Differences in Liver TFAM Binding to mtDNA and mtDNA Damage between Aged and Extremely Aged Rats

Abstract

While mitochondrial dysfunction is acknowledged as a major feature of aging, much less is known about the role of mitochondria in extended longevity. Livers from aged (28-month-old) and extremely aged (32-month-old) rats were analyzed for citrate synthase activity, mitochondrial transcription factor A (TFAM) amount, mitochondrial DNA (mtDNA), and 4.8 Kb "common deletion" contents. None of the assayed parameters differed significantly between age groups. TFAM-binding to mtDNA and the incidence of 8-oxo-deoxyguanosine in specific mtDNA regions, encompassing the origins of mtDNA replication (D-loop and Ori-L) and the 16-bp long direct repeat 1 (DR1) of the 4.8 Kb deletion, were determined. A decrease in TFAM binding was unveiled at all regions in extremely aged in comparison with aged rats. Reduced incidence of oxidized purines at all assayed regions was detected in 32-month-old rats compared with the 28-month-old group. A significant positive correlation between the incidence of 8-oxo-deoxoguanosine and TFAM-bound mtDNA was found at D-Loop and Ori-L regions only in 28-month-old rats. The absence of such correlation in 32-month-old rats indicates a different, fine-tuned regulation of TFAM binding in the two age groups and supports the existence of two different paces in aging and extended aging.

Keywords: 8-oxodG incidence; TFAM binding; longevity; mtDNA common deletion; mtDNA content; rat liver.

Figure 1

Citrate synthase activity and mitochondrial transcription factor A (TFAM) relative amount in liver from 28- and 32-month-old rats. (A) Citrate synthase activity. Bars represent the mean value and SEM of experiments performed in quadruplicate. (B) Relative amounts of TFAM. Bars represent the mean and standard error of the mean (SEM) of the relative TFAM/β-actin value, obtained by densitometry analysis of the results from triplicated western blot experiments, from each 28- and 32-month-old rat. The comparison was made with respect to the value of the 28-month-old rats, fixed as 1. Right panel: representative western blot carried out in two rats from each assayed group. The bands, from top to bottom, show the signals from β-actin and TFAM; n = number of analyzed animals.

Figure 2

Relative contents of mitochondrial DNA (mtDNA) and of the 4.8 Kb mtDNA deletion in liver from 28- and 32-month-old rats. (A) Relative mtDNA content and (B) relative 4.8 Kb deletion content determined by quantitative PCR (qPCR). (A,B) Bars represent the mean value and SEM of two independent experiments conducted in triplicate. The comparisons were made with respect to the value of the 28-month-old rats, fixed as 1; n = number of analyzed animals.

Figure 3

Localization and relative amount of TFAM-bound mtDNA at three mtDNA regions in liver from 28- and 32-month-old rats. (A) The amplified products enclose part of D-loop, Ori-L, and DR1-harboring regions, as depicted in the rat mtDNA map. (B) Bars represent the mean and SEM of the relative amounts of TFAM-bound mtDNA of two independent experiments performed in triplicate. Number of analyzed animals: 5 in the 28-month-old group, 4 in the 32-month-old group; * p < 0.05, Mann Whitney test. Bars not showing the same superscript differ significantly; p < 0.01, Dunn’s Multiple comparison test after Kruskal-Wallis test.

Figure 4

Oxidized purines-specific mtDNA damage at the D-loop, Ori-L, and DR1 regions in liver from 28- and 32-month-old rats. (A) Bars represent the mean and SEM of the values obtained for each of the five animals analyzed in both groups. Bars not showing the same superscript differ significantly; p < 0.05, Dunn’s Multiple comparison test after Kruskal-Wallis test. (B) Representative gel of formamidopyrimidine DNA glycosylase (Fpg)-treated and untreated total DNA from a 28-month-old rat and a 32-month-old animal; 2.5 and 5 ng total DNA were amplified using the D-loop long primer set (Table 1). An aliquot of each PCR amplification was loaded onto agarose ethidium bromide-stained gel and analyzed for band intensities (a: molecular weight marker (GeneRuler 100 bp DNA Ladder, Thermo Fisher Scientific); b, c, e, f, h, i, m, and n: 2.5 ng total DNA as PCR template; d, g, l, and o: 5 ng total DNA as PCR template).

Figure 5

Correlation analysis between Fpg-sensitive damage and TFAM-bound mtDNA amounts at the D-loop and Ori-L regions by age groups. Data in the graph are mean values, obtained at the D-loop and Ori-L regions, respectively, from each analyzed rat. Number of analyzed animals: 5 in the 28-month-old group, 4 in the 32-month-old group; p, r = Spearman correlation test.