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-monthold calorie-restricted rats were used to detect TFAM amount, TFAM-binding to mtDNA and mtDNA content. Results:We found an age-related increase in TFAMamount in the frontal cortex, not affected by CR, versus an agerelated 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 agerelated 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.

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 [PPAR gamma] coactivator-1 alpha [PGC-1 alpha], mitochondrial transcription factor A mitochondrial [TFAM], mitochondrial transcription factor 2B [TFB2]), as well as the protein content of PGC-1 alpha were determined. The results suggest that the ALCAR treatment in old rats activates PGC-1 alpha-dependent mitochondrial biogenesis, thus partially reverting the age-related mitochondrial decay.

Aging affects mitochondria in a tissue-specific manner and to date only calorie restriction (CR) is able to delay or prevent the onset of several age-related changes also in mitochondria. Mitochondrial Transcription Factor A (TFAM) is considered the histone-like protein of mitochondrial DNA (mtDNA), performing multiple functions for this genome including modulation of transcription, regulation of copy number and maintenance of mtDNA as well as constitution of mtDNA nucleoids. We used samples of frontal cortex and soleus skeletal muscle from 6- and 26-month-old ad libitum-fed and 26-month-old calorie-restricted rats and of livers from 18- and 28-month-old ad libitum-fed and 28-month-old calorie-restricted rats to determine TFAM amount, TFAM-binding to mtDNA and mtDNA content. We found an age-related increase in TFAM amount in frontal cortex, not affected by CR, versus an age-related decrease in 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, showed a marked age-dependent decrease in TFAM-binding activity in the frontal cortex, partially prevented by CR. Instead, an age-related increase in TFAM-binding to mtDNA, fully prevented by CR, was found in the soleus and liver. MtDNA content showed a common age-related decrease, completely prevented by CR in soleus and liver, but not in frontal cortex. We highlight for the first time a trend shared by skeletal muscle and liver, but not by the frontal cortex counterpart, in some relevant changes induced by aging and CR in mitochondria. Such trend involves the modulation of TFAM expression, TFAM-binding to mtDNA and mtDNA content and supports the existence of similar mitochondrial molecular mechanisms in skeletal muscle and liver but not in frontal cortex. Modulation of TFAM-binding to mtDNA, affecting mtDNA functions, appears to contribute to the tissue-specific alterations of mitochondrial biogenesis during aging as well as CR.

Aging markedly affects mitochondrial biogenesis and functions particularly in tissues highly dependent on the organelle’s bioenergetics capability such as the brain’s frontal cortex. Calorie restriction (CR) diet is, so far, the only intervention able to delay or prevent the onset of several age-related alterations in different organisms. We determined the contents of mitochondrial transcription factor A (TFAM), mitochondrial DNA (mtDNA), and the 4.8-kb mtDNA deletion in the frontal cortex from young (6-monthold) and aged (26-month-old), ad libitum-fed (AL) and calorie-restricted (CR), rats. We found a 70 % increase in TFAM amount, a 25 % loss in mtDNA content, and a 35 % increase in the 4.8-kb deletion content in theaged AL animals with respect to the young rats. TFAM-specific binding to six mtDNA regions was analyzed by mtDNA immunoprecipitation and semiquantitative polymerase chain reaction (PCR), showing a marked age-related decrease. Quantitative realtime PCR at two subregions involved in mtDNA replication demonstrated, in aged AL rats, a remarkable decrease (60–70 %) of TFAM-bound mtDNA. The decreased TFAM binding is a novel finding that may explain the mtDNA loss in spite of the compensatory TFAM increased amount. In aged CR rats, TFAM amount increased and mtDNA content decreased with respect to young rats’ values, but the extent of the changes was smaller than in aged AL rats. Attenuation of the age-related effects due to the diet in the CR animals was further evidenced by the unchanged content of the 4.8-kb deletion with respect to that of young animals and by the partial prevention of the age-related decrease in TFAM binding to mtDNA.

