Disruption of Mitochondrion-To-Nucleus Interaction in Deceased Cloned Piglets

Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA. Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones’ survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contrast, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones.     

Molecular genetic and clinical aspects of mitochondrial disorders in childhood

Mitochondrial OXPHOS disorders are caused by mutations in mitochondrial or nuclear genes, which directly or indirectly affect mitochondrial oxidative phosphorylation (OXPHOS). Primary mtDNA abnormalities in children are due to rearrangements (deletions or duplications) and point mutations or insertions. Mutations in the nuclear-encoded polypeptide subunits of OXPHOS result in complex I and II deficiency, whereas mutations in the nuclear proteins involved in the assembly of OXPHOS subunits cause defects in complexes I, III, IV, and V. Here, we review recent progress in the identification of mitochondrial and nuclear gene defects and the associated clinical manifestations of these disorders in childhood.     

Caenorhabditis elegans,a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown.Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C. elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.     

Genetic insights into OXPHOS defect and its role in cancer

Warburg proposed that cancer originates from irreversible injury to mitochondrial oxidative phosphorylation (mtOXPHOS), which leads to an increase rate of aerobic glycolysis in most cancers. However, despite several decades of research related to Warburg effect, very little is known about the underlying genetic cause(s) of mtOXPHOS impairment in cancers. Proteins that participate in mtOXPHOS are encoded by both mitochondrial DNA (mtDNA) as well as nuclear DNA. This review describes mutations in mtDNA and reduced mtDNA copy number, which contribute to OXPHOS defects in cancer cells. Maternally inherited mtDNA renders susceptibility to cancer, and mutation in the nuclear encoded genes causes defects in mtOXPHOS system. Mitochondria damage checkpoint (mitocheckpoint) induces epigenomic changes in the nucleus, which can reverse injury to OXPHOS. However, irreversible injury to OXPHOS can lead to persistent mitochondrial dysfunction inducing genetic instability in the nuclear genome. Together, we propose that "mitocheckpoint" led epigenomic and genomic changes must play a key role in reversible and irreversible injury to OXPHOS described by Warburg. These epigenetic and genetic changes underlie the Warburg phenotype, which contributes to the development of cancer.     

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 ρ(0) cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in ρ(0) cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.