Rosana Hernández-Sierra

Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.

Over‐expression of the catalytic core of mitochondrial DNA (mtDNA) polymerase in the nervous system of Drosophila melanogaster reduces median life span by inducing mtDNA depletion

DNA polymerase γ (pol γ) is the sole DNA polymerase devoted to mitochondrial DNA (mtDNA) replication. We have characterized the molecular and physiological effects of over‐expression of the catalytic subunit of pol γ, pol γ‐α, in the nervous system of Drosophila melanogaster using the upstream activation sequence (UAS)/yeast transcriptional activator by binding to UAS (GAL4) system. Tissue‐specific over‐expression of pol γ‐α was confirmed by immunoblot analysis, whereas the very low levels of endogenous protein are undetectable in UAS or GAL4 control lines. The transgenic flies over‐expressing pol γ‐α in the nervous system showed a moderate increase in pupal lethality, and a significant decrease in the median life span of adult flies. Moreover, these flies displayed a decrease in the rate of synthesis of mtDNA, which is accompanied by a significant mtDNA depletion, and a corresponding decrease in the levels of mitochondrial transcription factor A (mtTFA). Biochemical analysis showed an oxidative phosphorylation (OXPHOS) defect in transgenic flies, which were more susceptible to oxidative stress. Although we did not detect apoptosis in the nervous system of adult transgenic flies, brains of larvae over‐expressing pol γ‐α showed evidence of increased cell death that correlates with the observed phenotypes. Our data establish an animal model that mimics some of the features of human mtDNA depletion syndromes.

Coiled Coil Domain-containing Protein 56 (CCDC56) Is a Novel Mitochondrial Protein Essential for Cytochrome c Oxidase Function

Background: Cytochrome c oxidase (COX), the final enzyme of the mitochondrial electron transport chain, requires several assembly factors for its proper function. Results: ccdc56 knock-out flies showed developmental delay, lethality, and a dramatic decrease in the levels/activity of COX. Conclusion: CCDC56 protein is necessary for COX function and for viability in flies. Significance: Drosophila CCDC56 is a novel putative COX assembly factor conserved in humans. In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5′-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56D6 and ccdc56D11 alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/assembly.

Modeling pathogenic mutations of human twinkle in Drosophila suggests an apoptosis role in response to mitochondrial defects.

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations.

Evaluation of mitochondrial function and metabolic reprogramming during tumor progression in a cell model of skin carcinogenesis

Metabolic reprogramming from mitochondrial aerobic respiration to aerobic glycolysis is a hallmark of cancer. However, whether it is caused by a dysfunction in the oxidative phosphorylation pathway is still under debate. In this work, we have analyzed the bioenergetic cellular (BEC) index and the relative cell ability to grow in the presence of either galactose or glucose as sources of sugar (Gal/Glu index) of a system formed by four epidermal cell lines with increasing tumorigenic potentials, ranging from nontumorigenic to highly malignant. We find that the BEC index gradually decreases whereas the Gal/Glu index increases with tumorigenicity, indicating that a progressive metabolic adaptation to aerobic glycolysis occurs in tumor cells associated with malignancy. Interestingly, this metabolic adaptation does not appear to be caused by damaged respiration, since the expression and activity of components of the respiratory chain complexes were unchanged in the cell lines. Moreover, the corresponding mitochondrial ATP synthetic abilities of the cell lines were found similar. The production of reactive oxygen species was also measured. A shift in ROS generation was found when compared nontumorigenic with tumorigenic cell lines, the latter exhibiting about threefold higher ROS levels than nontumorigenic cells. This result indicates that oxidative stress is an early event during tumor progression.

Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity

The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Lebers hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

MUSCLE-SPECIFIC OVEREXPRESSION OF THE CATALYTIC SUBUNIT OF DNA POLYMERASE γ INDUCES PUPAL LETHALITY IN Drosophila melanogaster

We show the physiological effects and molecular characterization of overexpression of the catalytic core of mitochondrial DNA (mtDNA) polymerase (pol γ-α) in muscle of Drosophila melanogaster. Muscle-specific overexpression of pol γ-α using the UAS/GAL4 (where UAS is upstream activation sequence) system produced more than 90% of lethality at the end of pupal stage at 25°C, and the survivor adult flies showed a significant reduction in life span. The survivor flies displayed a decreased mtDNA level that is accompanied by a corresponding decrease in the levels of the nucleoid-binding protein mitochondrial transcription factor A (mtTFA). Furthermore, an increase in apoptosis is detected in larvae and adults overexpressing pol γ-α. We suggest that the pupal lethality and reduced life span of survivor adult flies are both caused mainly by massive apoptosis of muscle cells induced by mtDNA depletion.