Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis.

Diversity of respiratory chain spectrophotometric assays may lead to difficult comparison of results between centers. The French network of mitochondrial diseases diagnostic centers undertook comparison of the results obtained with different protocols in the French diagnostic centers. The diversity of protocols was shown to have striking consequences, which prompted the network to undertake standardization and optimization of the protocols with respect to clinical diagnosis, i.e. high velocity while maintaining linear kinetics relative to time and enzyme concentration. Assays were set up on animal tissues and verified on control human muscle and fibroblasts. Influence of homogenization buffer and narrow range of optimal concentration of phosphate, substrate and tissue were shown. Experimental data and proposed protocols have been posted on a free access website. Their subsequent use in several diagnostic centers has improved consistency for all assays.

Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts.

Mitochondria are dynamic organelles with continuous fusion and fission, the equilibrium of which results in mitochondrial morphology. Evidence points to there being an intricate relationship between mitochondrial dynamics and oxidative phosphorylation. We investigated the bioenergetics modulation of mitochondrial morphology in five control cultured primary skin fibroblasts and seven with genetic alterations of oxidative phosphorylation. Under basal conditions, control fibroblasts had essentially filamentous mitochondria. Oxidative phosphorylation inhibition with drugs targeting complex I, III, IV or V induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (D Psi m) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by D Psi m but loosely linked to mitochondrial oxidative phosphorylation. Its alteration by glycolysis inhibition points to a severe oxidative phosphorylation defect.

Impact on oxidative phosphorylation of immortalization with the telomerase gene.

Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells' dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.

Functional characterization of novel mutations in the human cytochrome b gene.

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Late-onset mitochondrial DNA depletion: DNA copy number, multiple deletions, and compensation.

Through a report of 4 late-onset cases with mitochondrial DNA (mtDNA) depletion, we address the specificity of the clinical entities associated with a very low residual amount of mtDNA. Three of the patients met the diagnostic criteria of Kearns Sayre syndrome, which has never been associated with mtDNA depletion. The fourth patient had an isolated skeletal myopathy. Deleted mtDNA molecules were found by long-range polymerase chain reaction (PCR) only in the Kearns Sayre syndromes, which strengthens the clinical differences between the two types of patients. All patients had extremely low residual amounts of mtDNA as shown by Southern blot analysis. Using an original method based on competitive PCR, we were able to measure the number of mtDNA copies per diploid genome. These results demonstrated the severity of the depletion in the patients by comparison not only to normal controls but also to patients with mtDNA disorders. Despite the severity of the depletion, in situ hybridization using two mtDNA transcripts revealed a normal steady-state level of transcription. Such compensation provides clues to the striking contrast between the severity of mtDNA depletion and the late onset and slowly progressive disease.

Functional mitochondrial heterogeneity in heteroplasmic cells carrying the mitochondrial DNA mutation associated with the MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes).

Most mitochondrial DNA (mtDNA) alterations associated with human disorders are heteroplasmic, i.e. mutant mtDNA molecules coexist with normal ones within the cell. We addressed the possibility of intermitochondrial exchanges through histologic analyses of cybrid clones with increasing proportion of the MELAS (A3243G) mtDNA transfer RNA point mutation. MtDNA-dependent cytochrome c oxidase activity and protein composition as well as mitochondrial membrane potential appeared heterogeneous in individual cells from clonal heteroplasmic cell populations on the basis of confocal and electron microscopy. The number of defective cells increased with increasing mutation load. We conclude that in the presence of a heteroplasmic mtDNA mutation in the cell type that we studied, intermitochondrial molecular exchanges cannot provide an efficient even distribution of the complementing molecules such as wild-type mtDNA, transfer RNA, or protein. Mitochondria in these heteroplasmic cells cannot, therefore, be considered a single functional unit.

["MELAS" (A3243G) mutation of mitochondrial DNA: a study of the relationships between the clinical phenotype in 19 patients and morphological and molecular data]. / Mutation <> (A3243G) de l'ADN mitochondrial: étude chez 19 patients des relations entre le phénotype clinique, les données morphologiques et moléculaires.

Nineteen patients were found to harbor the mitochondrial DNA A3243G mutation associated with MELAS syndrome (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes). Eight of them had presented with stroke-like episodes and therefore had a clinical diagnosis of MELAS syndrome. The other 11 patients had no strokes and presented with generally less severe multisystemic disease. In the two groups, we compared muscle morphology, biochemical activities of muscle respiratory chain, and genetic characteristics: proportion and tissue distribution of the mutation, sequence of the 22 transfer RNA genes of the mitochondrial DNA. The proportion of mutant mtDNA in muscle was always greater than in blood. The number of patients in the two groups was too low to reach significant values. However, the patients with a MELAS syndrome presented with more severe respiratory chain abnormalities and with a proportion of the A3243G mutation that was both higher and more uniformly distributed among tissues. For symptoms others than stroke-like episodes, we did not observe any correlation with the level of mutant mtDNA in muscle. The analysis of the 22 tRNA sequences did not show differences between the two groups, and no co-inherited modifying tRNA genes could explain the variability of severity in our patients.

The first pathogenic mitochondrial methionine tRNA point mutation is discovered in splenic lymphoma.

We describe a novel point mutation in the mitochondrial DNA transfer RNA methionine gene, a G-to-A transition at position 4450, in a patient with a splenic lymphoma with villous lymphocytes. The patient's lymphocytes were remarkable by the presence of large cytoplasmic inclusions demonstrated as abnormal mitochondria by electron microscopy and led to the discovery of the mutation using denaturing gradient gel electrophoresis as a screening procedure. The pathogenic potential of the mutation was clearly established by the following criteria. It was absent in a control population. It involves a nucleotide that is highly conserved along the phylogenetic tree. The mutation was heteroplasmic and, when present in a high proportion, was associated with morphological alterations of the mitochondria, with defects of respiratory chain complexes activities and with a decrease in the mitochondrially encoded cytochrome c oxidase subunit II. Transfer of the mutation in Rho0 cells allowed to demonstrate its association with a severe respiratory chain dysfunction. However, although the pathogenicity of the mutation was clearly demonstrated, its link with the patient disease remained disputable.

[Progressive external ophthalmoplegia of mitochondrial origin: contribution of morphological and molecular studies]. / Ophtalmoplégies externes progressives d'origine mitochondriale: apport des investigations morphologiques et moléculaires.

We performed a comparative analysis of the clinical, morphological and molecular characteristics of 62 patients affected with progressive external ophthalmoplegia with ragged-red fibres in muscle. Twenty-seven patients had only muscular disease, and 35 had a multisystemic disease with neurological, cardiac, sensory, or endocrine symptoms. Quantitation of mitochondrial accumulation and numbering of cytochrome c oxidase deficient muscle fibres were done in 43 patients. Muscle mitochondrial DNA deletions were detected, quantitated and localised by Southern Blot analysis. Point mutations were screened in five mitochondrial DNA transfer RNA genes by denaturing gradient gel electrophoresis technique. This study further emphasized the relationships between progressive external ophthalmoplegia and mitochondrial DNA mutations that were present in 46/62 patients (40 deletions, 4 h point mutations in the tRNA leucine gene and 2 further families with maternal inheritance but no mutation identified to-date). Family history was positive in 12 patients: 4 with a maternally inherited disease (2 of whom had an identified mitochondrial DNA mutation), and 4 with an autosomal dominant inherited disease, none of which was associated with multiple mitochondrial DNA deletions. Interestingly, 2 of our patients with an identified mitochondrial DNA mutation appeared as sporadic cases. No morphological or molecular parameters was correlated with the tissular extension of the disease. However, mitochondrial DNA deletions were significantly associated with ocular symptoms which had an earlier onset and were more severe. Clinical features of the patients with a multisystemic disease and a mitochondrial DNA deletion were essentially related to Kearns-Sayre syndrome. In particular, a cardiac conduction defect was present in 12 patients out of 18 with a multisystemic disease associated with a mitochondrial DNA deletion; it was never encountered in 17 patients with a multisystemic disease but no mitochondrial DNA deletion.

In vivo functional investigations of lactic acid in patients with respiratory chain disorders.

OBJECTIVE: To assess the prevalence of in vivo detectable abnormalities of lactate metabolism in mitochondrial disorders. DESIGN: Retrospective study in a metabolic investigation unit. PATIENTS: 28 patients with a respiratory chain disorder identified from biochemical or genetic analyses, or both, and 133 age matched controls. Controls were children in whom causes of secondary hyperlactataemia and/or disorders, affecting the energy pathways could be excluded. METHODS: Lactate and pyruvate were measured in blood, together with other intermediary metabolism indices, before and one hour after four meals each day. Lactate and creatinine in a 24 hour urine sample collected at the same time were analysed. When basal hyperlactataemia was not evident, an intravenous glucose or pyruvate loading test was performed as a provocative test. RESULTS: Abnormal lactate metabolism was found in 25 of 28 patients thus demonstrating the potential usefulness of these investigations in the diagnosis of mitochondrial disease. Moderate lactate accumulation was present in relatively mild disease, associated with a mitochondrial DNA mutation and combined respiratory complexes deficiency. By contrast, high lactate concentrations were observed in very young children, with severe disease, isolated complex deficiency, and no apparent mitochondrial DNA defect.

Clinical and molecular heterogeneity of cytochrome c oxidase deficiency in the newborn.

We report 8 cases of severe cytochrome c oxidase deficiency with onset in the neonatal period. Clinical symptoms were heterogeneous: antenatal cerebral malformations, neurological distress with ketoacidosis, severe myopathy, or isolated respiratory control failure. Lactic acid was elevated in blood and/or CSF in 7 cases. Muscle biopsy (7 patients), liver biopsy (4 patients), and cultured skin fibroblasts (7 patients) were used to assess the cytochrome c oxidase deficiency. Among the patients, the enzymatic defect differed in the level of residual activity, expression in different tissues and subunit composition in muscle (as analysed by immunohistochemistry). Southern blot analysis of the mitochondrial DNA was normal in 7 patients. The heterogeneity of cytochrome c oxidase deficiency was therefore demonstrated by these clinical presentations and by the biochemical assessment of the enzyme defect. This reflects, most probably, the diverse nature of the causal mutations.

Morphological studies of skeletal muscle in lactic acidosis.

This paper underscores the contribution of routine morphological examination of skeletal muscle in patients with lactic acidosis. Mitochondrial disorders are by far the most common causes of primary lactic acidosis, in which muscle biopsy analysis helps in diagnosis and in the search for the molecular anomalies. Thus, we focus our attention on one particular point: the contribution of the morphological study of muscle biopsy in primary lactic acidosis due to mitochondrial disorders, especially mitochondrial respiratory-chain diseases.

Chronic progressive external ophthalmoplegia with ragged-red fibers: clinical, morphological and genetic investigations in 43 patients.

The evaluation of the severity of progressive external ophthalmoplegia (PEO) with ragged-red fibers in muscle, at the onset of the disease, when PEO is most often the only presenting symptom, is a difficult problem in neurological practice. In order to address that issue, we have performed a comparative analysis of the clinical, morphological and molecular characteristics of 43 patients affected with that form of ocular myopathy. Quantification of mitochondrial accumulation was performed with an image analysis application on muscle sections stained with succinate dehydrogenase histochemical reaction. The proportion of muscle fibres appearing as cytochrome c oxidase deficient was used as an index of the muscle-energy defect. Muscle mitochondrial DNA deletions were detected, localized and quantitated by Southern blot analysis. Point mutations were screened in five transfer RNA genes in the mtDNA (tRNA(Leucine (UUR)), tRNA(Lysine), tRNA(Glutamine), tRNA(Isoleucine) and tRNA(Formylmethionine)) by a denaturing gradient gel electrophoresis technique. This investigation confirmed the high frequency of mtDNA deletions or point mutations in PEO. At the onset of the disease, no clinical, morphological or molecular features could predict whether PEO would remain isolated or become part of a more severe multisystem disease. However, patients with mtDNA deletions were characterized by more severe ophthalmoplegia of earlier onset. Their muscle alterations were roughly parallel in severity to the proportion of deleted mtDNA molecules in muscle. Patients with a multitissular disease and mtDNA deletions were always sporadic cases and their clinical presentation was, most often, closely related to Kearns Sayre syndrome.

