Rafael Garesse

Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes

Mitochondria play a pivotal role in cell physiology, producing the cellular energy and other essential metabolites as well as controlling apoptosis by integrating numerous death signals. The biogenesis of the oxidative phosphorylation system (OXPHOS) depends on the coordinated expression of two genomes, nuclear and mitochondrial. As a consequence, the control of mitochondrial biogenesis and function depends on extremely complex processes that require a variety of well orchestrated regulatory mechanisms. It is now clear that in order to provide cells with the correct number of structural and functional differentiated mitochondria, a variety of intracellular and extracellular signals including hormones and environmental stimuli need to be integrated. During the last few years a considerable effort has been devoted to study the factors that regulate mtDNA replication and transcription as well as the expression of nuclear-encoded mitochondrial genes in physiological and pathological conditions. Although still in their infancy, these studies are starting to provide the molecular basis that will allow to understand the mechanisms involved in the nucleo-mitochondrial communication, a cross-talk essential for cell life and death.

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana.

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3′ downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the C↔T substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.

Drosophila mitochondrial transcription factor B1 modulates mitochondrial translation but not transcription or DNA copy number in Schneider cells.

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knock-down of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.

THYROID HORMONE-REGULATED BRAIN MITOCHONDRIAL GENES REVEALED BY DIFFERENTIAL CDNA CLONING

Thyroid hormone (T3) plays a critical role in the development of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely unknown. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mitochondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit III). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-encoded mitochondrial cytochrome c oxidase subunits: subunits IV and VIc were also decreased in the brains of hypothyroid animals. Hypothyroidism-induced changes in mitochondrial RNAs were followed by a concomitant 40% decrease in cytochrome c oxidase activity. This study shows that T3 is an important regulator of mitochondrial function in the neonatal brain and, more importantly, provides a molecular basis for the specific action of this hormone in the developing brain.

Genetic basis of end‐stage hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by a heterogeneous presentation and clinical course. A minority of HCM patients develop end‐stage HCM and require cardiac transplantation. The genetic basis of end‐stage HCM is unknown but small series, isolated case reports and animal models have related the most aggressive heart failure course with the presence of multiple mutations.

A double mutation (A8296G and G8363A) in the mitochondrial DNA tRNALys gene associated with myoclonus epilepsy with ragged-red fibers

Objective: To define potential pathogenic mitochondrial DNA (mtDNA) point mutations in a patient with myoclonus epilepsy with ragged-red fibers (MERRF) syndrome. Background: MERRF syndrome is typically associated with point mutations in the mtDNA tRNALys gene. Methods: We performed morphologic, biochemical, and genetic analysis of muscle samples from the patient and four relatives. Molecular genetic studies included sequencing, PCR, and restriction enzyme analysis on whole muscle, blood, and single muscle fibers. Results: Muscle biopsy showed cytochrome c oxidase (COX), negative ragged-red fibers (RRF), and a defect of complex I of the mitochondrial respiratory chain. We found an A8296G transition and a G8363A mutation in the mtDNA tRNALys gene. The A8296G was almost homoplasmic in muscle and blood from the propositus and his oligosymptomatic maternal relatives. The G8363A mutation was heteroplasmic and more abundant in muscle than in blood, and its proportion correlated with clinical severity. Single muscle fiber analysis showed significantly higher levels of G8363A genomes in COX-negative than in normal fibers, and almost homoplasmic levels of mutant A8296G mtDNA in both COX-negative and normal fibers. The two mutations affect highly conserved nucleotides and were not found in controls. Conclusions: The G8363A mutation is pathogenic; the co-ocurrence of the A8296G mutation is of unclear significance and is likely to be a rare polymorphism.

Mitochondrial dysfunction associated with a mutation in the Notch3 gene in a CADASIL family

Background: Cerebral autosomal arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by recurrent subcortical ischemic strokes and dementia caused by mutations in the Notch3 gene. In Drosophila melanogaster, Notch signaling has a pleiotropic effect, affecting most tissues of the organism during development. Objective: To characterize a potential mitochondrial dysfunction associated with mutations in the Notch3 gene. Methods: Biochemical, histochemical, molecular, and genetic analyses were performed on muscle biopsy specimens and fibroblasts obtained from patients of a Spanish family with CADASIL. Additional biochemical and molecular analyses of the N55e11 mutant of D. melanogaster were performed. Results: In muscle biopsy specimens, a significant decrease was found in the activity of complex I (NADH [reduced form of nicotinamide adenine dinucleotide] dehydrogenase), and in one patient, histochemical analysis showed the presence of ragged-red fibers with abnormal cytochrome c oxidase staining. Reduced fibroblast activity of complex V (ATP synthase) was found. Supporting data on patients with CADASIL, it was found that the mutation N55e11 in Drosophila decreases the activity of mitochondrial respiratory complexes I and V. Conclusions: Mitochondrial respiratory chain activity responds, directly or indirectly, to the Notch signaling pathway. Mitochondrial dysfunction in patients with CADASIL may be an epiphenomenon, but results of this study suggest that the pathophysiology of the disease could include a defect in oxidative phosphorylation.

Microgravity effects on Drosophila melanogaster behavior and aging. Implications of the IML-2 experiment

Earlier Space experiments had indicated that young male Drosophila flies exposed to microgravity showed an acceleration in aging. In a 14.5-day Space Shuttle Flight we sent 300 young male flies with the purpose of confirming these findings and to establish whether changes in the behavior of the flies were responsible for the effect in accordance with the proposal that alterations in mitochondrial metabolism may be involved in the aging response. By repeatedly video-recording, we have found a very marked increase in the locomotor activity of the fruitflies in Space. The males showed an accelerated aging response upon recovery, both in terms of physiological vitality assays (mating and negative geotaxis) and of life-span curves. The involvement of mitochondrial metabolism is also suggested by the finding of a greater decrease in mitochondrial 16S ribosomal RNA in the microgravity exposed flies than in ground controls. On the other hand, a parallel 1 x g centrifuge control did not show such differences in the life-span curves when compared to flies exposed to a similar centrifugation on the ground. Drosophila females also increased their locomotor activity but did not show differential changes in the life-span curves. These results are discussed in terms of the current mechanisms of aging in multicellular eukaryotic organisms.

MidA is a putative methyltransferase that is required for mitochondrial complex I function

Dictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA− mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology.