Localization of abasic sites and single-strand breaks in mitochondrial DNA from brain of aged rat, treated or not with caloric restriction diet.

According to the "mitochondrial theory of aging" the lifelong accumulation of various kinds of damage to mitochondrial DNA (mtDNA) has been related to the age-dependent mitochondrial bioenergetic dysfunction. Caloric restriction (CR) diet is able to prevent or delay the onset of several age-related damages to mtDNA. The effects of aging and CR on the presence of abasic sites and single-strand breaks of the sugar-phosphate backbone in mtDNA have been analyzed by applying Ligation Mediated-PCR to a H strand region of brain mtDNA from young and old ad libitum-fed and old CR-treated rats. The region, encompassing the Direct Repeat 1 of the 4,834 bp-long deletion, is highly damaged in the old ad libitum-fed animals with respect to the young ones, whereas in the CR rats it shows a much lower extent of damage. The data confirm, at single nucleotide resolution, the protective effect of CR on the age-related mtDNA damage.

Acetyl-L-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis.

The expressional profile of mitochondrial transcripts and of genes involved in the mitochondrial biogenesis pathway induced by ALCAR daily supplementation in soleus muscle of control and unloaded 3-month-old rats has been analyzed. It has been found that ALCAR treatment is able to upregulate the expression level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA) in both control and unloaded animals. Interestingly, ALCAR feeding to unloaded rats resulted in the increase of transcript level for master factors involved in mitochondrial biogenesis (PGC-1alpha, NRF-1, TFAM). It also prevented the unloading-induced downregulation of mRNA levels for kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIbeta) into mitochondrial biogenesis. No significant effect on the expressional level of such genes was found in control ALCAR-treated rats. In addition, ALCAR feeding was able to prevent the loss of mitochondrial protein content due to unloading condition. Correlation analysis revealed a strong coordination in the expression of genes involved in mitochondrial biogenesis only in ALCAR-treated suspended animals, supporting a differentiated effect of ALCAR treatment in relation to the loading state of the soleus muscle. In conclusions, we demonstrated the ability of ALCAR supplementation to promote only in soleus muscle of hindlimb suspended rats an orchestrated expression of genes involved in mitochondrial biogenesis, which might counteract the unloading-induced metabolic changes, preventing the loss of mitochondrial proteins.

Tissue-specific effect of age and caloric restriction diet on mitochondrial DNA content.

The effect of age and caloric-restriction (CR) diet on mitochondrial DNA (mtDNA) content in different rat tissues was investigated. A decrease of the mtDNA content occurs with aging in liver and soleus muscle, whereas there is no age-related significant change of mtDNA content in brain. CR fully reverses the age-dependent loss of mtDNA in liver and soleus, whereas it results in a significant increase of mtDNA amount above the value of aged ad libitum fed rats in brain. These results further support the tissue-specific effect of CR, likely because of the different dependence of tissues on external nutrient uptake.

Measurement of the 4,834-bp mitochondrial DNA deletion level in aging rat liver and brain subjected or not to caloric restriction diet.

Several studies have demonstrated an age-related accumulation of the amount of a specific 4834-bp mitochondrial DNA (mtDNA) deletion in different tissues of rat (liver, brain, and skeletal muscle). We investigated the influence of a caloric restriction diet (CR) on a selected age-associated marker of mtDNA damage, as the 4834-bp deletion, using quantitative real-time PCR. The mtDNA deleted level has been determined with respect to the mitochondrial D-loop level, using specific primers and TaqMan probes for each target. In liver we found an age-related increase of the deletion level (twofold) that was reversed and brought back to the adult level by a CR diet. On the contrary, in the brain the age-related increase of the deletion level (eightfold) was not affected by CR at all. The different effect of the CR on the deletion level in liver and brain might be a further element supporting the tissue-specificity of the aging process.

Acetyl-L-carnitine dietary supplementation to old rats increases mitochondrial transcription factor A content in rat hindlimb skeletal muscles.

Acetyl-L-carnitine (ALCAR) fed to old rats has been reported to partially restore mitochondrial function and ambulatory activity. The results of the effect of ALCAR dietary supplementation to 28-month-old rats on mitochondrial transcription factor A (TFAM) content of rat hindlimb skeletal muscles are reported.

Rat hindlimb unloading: Soleus and Extensor Digitorum Longus histochemistry, mitochondrial DNA content and mitochondrial DNA deletions.

