human cell, mtDNAs from more distant primate species are functionally incompatible with human nuclear genes. Using microcell-mediated chromosome and mitochondria transfer, we introduced and maintained a functional orangutan mtDNA in a human nuclear background. However, partial oxidative phosphorylation function was restored only in the presence of most orangutan chromosomes, suggesting that human oxidative phosphorylation-related nuclear-coded genes are not able to replace many orangutan ones. The respiratory capacity of these hybrids was decreased by 65%-80%, and cytochrome c oxidase (COX) activity was decreased by 85%-95%. The function of other respiratory complexes was not significantly altered. The translation of mtDNA-coded COX subunits was normal, but their steady-state levels were approximately 10% of normal ones. Nuclear-coded COX subunits were loosely associated with mitochondrial membranes, a characteristic of COX assembly-defective mutants. Our results suggest that many human nuclear-coded genes not only cannot replace the orangutan counterparts, but also exert a specific interference at the level of COX assembly. This cellular model underscores the precision of COX assembly in mammals and sheds light on the nature of nuclear-mtDNA coevolutionary constraints.

A novel myopathy-associated mitochondrial DNA mutation altering the conserved size of the tRNA(Gln) anticodon loop.

We report a novel mitochondrial DNA alteration in a 12-year-old boy with myopathy. We identified a single nucleotide insertion (an adenine) in the mitochondrial tRNA-glutamine gene. This addition of an additional adenine in a polyadenine stretch (at mitochondrial DNA positions 4366-4369), alters the length of the evolutionary conserved anticodon loop from seven to eight bases. The nt-4370 addition was heteroplasmic and was abundant in the patient's muscle. Lower proportions of mutated mitochondrial DNA were observed in skin fibroblasts, but were below detectable levels in white blood cells. A muscle biopsy of the patient showed ragged red fibers and an unusually high percentage of cytochrome c oxidase-deficient fibers (89%). The pathogenicity of the mutation was also evident by the fact that fibers harboring lower levels of the mutation showed normal cytochrome c oxidase activity. The insertion in the anticodon loop of tRNA(Gln) gene identified in our patient may provide a unique tool to study protein synthesis in human mitochondria.

Functional constraints of nuclear-mitochondrial DNA interactions in xenomitochondrial rodent cell lines.

The co-evolution of nuclear and mitochondrial genomes in vertebrates led to more than 100 specific interactions that are crucial for an optimized ATP generation. These interactions have been examined by introducing rat mtDNA into mouse cells devoid of mitochondrial DNA (mtDNA). When mtDNA-less cells derived from the common mouse (Mus musculus domesticus) were fused to cytoplasts prepared from Mus musculus, Mus spretus, or rat (Rattus norvegicus), a comparable number of respiring clones could be obtained. Mouse xenomitochondrial cybrids harboring rat mtDNA had a slower growth rate in medium containing galactose as the carbon source, suggesting a defect in oxidative phosphorylation. These clones respired approximately 50% less than the parental mouse cells or xenomitochondrial cybrids harboring Mus spretus mtDNA. The activities of respiratory complexes I and IV were approximately 50% lower, but mitochondrial protein synthesis was unaffected. The defects in complexes I and IV were associated with decreased steady-state levels of respective subunits suggesting problems in assembly. We also showed that the presence of 10% mouse mtDNA co-existing with rat mtDNA was sufficient to restore respiration to normal levels. Our results suggest that evolutionary distance alone is not a precise predictor of nuclear-mitochondrial interactions as previously suggested for primates.

Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells.

We explored the role of low mitochondrial membrane potential (DeltaPsim) and the lack of oxidative phosphorylation in apoptosis by assessing the susceptibility of osteosarcoma cell lines with and without mitochondrial DNA to staurosporine-induced death. Our cells without mitochondrial DNA had low DeltaPsim and no functional oxidative phosphorylation. Contrary to our expectation, these cells were more resistant to staurosporine-induced death than were the parental cells. This reduced susceptibility was associated with decreased activation of caspase 3 but not with the mitochondrial permeability transition pore or cytochrome c release from the mitochondria. Apoptosis in both cell lines was associated with an increase in DeltaPsim. Bcl-x(L) could protect both cell types against caspase 3 activation and apoptosis by a mechanism that does not appear to be mediated by mitochondrial function or modulation of DeltaPsim. Nevertheless, we found that Bcl-x(L) expression can stimulate cell respiration in cells with mitochondrial DNA. Our results showed that the lack of functional oxidative phosphorylation and/or low mitochondrial membrane potential are associated with an antiapoptotic effect, possibly contributing to the development of some types of cancer. It also reinforces a model in which Bcl-x(L) can exert an antiapoptotic effect by stimulating oxidative phosphorylation and/or inhibiting caspase activation.

Mitochondrial function in heart muscle from patients with idiopathic dilated cardiomyopathy.

OBJECTIVE: To study the mitochondrial respiratory chain enzyme activities in patients with idiopathic dilated cardiomyopathy (IDC). METHODS: Mitochondrial respiratory chain enzyme activities were assessed spectrophotometrically in left ventricular tissue of 17 patients with IDC undergoing cardiac transplantation, as well as in two groups of controls: a group of six patients suffering from ischemic dilated cardiomyopathy (IC) also undergoing cardiac transplantation, and a group of 17 organ donors considered normal from a cardiac point of view. Cytochrome b gene from three IDC patients whose complex III activity was particularly low and from three controls was also sequenced. RESULTS: We found that complex III enzymatic activity was lower not only in IDC but also in IC patients when compared with normal controls. When analysing cytochrome b gene we only found neutral polymorphisms previously described. CONCLUSIONS: In view of such results, we believe that the decrease of respiratory chain complex III activity found in some cases of IDC is a secondary phenomenon, and not due to a primary mitochondrial disease.