Biomedical subjects
Publications and source records attributed to F Fracasso.
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.
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.
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.
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.
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.
Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria.
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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.
A system for studying RNA synthesis in isolated mitochondria from rat brain was set up to investigate the mechanisms responsible for the age-dependent reduction of mtRNA content. In the presence of an appropriate incubation buffer both synaptic and non-synaptic mitochondria from cerebral hemispheres were able to synthesize and process mtRNA in a way quantitatively and qualitatively similar to the in vivo transcription. The comparison of the electrophoretic pattern of mtRNAs synthesized by adult and senescent rat showed, in the senescent rat, a 50% reduction in the mtRNA synthesis rate relative to the adult value. This indicates that the age-dependent decrease of the mtRNA content is linked to a lower efficiency of the mt transcription.
The effect of acetyl-L-carnitine on the quantity of the messenger RNA for the subunit I of cytochrome oxidase in the liver mitochondria of hypothyroid rat was measured by Northern blot and solution hybridization. Three hours after pre-treatment of hypothyroid rat with acetyl-L-carnitine, the level of the transcript increased strongly. This effect was also obtained when acetyl-L-carnitine was administered to T3 pre-treated hypothyroid rats. These results add further evidence to the suggestion that acetyl-L-carnitine is able to stimulate mitochondrial transcription under altered metabolic conditions.
A quantitative study on the effect of senescence on mitochondrial DNA expression has been carried out by measuring the levels of the 12S rRNA and of the mRNA for the subunit I of cytochrome oxidase in several tissues of adult and senescent rats. The concentration of both RNA species/mitochondrial DNA molecule is significantly reduced in senescent brain and heart, as opposed to the respective adult tissues. No appreciable variation occurs in the liver. A 1-h pretreatment with acetyl-L-carnitine brings back the level of senescent brain and heart transcripts to that of adult tissues. The same treatment of adult rats does not cause significant changes in mitochondrial RNA content. These results suggest that the age-dependent impairment of both heavy-strand mitochondrial DNA transcription units is related to altered environmental conditions which acetyl-L-carnitine, a substance which acts by stimulating, directly or indirectly, the energy metabolism, is able to remove.
The content of DNA and of 16S rRNA and of two mRNAs, i.e., the mRNA for the cytochrome c oxidase subunit I and the mRNA for one subunit of the NADH dehydrogenase (ND4), in free (nonsynaptic) mitochondria of developing and adult rat cerebellum has been determined. During postnatal development, DNA content of free (nonsynaptic) mitochondria increases 10 times from 1 to 30 days of age whereas, in adult rats, it is about 60% compared to that found in 30-day-old rats. The total content of each RNA species studied also increases during development. However, when the content of each RNA is expressed per mtDNA molecule, rRNAs and mRNAs behave differently: 16S rRNA level does not change during development and it is not significantly different from that of the adult rat, whereas the level of mRNAs is higher during development than in the adult rat and changes with age. These results are discussed in light of mitochondrial biogenesis in rat cerebellum during development and of the regulation of the mitochondrial DNA transcription process.
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 (t1/2 approximately 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.
A quantitative study on the concentration of mitochondrial DNA and two species of mtRNA, the ribosomal (16S rRNA) and messenger (CoI mRNA) has been carried out in rat liver between -3 and 14 days of age. The cellular content of mitochondrial DNA begins to increase at one day of life and goes up linearly until 14 days of age. The cellular level of 16S rRNA and CoI mRNA changes during development: the 16S rRNA increases linearly after birth, whereas CoI mRNA shows a peak at birth and thereafter remains more or less constant. The concentration of 16S rRNA per mitochondrial DNA molecule remains substantially unchanged during development, whereas that of CoI mRNA increases before birth and, at birth, reaches values higher than in adults. These results support an independent regulation of mitochondrial rRNA and mRNA level in rat liver mitochondria during development.
A quantitative analysis of the frequency of the supercoiled mitochondrial DNA molecules containing the D-loop in rat heart and cerebral hemispheres, at different ages, is presented. Both tissues of aged animals exhibit a remarkable reduction in the content of super-coiled D-loop containing molecules compared to the adults. This alteration could be responsible for the age-dependent reduction of mitochondrial DNA transcription previously observed in rat brain and heart.