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Autism spectrum disorder (ASD) is a neurodevelopmental disorder displaying the modification of complex human behaviors, characterized by social interaction impairments, stereotypical/repetitive activities and emotional dysregulation. In this study, fecal microbiota transplant (FMT) via gavage from autistic children donors to mice, led to the colonization of ASD-like microbiota and autistic behaviors compared to the offspring of pregnant females exposed to valproic acid (VPA). Such variations seemed to be tightly associated with increased populations of Tenericutes plus a notable reduction (p 0.001) of Actinobacteria and Candidatus S. in the gastrointestinal region of FMT mice as compared to controls. Indeed altered behaviors of FMT mice was reported when evaluated in the different maze tests (light dark, novel object, three chamber tests, novel cage test). Contextually, FMT accounted for elevated expression levels of the pro-inflammatory factors IL-1β, IL-6, COX-1 and TNF-α in both brain and small intestine. Villous atrophy and inflammatory infiltration (Caspase 3 and Ki67) were increased in the small intestine of FMT and VPA mice compared to controls. Moreover, the observed FMT-dependent alterations were linked to a decrease in the methylation status. Overall, findings of the present study corroborate a key role of gut microbiota in ASD. However, further investigations are required before any possible manipulation of gut bacteria with appropriate diets or probiotics can be conducted in ASD individuals.

Aging and age-related diseases have been linked to microbial dysbiosis with changes in blood bacterial DNA concentration. This condition may promote chronic low-grade inflammation, which can be further aggravated by antioxidant nutrient deficiency. Low plasma carotenoids are associated with an increased risk of inflammation and cellular damage and predict mortality. However, no evidence is yet available on the relationship between antioxidants and the blood bacterial DNA (BB-DNA). Therefore, this study aimed to compare BB-DNA from (a) GO (nonagenarian offspring), (b) age-matched controls (Randomly recruited Age-Stratified Individuals from the General population [RASIG]), and (c) spouses of GO (SGO) recruited in the MARK-AGE project, as well as to investigate the association between BB-DNA, behavior habits, Charlson Comorbidity Index (CCI), leucocyte subsets, and the circulating levels of some antioxidants and oxidative stress markers. BB-DNA was higher in RASIG than GO and SGO, whereas GO and SGO participants showed similar values. BB-DNA increased in smokers and males with CCI ≥ 2 compared with those with CCI ≤ 1 within RASIG. Moreover, BB-DNA was positively associated with lymphocyte, neutrophil, and monocyte counts, but not with self-reported dietary habits. Higher quartiles of BB-DNA were associated with low lutein and zeaxanthin and elevated malondialdehyde plasma concentrations in RASIG. BB-DNA was also positively correlated with nitric oxide levels. Herein, we provide evidence of a reduced BB-DNA in individuals from long-living families and their spouses, suggesting a decreased microbial dysbiosis and bacterial systemic translocation. BB-DNA was also associated with smoking, CCI, leukocyte subsets, and some redox biomarkers in older participants.

Forensic DNA phenotyping refers to an emerging field of forensic sciences aimed at the prediction of externally visible characteristics of unknown sample donors directly from biological materials. The aging process significantly affects most of the above characteristics making the development of a reliable method of age prediction very important. Today, the so-called "epigenetic clocks" represent the most accurate models for age prediction. Since they are technically not achievable in a typical forensic laboratory, forensic DNA technology has triggered efforts toward the simplification of these models. The present study aimed to build an epigenetic clock using a set of methylation markers of five different genes in a sample of the Italian population of different ages covering the whole span of adult life. In a sample of 330 subjects, 42 selected markers were analyzed with a machine learning approach for building a prediction model for age prediction. A ridge linear regression model including eight of the proposed markers was identified as the best performing model across a plethora of candidates. This model was tested on an independent sample of 83 subjects providing a median error of 4.5 years. In the present study, an epigenetic model for age prediction was validated in a sample of the Italian population. However, its applicability to advanced ages still represents the main limitation in forensic caseworks.