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 agerelated 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 ageand 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.

The knowledge of the haplogroups of the analyzed subjects allows to prevent errors in the analysis of the association between mtDNA variations and some pathologies like diabetes. In this paper a cohort of type 2 diabetic Italian patients with known mitochondrial genetic background (haplogroup) were investigated for the presence in the blood of the 3243A>G mutation and other variants of close to tRNA(Leu(UUR)) gene (16S/ND1 region) of mitochondrial DNA (mtDNA). The 3243A>G mutation was present in 0.4% of analyzed patients in agreement with the reported frequencies of such mutation in other European groups. This result suggests a limited role for the 3243A>G mutation in the pathogenesis of diabetes also in the Italian population. In the 16S/ND1 region 15 base changes were found. The knowledge of the haplogroup of the analyzed individuals

Background: Mitochondrial dysfunction and oxidative stress are central mechanisms underlying the aging process and the pathogenesis of many age-related diseases. Selected antioxidants and specific combinations of nutritional compounds could target many biochemical pathways that affect both oxidative stress and mitochondrial function and, thereby, preserve or enhance physical performance. Methodology/Principal Findings: In this study, we evaluated the potential anti-aging benefits of a Q-terH based nutritional mixture (commercially known as EufortynH) mainly containing the following compounds: terclatrated coenzyme Q10 (QterH), creatine and a standardized ginseng extract. We found that EufortynH supplementation significantly ameliorated the age-associated decreases in grip strength and gastrocnemius subsarcolemmal mitochondria Ca2+ retention capacity when initiated in male Fischer344 x Brown Norway rats at 21 months, but not 29 months, of age. Moreover, the increases in muscle RNA oxidation and subsarcolemmal mitochondrial protein carbonyl levels, as well as the decline of total urine antioxidant power, which develop late in life, were mitigated by EufortynH supplementation in rats at 29 months of age. Conclusions/Significance: These data imply that EufortynH is efficacious in reducing oxidative damage, improving the agerelated mitochondrial functional decline, and preserving physical performance when initiated in animals at early midlife (21 months). The efficacy varied, however, according to the age at which the supplementation was provided, as initiation in late middle age (29 months) was incapable of restoring grip strength and mitochondrial function. Therefore, the EufortynH supplementation may be particularly beneficial when initiated prior to major biological and functional declines that appear to occur with advancing age.

Mitochondrial biogenesis is among the organelle’s features affected by aging in tissue-specific ways. The only intervention, so far, able to delay or prevent the onset of several age-related changes, also in mitochondria, is calorie restriction (CR). Aging and CR both influence in a relevant manner mitochondrial structure and function in rat liver. Using livers from 18-month-old, 28-month-old, 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. Also the proteins PGC-1 and NRF-1 as well as the nuclear-encoded cytochrome c oxidase subunit IV revealed an age-related decrease, confirming the reduction in mitochondrial biogenesis with aging, that was completely prevented by CR. The already described age-related increased oxidative stress was verified and also prevented by CR. We reported with aging a decrease in peroxiredoxin III (Prx III) and in mitochondrial superoxide dismutase 2 (SOD2) proteins, usually sensitive to an increased ROS presence and also involved in mitochondrial biogenesis, which suggested a prevailing relevance of the age-reduced mitochondrial biogenesis above the induction by ROS in the regulation of expression with aging. Furthermore, the full prevention of the decrease in Prx III and SOD2 by CR suggested that, in rat liver, the preservation of the mitochondrial biogenesis might be more relevant than the reduction of the oxidative stress in the anti-aging action of CR. We then analyzed the in vivo TFAM binding to specific mtDNA regions finding a marked increase in the TFAM-bound amounts of mtDNA at both origins of replication with aging, fully prevented by CR. Joining the data from the 18-month-old ad libitum-fed and the 28-month-old calorie-restricted samples we could find a novel, positive correlation between the coupled amounts of TFAM-bound mtDNA at the origins of replication, not present in the 28-month-old ad libitum-fed animals, thus suggesting a quite different modulation of TFAM binding at these sub-regions in aging and in CR, likely involved in the respective alterations of mitochondrial biogenesis.

