Mitochondrial Translation and Beyond: Processes Implicated in Combined Oxidative Phosphorylation Deficiencies

Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multisubunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders

The antioxidant Trolox restores mitochondrial membrane potential and Ca2+-stimulated ATP production in human complex I deficiency

Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca2+ content of the endoplasmic reticulum (ERCa) and bradykinin(Bk)-induced increases in cytosolic and mitochondrial free Ca2+ ([Ca2+]C; [Ca2+]M) and ATP ([ATP]C; [ATP]M) concentration. Here, we determined the mitochondrial membrane potential (Δψ) in patient skin fibroblasts and show significant correlations with cellular ROS levels and ERCa, i.e., the less negative Δψ, the higher these levels and the lower ERCa. Treatment with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) normalized Δψ and Bk-induced increases in [Ca2+]M and [ATP]M. These effects were accompanied by an increase in ERCa and Bk-induced increase in [Ca2+]C. Together, these results provide evidence for an integral role of increased ROS levels in complex I deficiency and point to the potential therapeutic value of antioxidant treatment

Global export partner concentration since 1980: Trends in dependency and globalisation

The deficiency of porin isoform 1 (HVDAC1) in human skeletal muscle has been associated with a pathological phenotype related to defects in the bioenergetic metabolism. In the best studied case, porin deficiency was not apparent in cultured fibroblasts: this observation raised the conclusion that no molecular defect was in the cDNA sequence coding for the protein. To get more insight in the pathogenetic mechanism that is involved in porin isoform 1 deficiency, we have determined the whole structure of the corresponding human gene. On the basis of the corresponding mouse gene structure and the human cDNA sequence, we designed long extension PCR amplifications using the whole genomic DNA as a template. Exonic/intronic regions were isolated and the exons and surrounding introns sequenced. The 5' and 3' extremities of the gene were determined by genome walking. The porin isoform 1 human gene is made up of 9 exons and spans about 33 kbp. A whole panel of PCR parameters was set and is now ready to be used for specific amplification upon patients' genomic DNA. The analysis of the putative promoter sequence was performed. It revealed the presence of a sterol Repressor element (SRE), an SRY, the testis-determining factor, and a nuclear respiratory factor 2 (NRF-2) binding site. These sites, according to results from literature, could be involved in the functional modulation of the gene expression. (C) 2000 Academic Press

A Drosophila Mitochondrial Complex I Deficiency Phenotype Array

Mitochondrial diseases are a group of rare life-threatening diseases often caused by defects in the oxidative phosphorylation system. No effective treatment is available for these disorders. Therapeutic development is hampered by the high heterogeneity in genetic, biochemical, and clinical spectra of mitochondrial diseases and by limited preclinical resources to screen and identify effective treatment candidates. Alternative models of the pathology are essential to better understand mitochondrial diseases and to accelerate the development of new therapeutics. The fruit fly Drosophila melanogaster is a cost- and time-efficient model that can recapitulate a wide range of phenotypes observed in patients suffering from mitochondrial disorders. We targeted three important subunits of complex I of the mitochondrial oxidative phosphorylation system with the flexible UAS-Gal4 system and RNA interference (RNAi): NDUFS4 (ND-18), NDUFS7 (ND-20), and NDUFV1 (ND-51). Using two ubiquitous driver lines at two temperatures, we established a collection of phenotypes relevant to complex I deficiencies. Our data offer models and phenotypes with different levels of severity that can be used for future therapeutic screenings. These include qualitative phenotypes that are amenable to high-throughput drug screening and quantitative phenotypes that require more resources but are likely to have increased potential and sensitivity to show modulation by drug treatment

Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency

AbstractIsolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. We reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency. Using video-imaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS-sensitive dyes 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein and hydroethidine for a cohort of 10 patient cell lines. On the other hand, video-imaging microscopy of cells expressing reduction–oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts. Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY581/591 demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production

