Julio Montoya

Human mtDNA Haplogroups Associated with High or Reduced Spermatozoa Motility

A variety of mtDNA mutations responsible for human diseases have been associated with molecular defects in the OXPHOS system. It has been proposed that mtDNA genetic alterations can also be responsible for sperm dysfunction. In addition, it was suggested that if sperm dysfunction is the main phenotypic consequence, these mutations could be fixed as stable mtDNA variants, because mtDNA is maternally inherited. To test this possibility, we have performed an extensive analysis of the distribution of mtDNA haplogroups in white men having fertility problems. We have found that asthenozoospermia, but not oligozoospermia, is associated with mtDNA haplogroups in whites. Thus, haplogroups H and T are significantly more abundant in nonasthenozoospermic and asthenozoospermic populations, respectively, and show significant differences in their OXPHOS performance.

Replication and transcription of mammalian mitochondrial DNA.

Mitochondria are subcellular organelles, devoted mainly to energy production in the form of ATP, that contain their own genetic system. Mitochondrial DNA codifies a small, but essential number of polypeptides of the oxidative phosphorylation system. The mammalian mitochondrial genome is an example of extreme economy showing a compact gene organization. The coding sequences for two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and 13 polypeptides are contiguous and without introns. The tRNAs are regularly interspersed between the rRNA and protein‐coding genes, playing a crucial role in RNA maturation from the polycistronic transcripts. A single major non‐coding region, called the D‐loop region, contains the main regulatory sequences for transcription and replication initiation. This genetic organization has its precise correspondence in the mode of expression and distinctive structural features of the RNAs. The basic mechanisms of mitochondrial DNA transcription and replication and the main cis‐acting elements playing a role in both processes have been determined. Many trans‐acting factors involved in mitochondrial gene expression, including the RNA and DNA polymerases, have been cloned or identified. However, the regulatory mechanisms participating in mitochondrial gene expression are still poorly understood. The interest to complete this knowledge is increased by the involvement of mitochondria in human diseases, in basic processes such as heat production, Ca2+ homeostasis and apoptosis, and by their potential role in ageing and carcinogenesis.

Direct Regulation of Mitochondrial RNA Synthesis by Thyroid Hormone

ABSTRACT We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter

Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.

Human mitochondrial haplogroup H: the highest VO2max consumer--is it a paradox?

Mitochondrial background has been demonstrated to influence maximal oxygen uptake (VO(2max), in mLkg(-1)min(-1)), but this genetic influence can be compensated for by regular exercise. A positive correlation among electron transport chain (ETC) coupling, ATP and reactive oxygen species (ROS) production has been established, and mitochondrial variants have been reported to show differences in their ETC performance. In this study, we examined in detail the VO(2max) differences found among mitochondrial haplogroups. We recruited 81 healthy male Spanish Caucasian individuals and determined their mitochondrial haplogroup. Their VO(2max) was determined using incremental cycling exercise (ICE). VO(2max) was lower in J than in non-J haplogroup individuals (P=0.04). The H haplogroup was responsible for this difference (VO(2max); J vs. H; P=0.008) and this group also had significantly higher mitochondrial oxidative damage (mtOD) than the J haplogroup (P=0.04). In agreement with these results, VO(2max) and mtOD were positively correlated (P=0.01). Given that ROS production is the major contributor to mtOD and consumes four times more oxygen per electron than the ETC, our results strongly suggest that ROS production is responsible for the higher VO(2max) found in the H variant. These findings not only contribute to a better understanding of the mechanisms underneath VO(2max), but also help to explain some reported associations between mitochondrial haplogroups and mtOD with longevity, sperm motility, premature aging and susceptibility to different pathologies.

Cerebral folate deficiency and leukoencephalopathy caused by a mitochondrial DNA deletion.

Our aim was to describe a child with an incomplete form of Kearns–Sayre syndrome who presented profound cerebrospinal fluid (CSF) folate deficiency and his response to folinic acid supplementation

Mitochondrial DNA Content of Human Spermatozoa

Abstract Sperm mitochondria play an important role in spermatozoa because of the high ATP demand of these cells. Different mitochondrial DNA (mtDNA) mutations and haplogroups influence sperm function. The mtDNA dose also contributes to genetic variability and pathology in different tissues and organs, but nothing is known about its relevance in the performance of spermatozoa. We estimated the variability in mtDNA content within a population of men. Different mtDNA:nuclear DNA ratios were characteristic of progressive and nonprogressive spermatozoa, confirming the influence of mtDNA content on sperm functionality. We also estimated that the absolute content of mtDNA was 700 and 1200 mtDNA copies per cell in progressive and nonprogressive human spermatozoa, respectively. These results suggest that a marked increase of mtDNA copy number per cell volume takes place during spermatogenesis.

Human mitochondrial variants influence on oxygen consumption

This work investigates if human mitochondrial variants influence on maximal oxygen consumption (VO(2max)). With this purpose we recruited, as a uniform population in term of nutritional habits and life style, 114 healthy male Spanish subjects that practiced fitness exercises 3-4 times a week. Once mtDNA haplogroups were determined, we found that J presents with lower VO(2max) (P=0.02) than nonJ variants. J has been related with a lower efficiency of electron transport chain (ETC), diminished ATP and ROS production. Thus, the difficult to compensate the mitochondrial energetic deficiency could explain the accumulation of J haplogroup in LHON and multiple sclerosis. Furthermore, the lower ROS production associated to J could also account for the accrual of this variant in elderly people consequent to a decreased oxidative damage.

Regulation of mitochondrial transcription by mitochondrial transcription factor A

In order to test the hypothesis that mitochondrial transcription factor A (mtTFA) regulates mitochondrial transcription in vivo, mtTFA was overexpressed in HeLa cells and imported into isolated rat liver mitochondria. Five hours after transfection with an eukaryotic expression vector, mitochondrial transcripts for cytochrome-c-oxidase subunit I and 12 S rRNA were increased over controls. In the presence of rat liver mitochondria, the 29 kDa mtTFA, generated by in vitro translation, was processed to a 24 kDa protein which was protected from protease digestion. This demonstrates that mtTFA was imported into the matrix. Incorporation of 32P-UTP into mitochondrial transcripts was stimulated following import of mtTFA. We conclude that the intracellular and intramitochondrial concentration of mtTFA, respectively, indeed regulates mitochondrial transcription. (Mol Cell Biochem 174: 227–230, 1997)

Bilateral striatal necrosis associated with a novel mutation in the mitochondrial ND6 gene

We report the molecular findings in two independent patients presenting with progressive generalized dystonia and bilateral striatal necrosis in whom we have identified a mutation (T14487C) in the mitochondrial ND6 gene. The mutation is heteroplasmic in all samples analyzed, and it fulfills all accepted criteria of pathogenicity. Transmitochondrial cell lines harboring 100% mutant mitochondrial DNA showed a marked decrease in the activity of complex I of the respiratory chain supporting the pathogenic role of T14487C.