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.     

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.     

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.     

Simian virus 40 T- and U-antigens: immunological characterization and localization in different nuclear subfractions of simian virus 40-transformed cells.

Simian virus 40 (SV40)-transformed cells and cells infected by the nondefective adenovirus 2(Ad2)-SV40 hybrid viruses Ad2+ND1 and Ad2+ND2 were analyzed for SV40 T- and U-antigens, respectively, using individual hamster SV40 tumor sera or serum for which U-antibodies were removd by absorption. These studies showed that (i) T- and U-antigens can be defined by separate classes of antigenic determinants and (ii) the U-antigenic determinants in SV40-transformed cells and in hybrid virus-infected cells are similar. The apparent discrepancy in the subcellular location of U-antigen in SV40-transformed cells (nuclear location) and in hybrid virus-infected cells (perinuclear location) as determined by immunofluorescence staining of methanol/acetone-fixed cells could be resolved by treating hybrid virus-infected cells with a hypotonic KCl solution before fixation. Upon this treatment hybrid virus-infected cells also showed nuclear U-antigen staining. The possibility of an association of T- and U-antigens with different nuclear subfractions in SV40-transformed cells was investigated. Detergent-cleaned nuclei of SV40-transformed cells were fractionated into nuclear matrices and a DNase-treated, high-salt nuclear extract. Analysis of the nuclear matrices by immunofluorescence microscopy with T+U+ and T+U- hamster SV40 tumor serum revealed that U-antigen remained associated with the nuclear matrices, whereas T-antigen could not be detected in this nuclear subfraction. T-antigen, however, could be immunoprecipitated from nuclear extracts of the SV40-transformed cells.     

Molecular research technologies in mitochondrial diseases: the microarray approach

Mitochondria are ubiquitous in eukaryotic cells where they generate much of the cellular energy by the process of oxidative phosphorylation (OXPHOS). The approximately 1500 genes of the mitochondrial genome are distributed between the cytoplasmic, maternally-inherited, mitochondrial DNA (mtDNA) which encodes 37 genes and the nuclear DNA (nDNA) which encompasses the remaining mitochondrial genes. The interplay between the mtDNA and nDNA encoded mitochondrial genes and their role in mitochondrial disorders is still largely unclear. One approach for elucidating the pathophysiology of mitochondrial diseases has been to look at changes in the expression of mtDNA and nDNA-encoded genes in response to specific mitochondrial genetic defects. Initial studies of gene expression changes in response to mtDNA defect employed blot technologies to analyze changes in the expression of individual genes one at a time. While Southern/Northern blot experiments confirmed the importance of nDNA-mtDNA interactions in the pathophysiology of mitochondrial myopathy, the methodology used limited the number of genes that could be analyzed from each patient. This barrier has been overcome, in part by the advent of DNA microarray technology. In DNA microarrays gene sequences or oligonucleotides homologous to gene sequences are arrayed on a solid support. The RNA from the subject is then isolated, the mRNA converted to cDNA and the cDNA labeled with a fluorescent probe. The labeled cDNA is hybridized on the microarray and the fluorescence bound to each array is then quantified. Recently, these technologies have been applied to mitochondrial disease patient tissues and the presence of coordinate changes in mitochondrial gene expression confirmed.     

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1–4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.     

Genetic characterization of the mitochondrial DNA from Lepeophtheirus salmonis (Crustacea; Copepoda). A new gene organization revealed

The mitochondrial DNA (mtDNA) from the salmon louse, Lepeophtheirus salmonis, is 15445 bp. It includes the genes coding for cytochrome B (Cyt B), ATPase subunit 6 and 8 (A6 and A8), NADH dehydrogenase subunits 1-6 and 4L (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), cytochrome c oxidase subunits I-III (COI, COII and COIII), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNAs. Two copies of tRNA-Lys are present in the mtDNA of L. salmonis, while tRNA-Cys was not identified. Both DNA strands contain coding regions in the salmon louse, in contrast to the other copepod characterized Tigriopus japonicus, but only a few genes overlap. In vertebrates, ND4 and ND4L are transcribed as one bicistronic mRNA, and are therefore localized together. The same organization is also found in crustaceans, with the exceptions of T. japonicus, Neocalanus cristatus and L. salmonis that deviate from this pattern. Another exception of the L. salmonis mtDNA is that A6 and A8 do not overlap, but are separated by several genes. The protein-coding genes have a bias towards AT-rich codons. The mitochondrial gene order in L. salmonis differs significantly from the copepods T. japonicus, Eucalanus bungii, N. cristatus and the other 13 crustaceans previously characterized. Furthermore, the mitochondrial rRNA genes are encoded on opposite strands in L. salmonis. This has not been found in any other arthropods, but has been reported in two starfish species. In a phylogenetic analysis, using an alignment of mitochondrial protein sequences, L. salmonis groups together with T. japonicus, being distant relatives to the other crustaceans.     

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.