Christophe Rocher

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases.

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

Prevalence of rare mitochondrial DNA mutations in mitochondrial disorders

Abstract Background Mitochondrial DNA (mtDNA) diseases are rare disorders whose prevalence is estimated around 1 in 5000. Patients are usually tested only for deletions and for common mutations of mtDNA which account for 5–40% of cases, depending on the study. However, the prevalence of rare mtDNA mutations is not known. Methods We analysed the whole mtDNA in a cohort of 743 patients suspected of manifesting a mitochondrial disease, after excluding deletions and common mutations. Both heteroplasmic and homoplasmic variants were identified using two complementary strategies (Surveyor and MitoChip). Multiple correspondence analyses followed by hierarchical ascendant cluster process were used to explore relationships between clinical spectrum, age at onset and localisation of mutations. Results 7.4% of deleterious mutations and 22.4% of novel putative mutations were identified. Pathogenic heteroplasmic mutations were more frequent than homoplasmic mutations (4.6% vs 2.8%). Patients carrying deleterious mutations showed symptoms before 16 years of age in 67% of cases. Early onset disease (<1 year) was significantly associated with mutations in protein coding genes (mainly in complex I) while late onset disorders (>16 years) were associated with mutations in tRNA genes. MTND5 and MTND6 genes were identified as ‘hotspots’ of mutations, with Leigh syndrome accounting for the large majority of associated phenotypes. Conclusions Rare mitochondrial DNA mutations probably account for more than 7.4% of patients with respiratory chain deficiency. This study shows that a comprehensive analysis of mtDNA is essential, and should include young children, for an accurate diagnosis that is now accessible with the development of next generation sequencing technology.

Base composition at mtDNA boundaries suggests a DNA triple helix model for human mitochondrial DNA large-scale rearrangements.

Different mechanisms have been proposed to account for mitochondrial DNA (mtDNA) instability based on the presence of short homologous sequences (direct repeats, DR) at the potential boundaries of mtDNA rearrangements. Among them, slippage-mispairing of the replication complex during the asymmetric replication cycle of the mammalian mitochondrial DNA has been proposed to account for the preferential localization of deletions. This mechanism involves a transfer of the replication complex from the first neo-synthesized heavy (H) strand of the DR1, to the DR2, thus bypassing the intervening sequence and producing a deleted molecule. Nevertheless, the nature of the bonds between the DNA strands remains unknown as the forward sequence of DR2, beyond the replication complex, stays double-stranded. Here, we have analyzed the base composition of the DR at the boundaries of mtDNA deletions and duplications and found a skewed pyrimidine content of about 75% in the light-strand DNA template. This suggests the possible building of a DNA triple helix between the G-rich neo-synthesized DR1 and the base-paired homologous G.C-rich DR2. In vitro experiments with the purified human DNA polymerase gamma subunits enabled us to show that the third DNA strand may be used as a primer for DNA replication, using a template with the direct repeat forming a hairpin, with which the primer could initiate DNA replication. These data suggest a novel molecular basis for mitochondrial DNA rearrangements through the distributive nature of the DNA polymerase gamma, at the level of the direct repeats. A general model accounting for large-scale mitochondrial DNA deletion and duplication is proposed. These experiments extend to a DNA polymerase from an eucaryote source the use of a DNA triple helix strand as a primer, like other DNA polymerases from phage and bacterial origins.

Novel mitochondrial DNA mutations responsible for maternally inherited nonsyndromic hearing loss

Some cases of maternally inherited isolated deafness are caused by mtDNA mutations, frequently following an exposure to aminoglycosides. Two mitochondrial genes have been clearly described as being affected by mutations responsible for this pathology: the ribosomal RNA 12S gene and the transfer RNA serine (UCN) gene. A previous study identified several candidate novel mtDNA mutations, localized in a variety of mitochondrial genes, found in patients with no previous treatment with aminoglycosides. Five of these candidate mutations are characterized in the present study. These mutations are localized in subunit ND1 of complex I of the respiratory chain (m.3388C>A [p.MT‐ND1:Leu28Met]), the tRNA for Isoleucine (m.4295A>G), subunit COII of complex IV (m.8078G>A [p.MT‐CO2:Val165Ile]), the tRNA of Serine 2 (AGU/C) (m.12236G>A), and Cytochrome B, subunit of complex III (m.15077G>A [p.MT‐CYB:Glu111Lys]). Cybrid cell lines have been constructed for each of the studied mtDNA mutations and functional studies have been performed to assess the possible consequences of these mutations on mitochondrial bioenergetics. This study shows that a variety of mitochondrial genes, including protein‐coding genes, can be responsible for nonsyndromic deafness, and that exposure to aminoglycosides is not required to develop the disease, giving new insights on the molecular bases of this pathology. Hum Mutat 33:681–689, 2012.

