Evaluation of DNA Methylation Profiles of LINE-1, Alu and Ribosomal DNA Repeats in Human Cell Lines Exposed to Radiofrequency Radiation.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as "possibly carcinogenic"; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied.

The epigenetics of inflammaging: The contribution of age-related heterochromatin loss and locus-specific remodelling and the modulation by environmental stimuli.

A growing amount of evidences indicates that inflammaging - the chronic, low grade inflammation state characteristic of the elderly - is the result of genetic as well as environmental or stochastic factors. Some of these, such as the accumulation of senescent cells that are persistent during aging or accompany its progression, seem to be sufficient to initiate the aging process and to fuel it. Others, like exposure to environmental compounds or infections, are temporary and resolve within a (relatively) short time. In both cases, however, a cellular memory of the event can be established by means of epigenetic modulation of the genome. In this review we will specifically discuss the relationship between epigenetics and inflammaging. In particular, we will show how age-associated epigenetic modifications concerned with heterochromatin loss and gene-specific remodelling, can promote inflammaging. Furthermore, we will recall how the exposure to specific nutritional, environmental and microbial stimuli can affect the rate of inflammaging through epigenetic mechanisms, touching also on the recent insight given by the concept of trained immunity.

Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells.

Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver availability can limit even this therapy. Identification of novel therapeutics for genetic-based liver diseases requires models that provide measurable hepatic functions and phenotypes. Advances in stem cell and genome editing technologies could provide models for the investigation of cell-based genetic diseases, as well as the platforms for drug discovery. This report demonstrates a practical, and widely applicable, approach that includes the successful reprogramming of somatic cells from a patient with a urea cycle defect, their genetic correction and differentiation into hepatic organoids, and the subsequent demonstration of genetic and phenotypic change in the edited cells consistent with the correction of the defect. While individually rare, there is a large number of other genetic-based liver diseases. The approach described here could be applied to a broad range and a large number of patients with these hepatic diseases where it could serve as an in vitro model, as well as identify successful strategies for corrective cell-based therapy.

The epigenetic landscape of age-related diseases: the geroscience perspective.

In this review, we summarize current knowledge regarding the epigenetics of age-related diseases, focusing on those studies that have described DNA methylation landscape in cardio-vascular diseases, musculoskeletal function and frailty. We stress the importance of adopting the conceptual framework of "geroscience", which starts from the observation that advanced age is the major risk factor for several of these pathologies and aims at identifying the mechanistic links between aging and age-related diseases. DNA methylation undergoes a profound remodeling during aging, which includes global hypomethylation of the genome, hypermethylation at specific loci and an increase in inter-individual variation and in stochastic changes of DNA methylation values. These epigenetic modifications can be an important contributor to the development of age-related diseases, but our understanding on the complex relationship between the epigenetic signatures of aging and age-related disease is still poor. The most relevant results in this field come from the use of the so called "epigenetics clocks" in cohorts of subjects affected by age-related diseases. We report these studies in final section of this review.

Increased hippocampal epigenetic age in the Ts65Dn mouse model of Down Syndrome.

Down syndrome (DS) is a segmental progeroid genetic disorder associated with multi-systemic precocious aging phenotypes, which are particularly evident in the immune and nervous systems. Accordingly, people with DS show an increased biological age as measured by epigenetic clocks. The Ts65Dn trisomic mouse, which harbors extra-numerary copies of chromosome 21 (Hsa21)-syntenic regions, was shown to recapitulate several progeroid features of DS, but no biomarkers of age have been applied to it so far. In this pilot study, we used a mouse-specific epigenetic clock to measure the epigenetic age of hippocampi from Ts65Dn and euploid mice at 20 weeks. Ts65Dn mice showed an increased epigenetic age in comparison with controls, and the observed changes in DNA methylation partially recapitulated those observed in hippocampi from people with DS. Collectively, our results support the use of the Ts65Dn model to decipher the molecular mechanisms underlying the progeroid DS phenotypes.

Epigenetic clocks suggest accelerated aging in patients with isolated REM Sleep Behavior Disorder.

Isolated REM Sleep Behavior Disorder (iRBD) is the strongest prodromal marker for α-synucleinopathies. Overt α-synucleinopathies and aging share several mechanisms, but this relationship has been poorly investigated in prodromal phases. Using DNA methylation-based epigenetic clocks, we measured biological aging in videopolysomnography confirmed iRBD patients, videopolysomnography-negative and population-based controls. We found that iRBDs tended to be epigenetically older than controls, suggesting that accelerated aging characterizes prodromal neurodegeneration.

Heterogeneity of Cellular Senescence: Cell Type-Specific and Senescence Stimulus-Dependent Epigenetic Alterations.

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

Quadrato Motor Training (QMT) is associated with DNA methylation changes at DNA repeats: A pilot study.

The control of non-coding repeated DNA by DNA methylation plays an important role in genomic stability, contributing to health and healthy aging. Mind-body practices can elicit psychophysical wellbeing via epigenetic mechanisms, including DNA methylation. However, in this context the effects of movement meditations have rarely been examined. Consequently, the current study investigates the effects of a specifically structured movement meditation, called the Quadrato Motor Training (QMT) on psychophysical wellbeing and on the methylation level of repeated sequences. An 8-week daily QMT program was administered to healthy women aged 40-60 years and compared with a passive control group matched for gender and age. Psychological well-being was assessed within both groups by using self-reporting scales, including the Meaning in Life Questionnaire [MLQ] and Psychological Wellbeing Scale [PWB]). DNA methylation profiles of repeated sequences (ribosomal DNA, LINE-1 and Alu) were determined in saliva samples by deep-sequencing. In contrast to controls, the QMT group exhibited increased Search for Meaning, decreased Presence of Meaning and increased Positive Relations, suggesting that QMT may lessen the automatic patterns of thinking. In the QMT group, we also found site-specific significant methylation variations in ribosomal DNA and LINE-1 repeats, consistent with increased genome stability. Finally, the correlations found between changes in methylation and psychometric indices (MLQ and PWB) suggest that the observed epigenetic and psychological changes are interrelated. Collectively, the current results indicate that QMT may improve psychophysical health trajectories by influencing the DNA methylation of specific repetitive sequences.

DNA Methylation Analysis of Ribosomal DNA in Adults With Down Syndrome.

Control of ribosome biogenesis is a critical aspect of the regulation of cell metabolism. As ribosomal genes (rDNA) are organized in repeated clusters on chromosomes 13, 14, 15, 21, and 22, trisomy of chromosome 21 confers an excess of rDNA copies to persons with Down syndrome (DS). Previous studies showed an alteration of ribosome biogenesis in children with DS, but the epigenetic regulation of rDNA genes has not been investigated in adults with DS so far. In this study, we used a targeted deep-sequencing approach to measure DNA methylation (DNAm) of rDNA units in whole blood from 69 adults with DS and 95 euploid controls. We further evaluated the expression of the precursor of ribosomal RNAs (RNA45S) in peripheral blood mononuclear cells (PBMCs) from the same subjects. We found that the rDNA promoter tends to be hypermethylated in DS concerning the control group. The analysis of epihaplotypes (the combination of methylated and unmethylated CpG sites along the same DNA molecule) showed a significantly lower intra-individual diversity in the DS group, which at the same time was characterized by a higher interindividual variability. Finally, we showed that RNA45S expression is lower in adults with DS. Collectively, our results suggest a rearrangement of the epigenetic profile of rDNA in DS, possibly to compensate for the extranumerary rDNA copies. Future studies should assess whether the regulation of ribosome biogenesis can contribute to the pathogenesis of DS and explain the clinical heterogeneity characteristic of the syndrome.

A Targeted Epigenetic Clock for the Prediction of Biological Age.

Epigenetic clocks were initially developed to track chronological age, but accumulating evidence indicates that they can also predict biological age. They are usually based on the analysis of DNA methylation by genome-wide methods, but targeted approaches, based on the assessment of a small number of CpG sites, are advisable in several settings. In this study, we developed a targeted epigenetic clock purposely optimized for the measurement of biological age. The clock includes six genomic regions mapping in ELOVL2, NHLRC1, AIM2, EDARADD, SIRT7 and TFAP2E genes, selected from a re-analysis of existing microarray data, whose DNA methylation is measured by EpiTYPER assay. In healthy subjects (n = 278), epigenetic age calculated using the targeted clock was highly correlated with chronological age (Spearman correlation = 0.89). Most importantly, and in agreement with previous results from genome-wide clocks, epigenetic age was significantly higher and lower than expected in models of increased (persons with Down syndrome, n = 62) and decreased (centenarians, n = 106; centenarians' offspring, n = 143; nutritional intervention in elderly, n = 233) biological age, respectively. These results support the potential of our targeted epigenetic clock as a new marker of biological age and open its evaluation in large cohorts to further promote the assessment of biological age in healthcare practice.

A Meta-Analysis of Brain DNA Methylation Across Sex, Age, and Alzheimer's Disease Points for Accelerated Epigenetic Aging in Neurodegeneration.

Alzheimer's disease (AD) is characterized by specific alterations of brain DNA methylation (DNAm) patterns. Age and sex, two major risk factors for AD, are also known to largely affect the epigenetic profiles in brain, but their contribution to AD-associated DNAm changes has been poorly investigated. In this study we considered publicly available DNAm datasets of four brain regions (temporal, frontal, entorhinal cortex, and cerebellum) from healthy adult subjects and AD patients, and performed a meta-analysis to identify sex-, age-, and AD-associated epigenetic profiles. In one of these datasets it was also possible to distinguish 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles. We showed that DNAm differences between males and females tend to be shared between the four brain regions, while aging differently affects cortical regions compared to cerebellum. We found that the proportion of sex-dependent probes whose methylation is modified also during aging is higher than expected, but that differences between males and females tend to be maintained, with only a few probes showing age-by-sex interaction. We did not find significant overlaps between AD- and sex-associated probes, nor disease-by-sex interaction effects. On the contrary, we found that AD-related epigenetic modifications are significantly enriched in probes whose DNAm varies with age and that there is a high concordance between the direction of changes (hyper or hypo-methylation) in aging and AD, supporting accelerated epigenetic aging in the disease. In summary, our results suggest that age-associated DNAm patterns concur to the epigenetic deregulation observed in AD, providing new insights on how advanced age enables neurodegeneration.

A geroscience approach for Parkinson's disease: Conceptual framework and design of PROPAG-AGEING project.

Advanced age is the major risk factor for idiopathic Parkinson's disease (PD), but to date the biological relationship between PD and ageing remains elusive. Here we describe the rationale and the design of the H2020 funded project "PROPAG-AGEING", whose aim is to characterize the contribution of the ageing process to PD development. We summarize current evidences that support the existence of a continuum between ageing and PD and justify the use of a Geroscience approach to study PD. We focus in particular on the role of inflammaging, the chronic, low-grade inflammation characteristic of elderly physiology, which can propagate and transmit both locally and systemically. We then describe PROPAG-AGEING design, which is based on the multi-omic characterization of peripheral samples from clinically characterized drug-naïve and advanced PD, PD discordant twins, healthy controls and "super-controls", i.e. centenarians, who never showed clinical signs of motor disability, and their offspring. Omic results are then validated in a large number of samples, including in vitro models of dopaminergic neurons and healthy siblings of PD patients, who are at higher risk of developing PD, with the final aim of identifying the molecular perturbations that can deviate the trajectories of healthy ageing towards PD development.

Age-related DNA methylation changes are sex-specific: a comprehensive assessment.

The existence of a sex gap in human health and longevity has been widely documented. Autosomal DNA methylation differences between males and females have been reported, but so far few studies have investigated if DNA methylation is differently affected by aging in males and females. We performed a meta-analysis of 4 large whole blood datasets, comparing 4 aspects of epigenetic age-dependent remodeling between the two sexes: differential methylation, variability, epimutations and entropy. We reported that a large fraction (43%) of sex-associated probes undergoes age-associated DNA methylation changes, and that a limited number of probes show age-by-sex interaction. We experimentally validated 2 regions mapping in FIGN and PRR4 genes and showed sex-specific deviations of their methylation patterns in models of decelerated (centenarians) and accelerated (Down syndrome) aging. While we did not find sex differences in the age-associated increase in epimutations and entropy, we showed that the number of probes having an age-related increase in methylation variability is 15 times higher in males compared to females. Our results can offer new epigenetic tools to study the interaction between aging and sex and can pave the way to the identification of molecular triggers of sex differences in longevity and age-related diseases prevalence.