Mitochondrial phenotypic alterations, mitochondrial DNA content and mitochondrial DNA deletions in a slow, Soleus, and a fast, Extensor Digitorum Longus, skeletal muscle of 3- and 15-month-old hindlimb suspended rats have been studied. Cytochrome c oxidase-negative fibers appeared after unloading in all examined animals and their percentage increased with increasing unloading time. After 14 days of suspension the mitochondrial DNA content did not change in 3-month-old but decreased significantly in 15-month-old rats. Soleus was much more affected by unloading than Extensor Digitorum Longus. The mitochondrial DNA deletion of 4834 bp as well as other mtDNA deletions, researched with Long Distance-PCR, were absent in both studied muscles before and after unloading.

Sea urchin mtDBP is a two-faced transcription termination factor with a biased polarity depending on the RNA polymerase.

The sea urchin mitochondrial displacement (D)-loop binding protein mtDBP has been previously identified and cloned. The polypeptide (348 amino acids) displays a significant homology with the human mitochondrial transcription termination factor mTERF. This similarity, and the observation that the 3' ends of mitochondrial RNAs coded by opposite strands mapped in correspondence of mtDBP-binding sites, suggested that mtDBP could function as transcription termination factor in sea urchin mitochondria. To investigate such a role we tested the capability of mtDBP bound to its target sequence in the main non-coding region to affect RNA elongation by mitochondrial and bacteriophage T3 and T7 RNA polymerases. We show that mtDBP was able to terminate transcription bidirectionally when initiated by human mitochondrial RNA polymerase but only unidirectionally when initiated by T3 or T7 RNA polymerases. Time-course experiments indicated that mtDBP promotes true transcription termination rather than transcription pausing. These results indicate that mtDBP is able to function as a bipolar transcription termination factor in sea urchin mitochondria. The functional significance of such an activity could be linked to the previously proposed dual role of the protein in modulating mitochondrial DNA transcription and replication.

Increased expression of mitochondrial transcription factor A and nuclear respiratory factor-1 in skeletal muscle from aged human subjects.

The expression of two factors involved in the nuclear-mitochondrial crosstalk, namely the mitochondrial transcription factor A (TFAM) and the nuclear respiratory factor-1 (NRF-1), was studied in human skeletal muscle biopsies of young and aged subjects. Aged subjects presented a 2.6-fold and an 11-fold increase of the levels of TFAM protein and TFAM mRNA, respectively. The increased expression of TFAM was associated to the doubling of NRF-1 DNA-binding affinity and to a 6-fold increase of NRF-1 mRNA level. The upregulation of TFAM and NRF-1, in aged skeletal muscle, appears involved in the pathway leading to the age-related increase of mitochondrial DNA content.

Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle.

To have a clearer picture of how mitochondrial damages are associated to aging, a comprehensive study of phenotypic and genotypic alterations was carried out, analyzing with histochemical and molecular biology techniques the same skeletal muscle specimens of a large number of healthy subjects from 13 to 92 years old. Histochemical data showed that ragged red fibers (RRF) appear at about 40 years of age and are mostly cytochrome c oxidase (COX)-positive, whereas they are almost all COX-negative thereafter. Molecular analyses showed that the 4977 bp deletion of mitochondrial DNA (mtDNA(4977)) and the 7436 bp deletion of mtDNA (mtDNA(7436)) are already present in individuals younger than 40 years of age, but their occurrence does not change with age. After 40 years of age the number of mtDNA deleted species, as revealed by Long Extension PCR (LX-PCR), increases, the 10422 bp deletion of mtDNA (mtDNA(10422)) appears, although with a very low frequency of occurrence, and mtDNA content is more than doubled. Furthermore, mtDNA(4977) level directly correlates with that of COX-negative fibers in the same analyzed subjects. These data clearly show that, after 40 years of age, the phenotypic and genotypic mitochondrial alterations here studied appear in human skeletal muscle and that they are closely related.

Regulation of the expression of the sea urchin mitochondrial D-loop binding protein during early development.

The Paracentrotus lividus mitochondrial D-loop binding protein (mtDBP) is a DNA-binding protein which is involved in the regulation of sea urchin mtDNA transcription. Immunoblots of Heparin Sepharose-bound proteins at selected early developmental stages, as well as electrophoretic mobility shift assay, show that mtDBP is present in the egg at a concentration of about 1 x 10(6) molecules/egg. Its level increases after fertilization of about twofold, remaining substantially unchanged between 16-h blastula stage and early pluteus stage and declines thereafter. The content of mtDBP mRNA, determined by RNase protection experiments, increases about sevenfold at the 16-h blastula stage compared to the egg. A considerable decrease occurs at the 40-h pluteus stage, which precedes that of the protein. These results suggest that the expression of mtDBP is regulated at transcriptional level up to blastula stage, while other factors, in addition to the level of the RNA, may control the content of this protein in the following stages of embryogenesis.