Mutation Rate Switch inside Eurasian Mitochondrial Haplogroups: Impact of Selection and Consequences for Dating Settlement in Europe

R-lineage mitochondrial DNA represents over 90% of the European population and is significantly present all around the planet (North Africa, Asia, Oceania, and America). This lineage played a major role in migration “out of Africa” and colonization in Europe. In order to determine an accurate dating of the R lineage and its sublineages, we analyzed 1173 individuals and complete mtDNA sequences from Mitomap. This analysis revealed a new coalescence age for R at 54.500 years, as well as several limitations of standard dating methods, likely to lead to false interpretations. These findings highlight the association of a striking under-accumulation of synonymous mutations, an over-accumulation of non-synonymous mutations, and the phenotypic effect on haplogroup J. Consequently, haplogroup J is apparently not a Neolithic group but an older haplogroup (Paleolithic) that was subjected to an underestimated selective force. These findings also indicated an under-accumulation of synonymous and non-synonymous mutations localized on coding and non-coding (HVS1) sequences for haplogroup R0, which contains the major haplogroups H and V. These new dates are likely to impact the present colonization model for Europe and confirm the late glacial resettlement scenario.

Adaptative Capacity of Mitochondrial Biogenesis and of Mitochondrial Dynamics in Response to Pathogenic Respiratory Chain Dysfunction

AIMS Cellular energy homeostasy relies on mitochondrial plasticity, the molecular determinants of which are multiple. Yet, the relative contribution of and possible cooperation between mitochondrial biogenesis and morphogenesis to cellular energy homeostasy remains elusive. Here we analyzed the adaptative capacity of mitochondrial content and dynamics in muscle biopsies of patients with a complex IV defect, and in skin fibroblasts challenged with complex IV inhibition. RESULTS We observed a biphasic variation of the mitochondrial content upon complex IV inhibition in muscle biopsies and in skin fibroblasts. Adjustment of mitochondrial content for respiratory maintenance was blocked by using a dominant negative form of CREB (CREB-M1) and by L-NAME, a blocker of NO production. Accordingly, cells treated with KCN 6 μM showed higher levels of phospho-CREB, PGC1α mRNA, eNOS mRNA, and mtTFA mRNA. We also observed the increased expression of the fission protein DRP1 during fibroblasts adaptation, as well as mitochondrial ultrastructural defects indicative of increased fission in patients muscle micrographs. Accordingly, the expression of a dominant negative form of DRP1 (K38A mutant) reduced the biogenic response in fibroblasts challenged with 6 μM KCN. INNOVATION Our findings indicate that mitochondrial biogenesis and mitochondrial fission cooperate to promote cellular adaptation to respiratory chain inhibition. CONCLUSIONS Our data show for the first time that DRP1 intervenes during the initiation of the mitochondrial adaptative response to respiratory chain defects. The evidenced pathway of mitochondrial adaptation to respiratory chain deficiency provides a safety mechanism against mitochondrial dysfunction.

Human testis-specific genes are under relaxed negative selection

Recent studies have suggested that selective forces and constraints acting on genes varied during human evolution depending on the organ in which they are expressed. To gain insight into the evolution of organ determined negative selection forces, we compared the non-synonymous SNP diversity of genes expressed in different organs. Based on a HAPMAP dataset, we determined for each SNP its frequency in 11 human populations and, in each case, predicted whether or not the change it produces is deleterious. We have shown that, for all organs under study, SNPs predicted to be deleterious are present at a significantly lower frequency than SNPs predicted to be tolerated. However, testis-specific genes contain a higher proportion of deleterious SNPs than other organs. This study shows that negative selection is acting on the whole human genome, but that the action of negative selection is relaxed on testis-specific genes. This result adds to and expands the hypothesis of a recent evolutionary change in the human male reproductive system and its behavior.