The Achilles heel of decision making system in termites

Mitochondrial beta-oxidation of long-chain fatty acids requires the concerted action of three tightly integrated membrane-bound enzymes (carnitine palmitoyltransferase I and II and carnitine/acylcarnitine translocase) that transport them into mitochondria. Neonatal onset of carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive, often lethal disorder of this transport. We describe a novel splice-site mutation in the CPT II gene, found in a Moroccan family, of which four out of five children have died from the neonatal form of CPT II deficiency. Mutation detection studies at the mRNA level in the CPT II gene implied that the affected children were homozygous for the previously reported 534T insertion followed by a 25-bp deletion (encompassing bases 534-558). Studies of genomic DNA, however, revealed all patients to be compound heterozygous for this 534T ins/del 25 mutation, and for a new g-->a splice-site mutation in the splice-acceptor site of intron 2. Because of these findings, prenatal diagnosis was performed in chorionic villi of three new pregnancies. This did not reveal new compound heterozygous genotypes, and, after uneventful pregnancies, all children appeared to be healthy. The new mutation is the first splice-site mutation ever identified in CPT II deficiency. The fact that it was not discovered in the patient's cDNA makes this study another example of the incompleteness of mutation detection at the mRNA level in cases where a mutation leads to aberrant splicing or nonsense-mediated messenger deca

Recovery, empowerment and rehabilitation: Do inpatient psychiatric rehabilitation services empower the individual?

Perceptions of the course and outcome from serious mental illness have changed over the last century and, more recently, the concept of recovery has gained prominence in this field. This paper reviews recent literature on recovery from serious mental illness and discusses both the meaning of the concept and the key contributing factors. Research suggests that empowerment is one of the most salient factors contributing to recovery and the relationship between recovery and empowerment is examined. Most research in the area of empowerment has, to date, focused on community settings and this paper considers the relevance of these ideas in other mental health settings. The relationship between empowerment, recovery and mental health services is discussed. Finally, conclusions are drawn and recommendations for further research are outlined

A urinary biosignature for mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS)

We used a comprehensive metabolomics approach to study the altered urinary metabolome of two mitochondrial myopathy, encephalopathy lactic acidosis and stroke like episodes (MELAS) cohorts carrying the m.3243A > G mutation. The first cohort were used in an exploratory phase, identifying 36 metabolites that were significantly perturbed by the disease. During the second phase, the 36 selected metabolites were able to separate a validation cohort of MELAS patients completely from their respective control group, suggesting usefulness of these 36 markers as a diagnostic set. Many of the 36 perturbed metabolites could be linked to an altered redox state, fatty acid catabolism and one-carbon metabolism. However, our evidence indicates that, of all the metabolic perturbations caused by MELAS, stalled fatty acid oxidation prevailed as being particularly disturbed. The strength of our study was the utilization of five different analytical platforms to generate the robust metabolomics data reported here. We show that urine may be a useful source for disease-specific metabolomics data, linking, amongst others, altered one-carbon metabolism to MELAS. The results reported here are important in our understanding of MELAS and might lead to better treatment options for the disease.Peer reviewe

The natural course of infantile Pompe's disease: 20 original cases compared with 133 cases from the literature

OBJECTIVE: Infantile Pompe's disease is a lethal cardiac and muscular disorder. Current developments toward enzyme replacement therapy are promising. The aim of our study is to delineate the natural course of the disease to verify endpoints of clinical studies. METHODS: A total of 20 infantile patients diagnosed by the collaborative Dutch centers and 133 cases reported in literature were included in the study. Information on clinical history, physical examination, and diagnostic parameters was collected. RESULTS: The course of Pompe's disease is essentially the same in the Dutch and the general patient population. Symptoms start at a median age of 1.6 months in both groups. The median age of death is 7.7 and 6 months, respectively. Five percent of the Dutch patients and 8% of all reported patients survive beyond 1 year of age. Only 2 patients from literature became older than 18 months. A progressive cardiac hypertrophy is characteristic for infantile Pompe's disease. The diastolic thickness of the left ventricular posterior wall and cardiac weight at autopsy increase significantly with age. Motor development is severely delayed and major d

NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4−/− mice and Leigh syndrome patients: A stabilizing role for NDUFAF2

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4−/− mouse tissues. Ndufs4−/− animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4−/− mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4−/− MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4−/− mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids