miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

BH3 mimetics activate multiple pro-autophagic pathways.

The BH3 mimetic ABT737 induces autophagy by competitively disrupting the inhibitory interaction between the BH3 domain of Beclin 1 and the anti-apoptotic proteins Bcl-2 and Bcl-X(L), thereby stimulating the Beclin 1-dependent allosteric activation of the pro-autophagic lipid kinase VPS34. Here, we examined whether ABT737 stimulates other pro-autophagic signal-transduction pathways. ABT737 caused the activating phosphorylation of AMP-dependent kinase (AMPK) and of the AMPK substrate acetyl CoA carboxylase, the activating phosphorylation of several subunits of the inhibitor of NF-κB (IκB) kinase (IKK) and the hyperphosphorylation of the IKK substrate IκB, inhibition of the activity of mammalian target of rapamycin (mTOR) and consequent dephosphorylation of the mTOR substrate S6 kinase. In addition, ABT737 treatment dephosphorylates (and hence likewise inhibits) p53, glycogen synthase kinase-3 and Akt. All these effects were shared by ABT737 and another structurally unrelated BH3 mimetic, HA14-1. Functional experiments revealed that pharmacological or genetic inhibition of IKK, Sirtuin and the p53-depleting ubiquitin ligase MDM2 prevented ABT737-induced autophagy. These results point to unexpected and pleiotropic pro-autophagic effects of BH3 mimetics involving the modulation of multiple signalling pathways.

A brain-specific isoform of mitochondrial apoptosis-inducing factor: AIF2.

Apoptosis-inducing factor (AIF) has important supportive as well as potentially lethal roles in neurons. Under normal physiological conditions, AIF is a vital redox-active mitochondrial enzyme, whereas in pathological situations, it translocates from mitochondria to the nuclei of injured neurons and mediates apoptotic chromatin condensation and cell death. In this study, we reveal the existence of a brain-specific isoform of AIF, AIF2, whose expression increases as neuronal precursor cells differentiate. AIF2 arises from the utilization of the alternative exon 2b, yet uses the same remaining 15 exons as the ubiquitous AIF1 isoform. AIF1 and AIF2 are similarly imported to mitochondria in which they anchor to the inner membrane facing the intermembrane space. However, the mitochondrial inner membrane sorting signal encoded in the exon 2b of AIF2 is more hydrophobic than that of AIF1, indicating a stronger membrane anchorage of AIF2 than AIF1. AIF2 is more difficult to be desorbed from mitochondria than AIF1 on exposure to non-ionic detergents or basic pH. Furthermore, AIF2 dimerizes with AIF1, thereby preventing its release from mitochondria. Conversely, it is conceivable that a neuron-specific AIF isoform, AIF2, may have been 'designed' to be retained in mitochondria and to minimize its potential neurotoxic activity.

Mitochondria and diabetes mellitus: untangling a conflictive relationship?

Diabetes mellitus is occasionally observed in patients with skeletal muscle respiratory chain deficiency, suggesting that skeletal muscle mitochondrial dysfunction might play a pathogenic role in type 2 diabetes (T2D). In support of this hypothesis, decreased muscle mitochondrial activity has been reported in T2D patients and in mouse models of diabetes. However, recent work by several groups suggests that decreased muscle mitochondrial function may be a consequence rather than a cause of diabetes, since decreased mitochondrial function in mice affords protection from diabetes and obesity. We review the data on this controversial but important issue of potential links between mitochondrial dysfunction and diabetes.

The molecular archaeology of a mitochondrial death effector: AIF in Drosophila.

Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (DeltaN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. DeltaN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that DeltaN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.

Succinate dehydrogenase deficiency in human.

Mitochondrial succinate dehydrogenase (SDH) consists merely of four nuclearly encoded subunits. It participates in the electron transfer in the respiratory chain and in succinate catabolism in the Krebs cycle. Mutations in the four genes, SDHA, B, C and D, have been reported, resulting in strikingly diverse clinical presentations. So far, SDHA mutations have been reported to cause an encephalomyopathy in childhood, while mutations in the genes encoding the other three subunits have been associated only with tumour formation. Following a brief description of SDH genes and subunits, we examine the properties and roles of SDH in the mitochondria. This allows further discussion of the several hypotheses proposed to account for the different clinical presentations resulting from impaired activity of the enzyme. Finally we stress the importance of SDH as a target and/or marker in a number of diseases and the need to better delineate the consequences of SDH deficiency in humans.

Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome.

Respiratory chain complex I deficiency represents a genetically heterogeneous group of diseases resulting from mutations in mitochondrial or nuclear genes. Mutations have been reported in 13 of the 14 subunits encoding the core of complex I (seven mitochondrial and six nuclear genes) and these result in Leigh or Leigh-like syndromes or cardiomyopathy. In this study, a combination of denaturing high performance liquid chromatography and sequence analysis was used to study the NDUFS3 gene in a series of complex I deficient patients. Mutations found in this gene (NADH dehydrogenase iron-sulphur protein 3), coding for the seventh and last subunit of complex I core, were shown to cause late onset Leigh syndrome, optic atrophy, and complex I deficiency. A biochemical diagnosis of complex I deficiency on cultured amniocytes from a later pregnancy was confirmed through the identification of disease causing NDUFS3 mutations in these cells. While mutations in the NDUFS3 gene thus result in Leigh syndrome, a dissimilar clinical phenotype is observed in mutations in the NDUFV2 and NDUFS2 genes, resulting in encephalomyopathy and cardiomyopathy. The reasons for these differences are uncertain.

Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency.

Starting from a cohort of 50 NADH-oxidoreductase (complex I) deficient patients, we carried out the systematic sequence analysis of all mitochondrially encoded complex I subunits (ND1 to ND6 and ND4L) in affected tissues. This approach yielded the unexpectedly high rate of 20% mutation identification in our series. Recurrent heteroplasmic mutations included two hitherto unreported (T10158C and T14487C) and three previously reported mutations (T10191C, T12706C and A13514G) in children with Leigh or Leigh-like encephalopathy. The recurrent mutations consistently involved T-->C transitions (p<10(-4)). This study supports the view that an efficient molecular screening should be based on an accurate identification of respiratory chain enzyme deficiency.

The mitochondrial DNA G13513A MELAS mutation in the NADH dehydrogenase 5 gene is a frequent cause of Leigh-like syndrome with isolated complex I deficiency.

Leigh syndrome is a subacute necrotising encephalomyopathy frequently ascribed to mitochondrial respiratory chain deficiency. This condition is genetically heterogeneous, as mutations in both mitochondrial (mt) and nuclear genes have been reported. Here, we report the G13513A transition in the ND5 mtDNA gene in three unrelated children with complex I deficiency and a peculiar MRI aspect distinct from typical Leigh syndrome. Brain MRI consistently showed a specific involvement of the substantia nigra and medulla oblongata sparing the basal ganglia. Variable degrees of heteroplasmy were found in all tissues tested and a high percentage of mutant mtDNA was observed in muscle. The asymptomatic mothers presented low levels of mutant mtDNA in blood leucocytes. This mutation, which affects an evolutionary conserved amino acid (D393N), has been previously reported in adult patients with MELAS or LHON/MELAS syndromes, emphasising the clinical heterogeneity of mitochondrial DNA mutations. Since the G13513A mutation was found in 21% of our patients with Leigh syndrome and complex I deficiency (3/14), it appears that this mutation represents a frequent cause of Leigh-like syndrome, which should be systematically tested for molecular diagnosis in affected children and for genetic counselling in their maternal relatives.

Prenatal diagnosis of respiratory chain deficiency by direct mutation screening.

Respiratory chain deficiency (RCD) is responsible for a clinically heterogeneous group of early-onset untreatable disorders. Enzymological prenatal diagnosis (PD) can only be offered to a fraction of families. Moreover, due to the two-fold genetic origin of the respiratory chain (nuclear and mitochondrial DNA) and owing to the large number of nuclear genes involved in the respiratory chain assembly, maintenance and functioning, the identification of the disease causing gene in a given family remains challenging. Here, we report on PD of RCD by direct screening of NDUFV1, SDH-Fp, SCO1 and SURF1 mutations in five unrelated families with complex I, II and IV deficiency, respectively. The identification of the disease-causing gene in a given family with RCD is a major issue to provide both adequate genetic counselling and early, reliable PD.

Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency.

Reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest complex of the mitochondrial respiratory chain and complex I deficiency accounts for approximately 30% cases of respiratory-chain deficiency in humans. Only seven mitochondrial DNA genes, but >35 nuclear genes encode complex I subunits. In an attempt to elucidate the molecular bases of complex I deficiency, we studied the six most-conserved complex I nuclear genes (NDUFV1, NDUFS8, NDUFS7, NDUFS1, NDUFA8, and NDUFB6) in a series of 36 patients with isolated complex I deficiency by denaturing high-performance liquid chromatography and by direct sequencing of the corresponding cDNA from cultured skin fibroblasts. In 3/36 patients, we identified, for the first time, five point mutations (del222, D252G, M707V, R241W, and R557X) and one large-scale deletion in the NDUFS1 gene. In addition, we found six novel NDUFV1 mutations (Y204C, C206G, E214K, IVS 8+41, A432P, and del nt 989-990) in three other patients. The six unrelated patients presented with hypotonia, ataxia, psychomotor retardation, or Leigh syndrome. These results suggest that screening for complex I nuclear gene mutations is of particular interest in patients with complex I deficiency, even when normal respiratory-chain-enzyme activities in cultured fibroblasts are observed.

Denaturing high-performance liquid chromatography (DHPLC)-based prenatal diagnosis for tuberous sclerosis.

Tuberous sclerosis (TSC) is a frequent autosomal-dominant condition (affecting 1 in 6000 individuals) caused by various mutations in either the hamartin (TSC1) or the tuberin gene (TSC2). This allelic and non-allelic heterogeneity makes genetic counseling and prenatal diagnosis difficult, especially as a significant proportion of TSC cases are due to de novo mutations. For this reason the identification of the disease causing mutation is mandatory for accurate counseling, yet current mutation detection methods such as single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) are labor intensive with limited detection efficiency. Denaturing high-performance liquid chromatography (DHPLC) is a high-throughput, semi-automated mutation detection system with a reported mutation detection rate close to 100% for PCR fragments of up to 800 bp. We used a recently described DHPLC assay allowing the efficient detection of mutations in TSC1 to analyze the DNA extracted from a chorion villus sample in order to perform a prenatal diagnosis for TSC. The fetus was found not to have inherited the deleterious mutation and the DHPLC diagnosis was confirmed by haplotype analysis. This represents the first DHPLC-based prenatal diagnosis of a genetic disease.

Mutation analysis of the hamartin gene using denaturing high performance liquid chromatography.

Denaturing high performance liquid chromatography (DHPLC) is a novel high-capacity technique for gene mutation scanning. We have assessed the sensitivity and specificity of this method for analysis of the full coding sequence of the hamartin (TSC1) gene in 20 tuberous sclerosis patients, whose TSC1 genes previously had been studied by single strand conformation polymorphism analysis and protein truncation assay. All eight sequence variants previously identified were adequately detected by DHPLC. Additionally, this approach picked up three polymorphisms, one of which (IVS13-55 C>G) was hitherto unreported, therefore serving as proof of principle for this technique. Thus, DHPLC appears to be a highly sensitive method with advantages in terms of flexibility, fragments size analysis, cost and time and labor sparing, compared to classical approaches of mutation scanning.

Protein truncation test for screening hamartin gene mutations and report of new disease-causing mutations.

Considering the prevalence of truncating mutations in the tuberous sclerosis (TSC) hamartin gene (TSC1), we devised a protein truncation test (PTT) to analyze the full length coding sequence of TSC1. Studying 12 sporadic cases and three familial forms by a combination of PTT and single-strand conformation polymorphism analysis (SSCA), we found 5/15 mutations while PTT alone detected 4/15 truncating mutations, two of which escaped SSCA analysis. SSCA alone picked up one missense mutation and two mutations also detected by PTT. Interestingly, a TSC1 mutation was identified in all three familial forms (3/3) while the rate of mutation detection was lower in sporadic cases (2/12). In conclusion, PTT proves to be a useful technique for the rapid detection of disease-causing mutations in the TSC1 gene.

