Angela Maria Serena Lezza

Mitochondrial DNA copy number and mitochondrial DNA deletion in adult and senescent rats.

In order to understand the cause of the reduced mitochondrial DNA transcription in heart and brain of senescent rat previously reported, we focused our attention on the content and structure of rat mitochondrial DNA in adult and senescent rats. The estimate of the mtDNA copy number in liver, heart and brain of adult and senescent rats showed that in all organs examined the senescent individuals have a mtDNA content higher than the adult counterparts. The analysis of mtDNA structural changes involved the search for point mutations and large deletions. As for the first case, the determination of the nucleotide sequence of many independent clones containing two mtDNA restriction fragments isolated from rat cerebral hemispheres did not show any sequence difference between adult and senescent individuals. However, analysis of mtDNA deletions by the polymerase chain reaction in liver and brain of adult and senescent rats identified a small population of mtDNA molecules harboring a deletion of 4834 bp. The estimate of the proportion of deleted molecules in the liver showed that they represent 0.02% and 0.0005% of total mtDNA in senescent and adult rat liver respectively. Therefore, a mtDNA deletion also accumulates in the rat during aging. This result supports the hypothesis of the accumulation of deleted mtDNA molecules in aging. However, the low percentage of deleted mtDNA molecules already found and the reversibility of the reduced mitochondrial DNA transcription in senescent rat raise doubts on the primary role of the irreversibly damaged mtDNA molecules in aging. Deleted mtDNA molecules along with changes caused by lipid peroxidation of mitochondrial membranes might contribute to the overall decline of mitochondrial function.

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.

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 Alzheimers 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.—Lezza, A. M. S., Mecocci, P., Cormio, A., Real, M. F., Cherubini, A., Cantatore, P., Senin, U., Gadaleta, M. N. Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimers disease patients. FASEB J. 13, 1083–1088 (1999)

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.

Regulation of mitochondrial biogenesis through TFAM-mitochondrial DNA interactions: Useful insights from aging and calorie restriction studies.

Mitochondrial biogenesis is regulated to adapt mitochondrial population to cell energy demands. Mitochondrial transcription factor A (TFAM) performs several functions for mtDNA and interactions between TFAM and mtDNA participate to regulation of mitochondrial biogenesis. Such interactions are modulated through different mechanisms: regulation of TFAM expression and turnover, modulation of TFAM binding activity to mtDNA through post-translational modifications and differential affinity of TFAM, occurrence of TFAM sliding on mtDNA filaments and of cooperative binding among TFAM molecules, modulation of protein-protein interactions. The tissue-specific regulation of mitochondrial biogenesis in aging and calorie restriction (CR) highlights the relevance of modulation of TFAM-mtDNA interactions.

Synthesis and turnover rates of four rat liver mitochondrial RNA species

The synthesis and turnover rates of the two 12 S and 16 S mt rRNAs and of the mt mRNAs for subunits I and III of cytochrome oxidase have been determined by measuring the kinetics of incorporation of [3H]uridine in the mtRNA of rat hepatocytes. All the RNA species examined have approximately the same turnover (t ~ 100 min) and therefore the rate of synthesis, which is about 10‐times higher for the rRNAs, seems to be the factor responsible for the different mt rRNA and mRNA steady‐state levels.

Measurement of the 4,834-bp Mitochondrial DNA Deletion Level in Aging Rat Liver and Brain Subjected or Not to Caloric Restriction Diet

Abstract: 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.

Aging and calorie restriction oppositely affect mitochondrial biogenesis through TFAM binding at both origins of mitochondrial DNA replication in rat liver.