Background The cline of human genetic diversity observable across Europe is recapitulated at a micro-geographic scale by variation within the Italian population. Besides resulting from extensive gene flow, this might be ascribable also to local adaptations to diverse ecological contexts evolved by people who anciently spread along the Italian Peninsula. Dissecting the evolutionary history of the ancestors of present-day Italians may thus improve the understanding of demographic and biological processes that contributed to shape the gene pool of European populations. However, previous SNP array-based studies failed to investigate the full spectrum of Italian variation, generally neglecting low-frequency genetic variants and examining a limited set of small effect size alleles, which may represent important determinants of population structure and complex adaptive traits. To overcome these issues, we analyzed 38 high-coverage whole-genome sequences representative of population clusters at the opposite ends of the cline of Italian variation, along with a large panel of modern and ancient Euro-Mediterranean genomes. Results We provided evidence for the early divergence of Italian groups dating back to the Late Glacial and for Neolithic and distinct Bronze Age migrations having further differentiated their gene pools. We inferred adaptive evolution at insulin-related loci in people from Italian regions with a temperate climate, while possible adaptations to pathogens and ultraviolet radiation were observed in Mediterranean Italians. Some of these adaptive events may also have secondarily modulated population disease or longevity predisposition. Conclusions We disentangled the contribution of multiple migratory and adaptive events in shaping the heterogeneous Italian genomic background, which exemplify population dynamics and gene-environment interactions that played significant roles also in the formation of the Continental and Southern European genomic landscapes.

Patterns of DNA methylation, the best characterized epigenetic modification, are modulated by aging. In humans, different studies at both site-specific and genome-wide levels have reported that modifications of DNA methylation are associated with the chronological aging process but also with the quality of aging (or biological aging), providing new perspectives for establishing powerful biomarkers of aging.:In this article, the role of DNA methylation in aging and longevity has been reviewed by analysing literature data about DNA methylation variations occurring during the lifetime in response to environmental factors and genetic background, and their association with the aging process and, in particular, with the quality of aging. Special attention has been devoted to the relationship between nuclear DNA methylation patterns, mitochondrial DNA epigenetic modifications, and longevity. Mitochondrial DNA has recently been reported to modulate global DNA methylation levels of the nuclear genome during the lifetime, and, in spite of the previous belief, it has been found to be the target of methylation modifications.:Analysis of DNA methylation profiles across lifetime shows that a remodeling of the methylome occurs with age and/or with age-related decline. Thus, it can be an excellent biomarker of aging and of the individual decline and frailty status. The knowledge about the mechanisms underlying these modifications is crucial since it might allow the opportunity for targeted treatment to modulate the rate of aging and longevity.

The mitochondrial quality control network includes several epigenetically-regulated genes involved in mitochondrial dynamics, mitophagy, and mitochondrial biogenesis under physiologic conditions. Dysregulated expression of such genes has been reported in various disease contexts, including cancer. However, their expression pattern and the possible underlying epigenetic modifications remain to be defined within plasma cell (PC) dyscrasias. Herein, we compared the mRNA expression of mitochondrial quality control genes from multiple myeloma, plasma cell leukemia patients and human myeloma cell lines (HMCLs) with healthy plasma cells; moreover, by applying the Sequenom MassARRAY EpiTYPER technology, we performed a pilot investigation of their CpG methylation status in HMCLs. Overall, the results provided indicate dysregulated expression of several mitochondrial network’s genes, and alteration of the CpG methylation profile, underscoring novel potential myeloma biomarkers deserving in-depth functional investigation in the future.

Extreme longevity is the paradigm of healthy aging as individuals who reached the extreme decades of human life avoided or largely postponed all major age-related diseases. In this study, we sequenced at high coverage (90X) the whole genome of 81 semi-supercentenarians and supercentenarians [105+/110+] (mean age: 106.6 ± 1.6) and of 36 healthy unrelated geographically matched controls (mean age 68.0 ± 5.9) recruited in Italy. The results showed that 105+/110+ are characterized by a peculiar genetic background associated with efficient DNA repair mechanisms, as evidenced by both germline data (common and rare variants) and somatic mutations patterns (lower mutation load if compared to younger healthy controls). Results were replicated in a second independent cohort of 333 Italian centenarians and 358 geographically matched controls. The genetics of 105+/110+ identified DNA repair and clonal haematopoiesis as crucial players for healthy aging and for the protection from cardiovascular events.

A number of epigenetic studies have demonstrated that DNA methylation patterns exhibit a tissue specificity, but not much has been done to highlight the extent of this phenomenon. Moreover, it is unknown how external factors modulate the plasticity of the tissue specific epigenetic profile. We examined global DNA methylation profiles in tissues from rats of different age, fed with standard or low-calorie diet, and evaluated their association with aging and nutrition. Tissue-specific variations occur during aging with hyper-methylation taking place in all tissues except for liver. The expression of enzymes involved in methylation reactions (DNMTs and TETs) was consistent with the methylation patterns. Nutrition affects global DNA methylation status throughout lifespan. Interestingly, the differences among different tissues are magnified in 96 weeks old rats fed with low calorie diet. Moreover, the low-calorie diet appears to affect the offspring's epigenetic status more strongly if administered during the maternal pre-gestational period than the gestational and lactation time. Therefore, we propose that changes in the global DNA methylation status may represent an epigenetic mechanism by which age and nutrition intersect each other and, in turn, influence the aging plasticity.

Given the role of amino acid regulation for physiological and pathological cell proliferation, we investigated whether the variability of solute carrier family 1, member 5 (SLC1A5, namely ASCT2), encoding for ASCT2 protein, a major glutamine transporter, is related to longevity. A total of 607 differently aged unrelated individuals, 351 very old subjects (≥85 years, range 85-106 years, mean age 93.82 ± 4.44 years) and 256 younger controls (

An increasing body of data is progressively indicating that the comprehension of the epigenetic landscape, actively integrated with the genetic elements, is crucial to delineate the molecular basis of the inter-individual complexity of ageing process. Indeed, it has emerged that DNA methylation changes occur during ageing, consisting mainly in a progressive process of genome demethylation, in a hypermethylation of gene-specific CpG dinucleotides, as well as in an inter-individual divergence of the epigenome due to stochastic events and environmental exposures throughout life, namely as epigenetic drift. Additionally, it has also come to light an implication of the mitochondrial genome in the regulation of the intracellular epigenetic landscape, as demonstrated by the being itself object of epigenetic modifications. An overview of DNA methylation changes occurring during ageing process at both nuclear and mitochondrial level will be described in this review, also taking into account the recent and promising data available on the 5-hydroxymethylcytosine.

Summary The transcription of ribosomal RNA genes (rDNA) is subject to epigenetic regulation, as it is abrogated by the methylation of CpG dinucleotides within their promoter region. Here, we investigated, through Sequenom platform, the age-related methylation status of the CpG island falling into the rDNA promoter in 472 blood samples from 20- to 105-year-old humans and in different tissues (blood, heart, liver, kidney, and testis) of 15 rats 3–96 weeks old. In humans, we did not find a consistently significant correlation between CpG site methylation and chronological age. Furthermore, the methylation levels of one of the analyzed CpG sites were negatively associated with both cognitive performance and survival chance measured in a 9-year follow-up study. We consistently confirmed such result in a replication sample. In rats, the analysis of the homologous region in the tissues revealed the existence of increased methylation in old rats. rRNA expression data, in both humans and rats, were consistent with observed methylation patterns, with a lower expression of rRNA in highly methylated samples. As chronological and biological ages in rats of a given strain are likely to be much closer to each other than in humans, these results seem to provide the first evidence that epigenetic modifications of rDNA change over time according to the aging decline. Thus, the methylation profile of rDNA may represent a potential biomarker of aging.

Sirtuins are conserved NAD+ -dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1-SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1-SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1-silenced cell lines. Our results demonstrate that SIRT1-silenced cells are more resistant to H2 O2 and etoposide treatment showing decreased ROS accumulation, γ-H2AX phosphorylation, caspase-3 activation and PARP cleavage. Interestingly, we observed that SIRT1-silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1-silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co-immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1-silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835-1844, 2017. © 2016 Wiley Periodicals, Inc.

Multiple Myeloma (MM) is a clonal late B-cell disorder accounting for about 13% of hematological cancers and 1% of all neoplastic diseases. Recent studies on the molecular pathogenesis and biology of MM have highlighted a complex epigenomic landscape contributing to MM onset, prognosis and high individual variability. Areas covered: We describe here the current knowledge on epigenetic events characterizing MM initiation and progression, focusing on the role of DNA and histone methylation and on the most promising epi-therapeutic approaches targeting the methylation pathway. Expert opinion: Data published so far indicate that alterations of the epigenetic framework, which include aberrant global or gene/non-coding RNA specific methylation profiles, feature prominently in the pathobiology of MM. Indeed, the aberrant expression of components of the epigenetic machinery as well as the reversibility of the epigenetic marks make this pathway druggable, providing the basis for the design of epigenetic therapies against this still fatal malignancy.

The plasticity of the individual epigenetic landscape that goes to countless rearrangements throughout life is closely the reflection of environmental factors such as chemical exposure, socio-economic status and nutrient intakes both early and late in life. The Mini Nutritional Assessment (MNA) is a well-validated tool for assessing malnutrition in old people. It includes 6 (MNA-SF) or 18 (MNA-LF) self-reported questions derived from general, anthropometric, dietary, and self- assessment. We evaluated the association between the nutritional status, as measured by MNA, and methylation biomarkers we previously demonstrated to be associated with chronological and biological age in human. We found that malnutrition is positively correlated with DNA methylation status at the global level, in line with our previous reports. On the contrary, most of the sites located within specific genes, which were previously reported to be correlated with chronological and biological aging, showed to be not affected by malnutrition, or even to have correlations with malnutrition opposite to those previously reported with frailty. These results may suggest that malnutrition is among the first effects of disability and other age- related problems and a generalized non-specific epigenetic remodeling may be the initial response of the organism. By contrast, the fine remodeling of specific genomic sites is scarcely affected by malnutrition and may respond to a more complex interaction of different factors. Therefore, although malnutrition in the elderly is certainly a risk factor for survival, this is partially independent of the aging process of the organism which leads to the methylation remodeling previously described to measure chronological and biological aging.

Maintenance of functional mitochondria is essential to prevent damage leading to aging and diseases. What is more, the research of biomarkers of aging is focusing on better predicting functional capability along the lifetime beyond chronological age. Aim of this study was to identify novel CpG sites the methylation of which might be correlated to the chronological and biological age. We performed methylation analyses of the CpG sites in candidate genes involved in mitochondrial biogenesis, mitophagy, fusion, and fission, all key quality control mechanisms to ensure maintenance of healthy mitochondria and homeostasis during aging, using DNA samples from two independent datasets composed by 381 and 468 differently-aged individuals, respectively. Twelve potential CpG predictors resulted associated with aging in the discovery dataset. Of these, two sites located within RAB32 and RHOT2 genes were replicated in the second dataset. What is more, individuals exhibiting methylation levels of the RAB32 CpG site higher than 10% were observed more prone to disability than people with lower levels. These results seem to provide the first evidence that epigenetic modifications of genes involved in mitochondrial quality control occur over time according to the aging decline, and may then represent potential biomarkers of both chronological and biological age.