Mitochondrial biogenesis and remodeling occur in response to exercise and redox state (reviewed in Scarpulla et al. 2012, Handy and Loscalzo 2012, Piantadosi and Suliman 2012, Scarpulla 2011, Wenz et al. 2011, Bo et al. 2010, Jornayvaz and Shulman 2010, Ljubicic et al. 2010, Hock and Kralli 2009, Canto and Auwerx 2009, Lin 2009, Scarpulla 2008, Ventura-Clapier et al. 2008). It is hypothesized that calcium influx and energy depletion are the signals that initiate changes in gene expression leading to new mitochondrial proteins. Energy depletion causes a reduction in ATP and an increase in AMP which activates AMPK. AMPK in turn phosphorylates the coactivator PGC-1alpha (PPARGC1A), one of the master regulators of mitochondrial biosynthesis. Likewise, p38 MAPK is activated by muscle contraction (possibly via calcium and CaMKII) and phosphorylates PGC-1alpha. CaMKIV responds to intracellular calcium by phosphorylating CREB, which activates expression of PGC-1alpha. Deacetylation of PGC-1alpha by SIRT1 may also play a role in activation (Canto et al. 2009, Gurd et al. 2011), however Sirt11 deacetylation of Ppargc1a in mouse impacted genes related to glucose metabolism rather than mitochondrial biogenesis (Rodgers et al. 2005) and mice lacking SIRT1 in muscle had normal levels of mitochondrial biogenesis in response to exercise (Philp et al. 2011) so the role of deacetylation is not fully defined. PGC-1beta and PPRC appear to act similarly to PGC-1alpha but they have not been as well studied. Phosphorylated PGC-1alpha does not bind DNA directly but instead interacts with other transcription factors, notably NRF1 and NRF2 (via HCF1). NRF1 and NRF2 together with PGC-1alpha activate the transcription of nuclear-encoded, mitochondrially targeted proteins such as TFB2M, TFB1M, and TFAM.

Mitochondrial Transcription Factor A (TFAM) is a nuclear-encoded factor present in mitochondria from all kinds of animals, even with a homologous form in yeast, which has strongly and progressively gained an eminent relevance in the regulation of mitochondrial DNA (mtDNA) functions. This is due to the various functions performed by the protein, which cover a very wide scenario including: replication, transcription, maintenance and eventually repair of mtDNA molecules. In consideration of so many different roles it plays, it can be understood there is a deep interest in its regulation of expression and activity as well as its eventual involvement in pathologies related to a decreased functionality/presence of the mitochondrial genome and the consequences of TFAM induced overexpression.

According to the mitochondrial theory of aging mitochondria are involved with aging through endogenous production of oxidants. Recently it has become clear that in all species there is a marked decline in mitochondrial (mt) DNA, but the mechanisms are unknown. Calorie restriction (CR) is, so far, the only treatment shown to delay or avoid the onset of many age-related features in various species. Mitochondrial transcription factor A (TFAM) is a nuclear-encoded protein that plays a critical role in maintaining mtDNA structural integrity and functionality. Based on TFAM function in mtDNA maintenance and on the known accumulation of mtDNA oxidative DNA damage during aging we have tested whether the efficiency and/or the fidelity of TFAM binding to mtDNA changes with age in rat brain and liver tissues. We have measured TFAM binding to several functionally relevant mtDNA regions by mtDNA immunoprecipitation with TFAM policlonal antibody. We found tissue-specific changes in TFAM binding to mtDNA as a function of aging and CR treatment. These changes vary depending on the functional relevance of the DNA region bound, suggesting that TFAM functions in mtDNA maintenance processes (i.e. in replication and transcription) may be compromised during aging.