The effect of aging and acetyl-L-carnitine on the pyruvate transport and oxidation in rat heart mitochondria.

The effect of aging and acute treatment with acetyl-L-carnitine on the pyruvate transport and oxidation in rat heart mitochondria was studied. The activity of the pyruvate carrier as well as the rates of pyruvate-supported respiration were both depressed (around 40%) in heart mitochondria from aged rats, the major decrease occurring during the second year of life. Administration of acetyl-L-carnitine to aged rats almost completely restored the rates of these metabolic functions to the level of young control rats. This effect of acetyl-L-carnitine was not due to changes in the content of pyruvate carrier molecules. The heart mitochondrial content of cardiolipin, a key phospholipid necessary for mitochondrial substrate transport, was markedly reduced (approximately 40%) in aged rats. Treatment of aged rats with acetyl-L-carnitine reversed the age-associated decline in cardiolipin content. As the changes in cardiolipin content were correlated with changes in rates of pyruvate transport and oxidation, it is suggested that acetyl-L-carnitine reverses the age-related decrement in the mitochondrial pyruvate metabolism by restoring the normal cardiolipin content.

Identification of human GC-box-binding zinc finger protein, a new Krüppel-like zinc finger protein, by the yeast one-hybrid screening with a GC-rich target sequence.

A new human zinc finger DNA-binding protein was identified by using a yeast one-hybrid selection system. Two versions of the cDNA, encoding the same protein, were detected that differ for a 584 bp extension at the 5' region. Sequence analysis showed that the longer clone is a full length version containing part of the 5' untranslated region. The smaller version was fused in frame with the yeast GAL4 activation domain whereas the 5' region of the longer clone displayed a stop codon interrupting the fusion with the GAL4 domain. Nevertheless, this clone activated the yeast HIS3 reporter gene with the same efficiency as the smaller version. Sequence comparison of the derived protein with the database showed that it belongs to a family of zinc finger DNA-binding proteins which regulate the expression of genes involved in cell proliferation. Expression of the protein in an in vitro system, DNA-binding studies and genetic experiments identify this factor as a new zinc finger DNA-binding protein which binds GC-rich sequences and contains a domain probably functioning as a transcriptional activator. The new human protein identified in this study was therefore named GC-box-binding zinc finger protein).

Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimer's disease patients.

The levels of mitochondrial DNA 4977 bp deletion (mtDNA4977) and mitochondrial DNA 8'-hydroxy-2'-deoxyguanosine (OH8dG) were determined in the same samples from two brain areas of healthy subjects and Alzheimer's disease (AD) patients. A positive correlation between the age-related increases of mtDNA4977 and of OH8dG levels was found in the brain of healthy individuals. On the contrary, in both brain areas of AD patients, mtDNA4977 levels were very low in the presence of high OH8dG amounts. These results might be explained assuming that the increase of OH8dG above a threshold level, as in AD patients, implies consequences for mtDNA replication and neuronal cell survival.

Cloning and characterisation of mtDBP, a DNA-binding protein which binds two distinct regions of sea urchin mitochondrial DNA.

The cDNA for the sea urchin mitochondrial D-loop-binding protein (mtDBP), a 40 kDa protein which binds two homologous regions of mitochondrial DNA (the D-loop region and the boundary between the oppositely transcribed ND5 and ND6 genes), has been cloned. Four different 3'-untranslated regions have been detected that are related to each other in pairs and do not contain the canonical polyadenylation signal. The in vitro synthesised mature protein (348 amino acids), deprived of the putative signal sequence, binds specifically to its DNA target sequence and produces a DNase I footprint identical to that given by the natural protein. mtDBP contains two leucine zippers, one of which is bipartite, and two small N- and C-terminal basic domains. A deletion mutation analysis of the recombinant protein has shown that the N-terminal region and the two leucine zippers are necessary for the binding. Furthermore, evidence was provided that mtDBP binds DNA as a monomer. This rules out a dimerization role for the leucine zippers and rather suggests that intramolecular interactions between leucine zippers take place. A database search has revealed as the most significative homology a match with the human mitochondrial transcription termination factor (mTERF), a protein that also binds DNA as a monomer and contains three leucine zippers forming intramolecular interactions. These similarities, and the observation that mtDBP-binding sites contain the 3'-ends of mtRNAs coded by opposite strands and the 3'-end of the D-loop structure, point to a dual function of the protein in modulating sea urchin mitochondrial DNA transcription and replication.

In vivo mitochondrial DNA-protein interactions in sea urchin eggs and embryos.