The involvement of mitochondrial content, structure and function as well as of the mitochondrial genome (mtDNA) in cell biology, by participating in the main processes occurring in the cells, has been a topic of intense interest for many years. More specifically, the progressive accumulation of variations in mtDNA of post-mitotic tissues represents a major contributing factor to both physiological and pathological phenotypes. Recently, an epigenetic overlay on mtDNA genetics is emerging, as demonstrated by the implication of the mitochondrial genome in the regulation of the intracellular epigenetic landscape being itself object of epigenetic modifications. Indeed, in vitro and population studies strongly suggest that, similarly to nuclear DNA, also mtDNA is subject to methylation and hydroxymethylation. It follows that the mitochondrial-nucleus cross talk and mitochondrial retrograde signaling in cellular properties require a concerted functional cooperation between genetic and epigenetic changes. The present paper aims to review the current advances in mitochondrial epigenetics studies and the increasing indication of mtDNA methylation status as an attractive biomarker for peculiar pathological phenotypes and environmental exposure.

Summary The transcription of ribosomal RNA genes (rDNA) is subject to epigenetic regulation, as it is abrogated by the methylation of CpG dinucleotides within their promoter region. Here, we investigated, through Sequenom platform, the age‐related methylation status of the CpG island falling into the rDNA promoter in 472 blood samples from 20‐ to 105‐year‐old humans and in different tissues (blood, heart, liver, kidney, and testis) of 15 rats 3–96 weeks old. In humans, we did not find a consistently significant correlation between CpG site methylation and chronological age. Furthermore, the methylation levels of one of the analyzed CpG sites were negatively associated with both cognitive performance and survival chance measured in a 9‐year follow‐up study. We consistently confirmed such result in a replication sample. In rats, the analysis of the homologous region in the tissues revealed the existence of increased methylation in old rats. rRNA expression data, in both humans and rats, were consistent with observed methylation patterns, with a lower expression of rRNA in highly methylated samples. As chronological and biological ages in rats of a given strain are likely to be much closer to each other than in humans, these results seem to provide the first evidence that epigenetic modifications of rDNA change over time according to the aging decline. Thus, the methylation profile of rDNA may represent a potential biomarker of aging.

Oxidative stress is both the cause and consequence of impaired functional homeostasis characterizing human aging. The worsening efficiency of stress response with age represents a health risk and leads to the onset and accrual of major age-related diseases. In contrast, centenarians seem to have evolved conservative stress response mechanisms, probably derived from a combination of a diet rich in natural antioxidants, an active lifestyle and a favorable genetic background, particularly rich in genetic variants able to counteract the stress overload at the level of both nuclear and mitochondrial DNA. The integration of these factors could allow centenarians to maintain moderate levels of free radicals that exert beneficial signaling and modulator effects on cellular metabolism. Considering the hot debate on the efficacy of antioxidant supplementation in promoting healthy aging, in this review we gathered the existing information regarding genetic variability and lifestyle factors which potentially modulate the stress response at old age. Evidence reported here suggests that the integration of lifestyle factors (moderate physical activity and healthy nutrition) and genetic background could shift the balance in favor of the antioxidant cellular machinery by activating appropriate defense mechanisms in response to exceeding external and internal stress levels, and thus possibly achieving the prospect of living a longer life.

DNA methylation is a common epigenetic modification of the mammalian genome. Conflicting data regarding the possible presence of methylated cytosines within mitochondrial DNA (mtDNA) have been reported. To clarify this point, we analysed the methylation status of mtDNA control region (D-loop) on human and murine DNA samples from blood and cultured cells by bisulphite sequencing and methylated/hydroxymethylated DNA immunoprecipitation assays. We found methylated and hydroxymethylated cytosines in the L-strand of all samples analysed. MtDNA methylation particularly occurs within non-C-phosphate-G (non-CpG) nucleotides, mainly in the promoter region of the heavy strand and in conserved sequence blocks, suggesting its involvement in regulating mtDNA replication and/or transcription. We observed DNA methyltransferases within the mitochondria, but the inactivation of Dnmt1, Dnmt3a, and Dnmt3b in mouse embryonic stem (ES) cells results in a reduction of the CpG methylation, while the non-CpG methylation shows to be not affected. This suggests that D-loop epigenetic modification is only partially established by these enzymes. Our data show that DNA methylation occurs in the mtDNA control region of mammals, not only at symmetrical CpG dinucleotides, typical of nuclear genome, but in a peculiar non-CpG pattern previously reported for plants and fungi. The molecular mechanisms responsible for this pattern remain an open question. © The Author 2013.