Immunization with a peptide having both T cell and conformationally restricted B cell epitopes elicits neutralizing antisera against a snake neurotoxin.

We have synthesized a free peptide capable of eliciting antibodies that neutralize toxin alpha from Naja nigricollis, a protein that binds specifically to the acetylcholine nicotinic receptor. Of the five tested fragments that encompassed the whole toxin sequence, only fragment 24-41 stimulated T cells from BALB/c mice primed with the whole toxin and conversely, only T cells from mice primed with fragment 24-41 could be stimulated by both the toxin and priming peptide. No other peptides had such properties, indicating that only fragment 24-41 possessed T determinant(s) in BALB/c mice (H-2d haplotype). In agreement with the current view that B cell proliferation requires specific T cell stimulation, only fragment 24-41 elicited an antibody response. However, the antipeptide antisera failed to bind to the native toxin and thereby to neutralize it. Instead, it recognized an unfolded form of the toxin. The peptide 24-41 was then made cyclic. A circular dichroism analysis revealed that, in organic solvent, this peptide had a tendency to adopt a beta-sheet structure, as in the folded toxin, whereas the linear peptide adopted an helical structure. The cyclic peptide not only remained T stimulating but elicited antisera that recognized and neutralized the native toxin. Furthermore, the antisera cross-reacted with several toxin variants. Our data show, therefore, that it is possible to give an appropriate B cell specificity directly to a T cell-stimulating peptide, an approach that may be of value for the design of synthetic vaccines.

Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus.

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A2 (PLA2) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti-notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA2 or PLA2-containing venoms from other origins. Substitutions or chemical modifications occurring in the C-terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.

Evidence that the anti-coagulant and lethal properties of a basic phospholipase A2 from snake venom are unrelated.

The basic phospholipase A2 (PLA2) from venom of the African elapid Naja nigricollis was previously shown to have anti-coagulant and lethal properties, both of which were abolished by treatment with p-bromophenacyl bromide (pBP). In the present paper we first report that pBP-treated PLA2 is capable of inhibiting the anti-coagulant activity but not the lethal activity of native PLA2, thus suggesting that both properties might be independent. We then confirm this evidence using PLA2-specific monoclonal immunoglobulins. One of these, called HSF, neutralized the lethal activity but not the anti-coagulant activity, whereas another antibody, called HSP2, inhibited the anti-coagulant activity but not the lethal activity of the PLA2. The data presented in this paper are taken as evidence that the anti-coagulant activity is not implicated in the lethal effects of basic PLA2 from Naja nigricollis.

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration.

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.

Calcium uptake by cholinergic synaptic vesicles.

Pure synaptic vesicles have been isolated in sucrose-KCl media. They are able to take up calcium in the presence of ATP and Mg. This is based on the following evidence. First, the synaptic vesicle fraction is the gradient peak for calcium uptake. Second, it was not possible to separate ACh and ATP from the uptake peak after refractionation of synaptic vesicles. Third, the fraction appears very pure on morphological and biochemical grounds. The physiological significance of the calcium uptake by synaptic vesicles is discussed.

ATP-dependent calcium uptake by cholinergic synaptic vesicles isolated from Torpedo electric organ.

Cholinergic synaptic vesicles were purified from Torpedo electric organ to near morphological homogeneity. They were isolated in a K+ environment. A method is described for the preparation of concentrated synaptic vesicles that allows uptake studies by conventional techniques. An ATP-Mg-dependent calcium uptake associated with synaptic vesicles is characterized. The uptake system transports calcium against a high concentration gradient. The maximum accumulation rate is obtained for the calcium, Mg++ and ATP concentrations likely to be found in the nerve terminal cytoplasm. It is suggested that synaptic vesicles are implicated in the removal of the calcium entering the nerve terminal during synaptic activity.