Footprinting studies with the purine-modifying agent dimethyl sulphate were performed in Paracentrotus lividus eggs and embryos to analyze in vivo the interactions between protein and mitochondrial DNA. Footprinting in the small non-coding region and at the boundary between the ND5 and ND6 genes revealed two strong contact sites corresponding with the in vitro binding sequences of mitochondrial D-loop-Binding Protein (mtDBP). The analysis of the pause region of mtDNA replication showed a strong footprint corresponding with the binding site of the mitochondrial Pause region-Binding Protein-2 (mtPBP-2), but only a very weak signal at the binding site of the mitochondrial Pause region-Binding Protein-1 (mtPBP-1), which in vitro binds DNA with high efficiency. In vitro and in vivo analysis of the 3' end-region of the two rRNA genes showed no significant protein-DNA interactions, suggesting that, in contrast to mammals, the 3' ends of sea urchin mitochondrial rRNAs are not generated by a protein-dependent transcription termination event. These and other data support a model in which expression of mitochondrial genes in sea urchins is regulated post-transcriptionally. Footprinting at the five AT-rich consensus regions allowed the detection of a binding site in the non-coding region for an as-yet unidentified protein, mtAT-1BP. The occupancy of this site appears to be developmentally regulated, being detectable in the pluteus larval stage, but not in unfertilized eggs.

Alteration of mitochondrial DNA and RNA level in human fibroblasts with impaired vitamin B12 coenzyme synthesis.

Alterations of mitochondrial (mt) nucleic acid metabolism in methylmalonic aciduria (MMA) were studied in two cell lines from skin fibroblasts of patients with mitochondrial (GM00595) or cytosolic (GM10011) defects in the biosynthesis pathways of cobalamin coenzymes. The mtDNA level increased two-fold in GM00595 cells, which carry a mt defect in the adenosylcobalamin synthesis, whereas no appreciable change was found in GM10011 cells. The content of the two rRNAs 16S and 12S mtRNAs, normalized for the mtDNA copy number, decreased by 70% and 50% in GM00595 and GM10011, respectively. The normalized content of ND1, ND2 and CO I mRNAs decreased in GM00595, but was unchanged in GM10011. Respiratory chain complex activities measured in these two cell lines were not different from control activities. These data suggest that the maintenance of the mt function is due to doubling of mtDNA and that this compensatory response takes place only in those cells in which the greater reduction of the level of rRNA might have brought the content of these transcripts below the threshold value for optimal expression of the mt genome.

Multiple protein-binding sites in the TAS-region of human and rat mitochondrial DNA.

To study the molecular mechanisms responsible for the regulation of mitochondrial DNA copy number, in vivo and in organello dimethyl sulfate footprinting experiments in human fibroblasts and rat liver mitochondria were carried out. By this approach we identified in both species two specific protein binding sites in the 3' region of the displacement loop of mitochondrial DNA. One site contains the TAS-D element of human and rat mitochondrial DNA; the other covers TAS-C and TAS-B in human, whereas in rat it comprises part of TAS-B. We suggest that the protected sequences might be the site of action of protein factors involved in the premature termination of mitochondrial DNA heavy-strand synthesis.

Aging and mitochondria.

Aging is a complex physiological phenomenon and several different theories have been elaborated about its origin. Among such theories, the 'mitochondrial theory of aging', which has gained a large support, indicates the accumulation of somatic mutations of mitochondrial DNA leading to the decline of mitochondrial functionality as one of the driving forces for the process itself. In this review data on rat and man from our laboratory and from recent literature have been thoroughly examined and compared in order to provide the 'state-of-the-art' on the role of mitochondria in aging. Alterations of structure and expression of mitochondrial genome with aging, to find out the eventual relevant changes of mitochondrial biogenesis, have been studied in rat whereas the relationship between cytochrome c oxidase activity and 'common deletion' has been studied in man. Results on the effect of acetyl-L-carnitine on the mitochondrial functionality are also reported.

Mitochondrial DNA deletions in oculopharyngeal muscular dystrophy.

The deletions in the mitochondrial DNA from skeletal muscle samples of two oculopharyngeal muscular dystrophy cases were studied using polymerase chain reaction techniques. The 4977 bp 'common deletion' was present in both specimens, exceeding the corresponding values of similarly aged, healthy controls. In the two samples multiple different mitochondrial DNA deletions, some case-specific and present at quite high, although not pathogenetic levels, were observed. The results suggest that mitochondrial DNA deletions, and the 'common deletion' in particular, might be a sensitive and early marker of a generalized mitochondrial suffering, due to a variety of pathological and physiological causes.