Over the past two decades, a growing interest on the research of the biological basis of human longevity has emerged, in order to clarify the intricacy of biological and environmental factors affecting (together with stochastic factors) the quality and the rate of human aging. These researches have outlined a complex scenario in which epigenetic marks, such as DNA methylation and numerous histone modifications, are emerging as important factors of the overall variation in life expectancy. In fact, epigenetic marks, that are responsible of the establishment of specific expression programs and of genome stability, represent a "drawbridge" across genetic, environmental and stochastic factors. In this review we provide an overview on the current knowledge and the general features of the epigenetic modifications characterizing the aging process.

Sirtuins are conserved NAD

Signaling pathways between mitochondrial and nuclear genomes are activated to preserve cellular homeostasis, especially in the event of stress. Using cybrid cell lines, we investigated whether inherited mitochondrial DNA (mtDNA) variants modulate the expression profiles of mammalian sirtuins (SIRT1-7) under oxidative stress conditions. We found that the expression of the SIRT3 gene was down-regulated in cybrids harboring mtDNA of the J haplogroup, which correlated with mitochondrial function, resulting in a decline of NAD(+)/NADH and ATP levels. Overall, the data reported here highlight a link between SIRT3, mitochondrial DNA variability and mitochondrial functionality, three fundamental components of the cellular stress response.

Aim: In the present study, we investigated whether global DNA methylation levels are affected by mitochondrial DNA (mtDNA) variants, which are known to modulate mitochondrial functions. Materials & methods: Global DNA methylation levels were evaluated in peripheral blood DNA collected from adult subjects and in vitro using the DNA of cybrid cells harboring mtDNAs of different haplogroups. In these cells, mRNA expression of genes involved in DNA methylation processes, and ATP and reactive oxygen species levels were also analyzed. Results: The analysis revealed that methylation levels were higher in the subjects carrying the J haplogroup than in non-J carriers. Consistently, cybrids with J haplogroup mtDNA showed higher methylation levels than other cybrids. Interestingly, we observed overexpression of the MAT1A gene and low ATP and ROS levels in J cybrids. Conclusion: Our findings indicate that mtDNA-specific interactions between mitochondria and the nucleus regulate epigenetic changes, possibly by affecting oxidative phosphorylation efficiency.

The brown fat specific UnCoupling Protein 1 (UCP1) is involved in thermogenesis, a process by which energy is dissipated as heat in response to cold stress and excess of caloric intake. Thermogenesis has potential implications for body mass control and cellular fat metabolism. In fact, in humans, the variability of the UCP1 gene is associated with obesity, fat gain and metabolism. Since regulation of metabolism is one of the key-pathways in lifespan extension, we tested the possible effects of UCP1 variability on survival. Two polymorphisms (A-3826G and C-3740A), falling in the upstream promoter region of UCP1 , were analyzed in a sample of 910 subjects from southern Italy (475 women and 435 men; age range 40–109). By analyzing haplotype specific survival functions we found that the A-C haplotype favors survival in the elderly. Consistently, transfection experiments showed that the luciferase activity of the construct containing the A-C haplotype was significantly higher than that containing the G-A haplotype. Interestingly, the different UCP1 haplotypes responded differently to hormonal stimuli. The results we present suggest a correlation between the activity of UCP1 and human survival, indicating once again the intricacy of mechanisms involved in energy production, storage and consumption as the key to understanding human aging and longevity.