Aging affects mitochondria in a tissue-specific manner. Calorie restriction (CR) is, so far, the only intervention able to delay or prevent the onset of several age-related changes also in mitochondria. Using livers from middle age (18-month-old), 28-month-old and 32-month-old ad libitum-fed and 28-month-old calorie-restricted rats we found an age-related decrease in mitochondrial DNA (mtDNA) content and mitochondrial transcription factor A (TFAM) amount, fully prevented by CR. We revealed also an age-related decrease, completely prevented by CR, for the proteins PGC-1α NRF-1 and cytochrome c oxidase subunit IV, supporting the efficiency of CR to forestall the age-related decrease in mitochondrial biogenesis. Furthermore, CR counteracted the age-related increase in oxidative damage to proteins, represented by the increased amount of oxidized peroxiredoxins (PRX-SO3) in the ad libitum-fed animals. An unexpected age-related decrease in the mitochondrial proteins peroxiredoxin III (Prx III) and superoxide dismutase 2 (SOD2), usually induced by increased ROS and involved in mitochondrial biogenesis, suggested a prevailing relevance of the age-reduced mitochondrial biogenesis above the induction by ROS in the regulation of expression of these genes with aging. The partial prevention of the decrease in Prx III and SOD2 proteins by CR also supported the preservation of mitochondrial biogenesis in the anti-aging action of CR. To investigate further the age- and CR-related effects on mitochondrial biogenesis we analyzed the in vivo binding of TFAM to specific mtDNA regions and demonstrated a marked increase in the TFAM-bound amounts of mtDNA at both origins of replication with aging, fully prevented by CR. A novel, positive correlation between the paired amounts of TFAM-bound mtDNA at these sub-regions was found in the joined middle age ad libitum-fed and 28-month-old calorie-restricted groups, but not in the 28-month-old ad libitum-fed counterpart suggesting a quite different modulation of TFAM binding at both origins of replication in aging and CR.

Acetyl-L-carnitine supplementation to old rats partially reverts the age-related mitochondrial decay of soleus muscle by activating peroxisome proliferator-activated receptor γ coactivator-1α-dependent mitochondrial biogenesis.

The age-related decay of mitochondrial function is a major contributor to the aging process. We tested the effects of 2-month-daily acetyl-L-carnitine (ALCAR) supplementation on mitochondrial biogenesis in the soleus muscle of aged rats. This muscle is heavily dependent on oxidative metabolism. Mitochondrial (mt) DNA content, citrate synthase activity, transcript levels of some nuclear- and mitochondrial-coded genes (cytochrome c oxidase subunit IV [COX-IV], 16S rRNA, COX-I) and of some factors involved in the mitochondrial biogenesis signaling pathway (peroxisome proliferator-activated receptor gamma [PPARgamma] coactivator-1alpha [PGC-1alpha], mitochondrial transcription factor A mitochondrial [TFAM], mitochondrial transcription factor 2B [TFB2]), as well as the protein content of PGC-1alpha were determined. The results suggest that the ALCAR treatment in old rats activates PGC-1alpha-dependent mitochondrial biogenesis, thus partially reverting the age-related mitochondrial decay.

A comparison among the tissue-specific effects of aging and calorie restriction on TFAM amount and TFAM-binding activity to mtDNA in rat

BACKGROUND Mitochondrial Transcription Factor A (TFAM) is regarded as a histone-like protein of mitochondrial DNA (mtDNA), performing multiple functions for this genome. Aging affects mitochondria in a tissue-specific manner and only calorie restriction (CR) is able to delay or prevent the onset of several age-related changes also in mitochondria. METHODS Samples of the frontal cortex and soleus skeletal muscle from 6- and 26-month-old ad libitum-fed and 26-month-old calorie-restricted rats and of the livers from 18- and 28-month-old ad libitum-fed and 28-month-old calorie-restricted rats were used to detect TFAM amount, TFAM-binding to mtDNA and mtDNA content. RESULTS We found an age-related increase in TFAM amount in the frontal cortex, not affected by CR, versus an age-related decrease in the soleus and liver, fully prevented by CR. The semi-quantitative analysis of in vivo binding of TFAM to specific mtDNA regions, by mtDNA immunoprecipitation assay and following PCR, showed a marked age-dependent decrease in TFAM-binding activity in the frontal cortex, partially prevented by CR. An age-related increase in TFAM-binding to mtDNA, fully prevented by CR, was found in the soleus and liver. MtDNA content presented a common age-related decrease, completely prevented by CR in the soleus and liver, but not in the frontal cortex. CONCLUSIONS The modulation of TFAM expression, TFAM-binding to mtDNA and mtDNA content with aging and CR showed a trend shared by the skeletal muscle and liver, but not by the frontal cortex counterpart. GENERAL SIGNIFICANCE Aging and CR appear to induce similar mitochondrial molecular mechanisms in the skeletal muscle and liver, different from those elicited in the frontal cortex.