The mutant genotype is the main determinant of the metabolic phenotype in phenylalanine hydroxylase deficiency.

Phenylketonuria and mild hyperphenylalaninemias are allelic disorders caused by mutations in the phenylalanine hydroxylase (PAH) gene. Following identification of the disease-causing mutation in 11 PAH-deficient patients, we tested the activity of the mutant gene products in an eukaryotic expression system. Two mutations markedly reduced PAH activity (A259V and L333F), one mutation mildly altered the enzyme activity (E390G), while the majority of mutant genotypes reduced the in vitro expression of PAH activity to 15-30% of controls. Comparing the predicted residual activity derived from expression studies to the clinical phenotypes of our PAH-deficient patients, we found that homozygosity for the L333F and E390G mutations resulted in severe and mild PAH deficiencies, respectively, both in vivo and in vitro, while compound heterozygosity (L333F/E390G) resulted in an intermediate dietary tolerance. Similarly, in vitro expression studies largely predicted dietary tolerance in compound heterozygotes for the A259V/IVS12nt1 (typical PKU), A259V/A403V, G218V/I65T, and G218V/R158Q mutations (mild variants). Taken together, these results support the view that expression studies are useful in predicting residual enzyme activity and that the mutant genotype at the PAH locus is the major determinant of metabolic phenotype in hyperphenylalaninemias.

Familial hyperproinsulinaemia due to a mutation substituting histidine for arginine at position 65 in proinsulin: identification of the mutation by restriction enzyme mapping.

UNLABELLED: Familial hyperproinsulinaemia is a rare genetic disorder characterized by point mutations in the insulin gene which impair the conversion of proinsulin to insulin. We report here three members of a two-generation Caucasian family in whom this syndrome was identified by unexplained hyperinsulinism associated with normal glucose tolerance and normal insulin sensitivity. Plasma insulin immunoreactivity showed a reduced affinity for the insulin receptor and eluted mainly, on Biogel chromatography, at the position of proinsulin. Analysis of the PCR-amplified insulin gene by restriction enzyme mapping revealed a new recognition site for the enzyme Nla III, indicating a Arg65 to His mutation. Sequence analysis of exon 3 confirmed this mutation in one allele of the gene. CONCLUSION: This study reports a two-generation European-Caucasian family with hyperproinsulinaemia due to a substitution of His for Arg at position 65 in proinsulin, the seventh now identified worldwide and the second from Europe. The mutation generated a new restriction site on the insulin gene suggesting the usefulness of restriction enzyme mapping as a screening procedure.

Mutations of the TWIST gene in the Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome (acrocephalo-syndactyly type III, ACS III) is an autosomal dominant craniosynostosis with brachydactyly, soft tissue syndactyly and facial dysmorphism including ptosis, facial asymmetry and prominent ear crura. ACS III has been mapped to chromosome 7p21-22. Of interest, TWIST, the human counterpart of the murine Twist gene, has been localized on chromosome 7p21 as well. The Twist gene product is a transcription factor containing a basic helix-loop-helix (b-HLH) domain, required in head mesenchyme for cranial neural tube morphogenesis in mice. The co-localisation of ACS III and TWIST prompted us to screen ACS III patients for TWIST gene mutations especially as mice heterozygous for Twist null mutations displayed skull defects and duplication of hind leg digits. Here, we report 21-bp insertions and nonsense mutations of the TWIST gene (S127X, E130X) in seven ACS III probands and describe impairment of head mesenchyme induction by TWIST as a novel pathophysiological mechanism in human craniosynostoses.

Sequence of the PHO2-POL3 (CDC2) region of chromosome IV of Saccharomyces cerevisiae.

The nucleotide sequence of a 5 kb EcoRI-NcoI fragment of chromosome IV, contiguous to gene POL3 (CDC2), has been determined. It contains three open reading frames: QRI1, QRI2 and QRI7. Two of them are essential genes. QRI7 is homologous to the Escherichia coli orfx gene.