Epigenetic variations have been widely described to occur during the aging process. To verify if these modifications are correlated with the inter-individual phenotypic variability of elderly people, we searched for a correlation between global DNA methylation levels and frailty. We found that the global DNA methylation levels were correlated to the frailty status in middle/advanced-aged subjects but not with age. A 7-year follow-up study also revealed that a worsening in the frailty status was associated to a significant decrease in the global DNA methylation levels. These results suggest that the relaxation of the epigenetic control in aging is specifically associated with the functional decline rather than with the chronological age of individuals. Thus, the modifications of DNA methylation, representing a drawbridge between the genetic and the environmental factors affecting the age-related decay of the organism, may play an important role in determining physiological changes over old age.

Aim: We evaluated the methylation of two CpG sites located within human mitochondrial 12S and 16S ribosomal RNA (MT-RNR1 and MT-RNR2) genes. Materials & methods: Methylation was measured through bisulfite sequencing and qPCR assays on DNA samples collected from 381 differently aged human subjects. Results: Analyses revealed the methylation of the site in the MT-RNR1 gene and the co-presence of both unmethylated and methylated cytosines in most samples. High methylation levels (>10%) were more frequent in old women with respect to younger controls. A 9-year-long follow-up survey showed that subjects with high methylation levels exhibit a mortality risk significantly higher than subjects with low levels. Conclusion: Our data further support the presence of methylation within human mitochondrial DNA and suggest that high levels of methylation of the MT-RNR1 site may reflect a condition of the cell or of the organism unfavorable to survival.

Given the dramatic increase in ageing populations, it is of great importance to understand the genetic and molecular determinants of healthy ageing and longevity. Semi-supercentenarians (subjects who reached an age of 105-109 years) arguably represent the gold standard of successful human ageing because they managed to avoid or postpone the onset of major age-related diseases. Relatively few studies have looked at epigenetic determinants of extreme longevity in humans. Here we test whether families with extreme longevity are epigenetically distinct from controls according to an epigenetic biomarker of ageing which is known as "epigenetic clock". We analyze the DNA methylation levels of peripheral blood mononuclear cells (PBMCs) from Italian families constituted of 82 semi-supercentenarians (mean age: 105.6 ± 1.6 years), 63 semi-supercentenarians' offspring (mean age: 71.8 ± 7.8 years), and 47 age-matched controls (mean age: 69.8 ± 7.2 years). We demonstrate that the offspring of semi-supercentenarians have a lower epigenetic age than age-matched controls (age difference=5.1 years, p=0.00043) and that centenarians are younger (8.6 years) than expected based on their chronological age. By contrast, no significant difference could be observed for estimated blood cell counts (such as naive or exhausted cytotoxic T cells or helper T cells). Future studies will be needed to replicate these findings in different populations and to extend them to other tissues. Overall, our results suggest that epigenetic processes might play a role in extreme longevity and healthy human ageing.

The rate/quality of human aging and the development/progression of diseases depend on a complex interplay among genetics, epigenetics and environment. In this scenario, mitochondrial function (or dysfunction) and mitochondrial DNA have emerged as major players. This is mainly due to their crucial role in energetic balance, in modulating epigenetic programs and in influencing cell stress response. Moreover, it is also emerging the existence of epigenetic changes in mitochondrial DNA and of non coding mitochondrial RNAs which, together with the nuclear ones, play regulatory roles in numerous human phenotypes. In this review we will provide an overview on "mitochondrial epigenetics" state of the art, by summarizing the involvement of mitochondrial function and of mitochondria-nucleus communication in regulating nuclear epigenome, as well as the key aspects of the epigenetic marks related to mitochondrial DNA. Despite the limited data available in the literature to date, mainly due to the novelty of the topic, the intriguing interplay of the mitochondrial epigenetic changes in both physiological and pathological conditions will also be presented.