Aging and Caloric Restriction Modulate the DNA Methylation Profile of the Ribosomal RNA Locus in Human and Rat Liver.

A growing amount of evidence suggests that the downregulation of protein synthesis is an adaptive response during physiological aging, which positively contributes to longevity and can be modulated by nutritional interventions like caloric restriction (CR). The expression of ribosomal RNA (rRNA) is one of the main determinants of translational rate, and epigenetic modifications finely contribute to its regulation. Previous reports suggest that hypermethylation of ribosomal DNA (rDNA) locus occurs with aging, although with some species- and tissue- specificity. In the present study, we experimentally measured DNA methylation of three regions (the promoter, the 5' of the 18S and the 5' of 28S sequences) in the rDNA locus in liver tissues from rats at two, four, 10, and 18 months. We confirm previous findings, showing age-related hypermethylation, and describe, for the first time, that this gain in methylation also occurs in human hepatocytes. Furthermore, we show that age-related hypermethylation is enhanced in livers of rat upon CR at two and 10 months, and that at two months a trend towards the reduction of rRNA expression occurs. Collectively, our results suggest that CR modulates age-related regulation of methylation at the rDNA locus, thus providing an epigenetic readout of the pro-longevity effects of CR.

Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts.

Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105-107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors. In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases.

Molecular Aging of Human Liver: An Epigenetic/Transcriptomic Signature.

The feasibility of liver transplantation from old healthy donors suggests that this organ is able to preserve its functionality during aging. To explore the biological basis of this phenomenon, we characterized the epigenetic profile of liver biopsies collected from 45 healthy liver donors ranging from 13 to 90 years old using the Infinium HumanMethylation450 BeadChip. The analysis indicates that a large remodeling in DNA methylation patterns occurs, with 8,823 age-associated differentially methylated CpG probes. Notably, these age-associated changes tended to level off after the age of 60, as confirmed by Horvath's clock. Using stringent selection criteria, we further identified a DNA methylation signature of aging liver including 75 genomic regions. We demonstrated that this signature is specific for liver compared to other tissues and that it is able to detect biological age-acceleration effects associated with obesity. Finally, we combined DNA methylation measurements with available expression data. Although the intersection between the two omic characterizations was low, both approaches suggested a previously unappreciated role of epithelial-mesenchymal transition and Wnt-signaling pathways in the aging of human liver.

Age-Related Epigenetic Derangement upon Reprogramming and Differentiation of Cells from the Elderly.

Aging is a complex multi-layered phenomenon. The study of aging in humans is based on the use of biological material from hard-to-gather tissues and highly specific cohorts. The introduction of cell reprogramming techniques posed promising features for medical practice and basic research. Recently, a growing number of studies have been describing the generation of induced pluripotent stem cells (iPSCs) from old or centenarian biologic material. Nonetheless, Reprogramming techniques determine a profound remodelling on cell epigenetic architecture whose extent is still largely debated. Given that cell epigenetic profile changes with age, the study of cell-fate manipulation approaches on cells deriving from old donors or centenarians may provide new insights not only on regenerative features and physiology of these cells, but also on reprogramming-associated and age-related epigenetic derangement.

Responders and non-responders to influenza vaccination: A DNA methylation approach on blood cells.

Several evidences indicate that aging negatively affects the effectiveness of influenza vaccination. Although it is well established that immunosenescence has an important role in vaccination response, the molecular pathways underlying this process are largely unknown. Given the importance of epigenetic remodeling in aging, here we analyzed the relationship between responsiveness to influenza vaccination and DNA methylation profiles in healthy subjects of different ages. Peripheral blood mononuclear cells were collected from 44 subjects (age range: 19-90 years old) immediately before influenza vaccination. Subjects were subsequently classified as responders or non-responders according to hemagglutination inhibition assay 4-6 weeks after the vaccination. Baseline whole genome DNA methylation in peripheral blood mononuclear cells was analyzed using the Illumina® Infinium 450 k microarray. Differential methylation analysis between the two groups (responders and non-responders) was performed through an analysis of variance, correcting for age, sex and batch. We identified 83 CpG sites having a nominal p-value <.001 and absolute difference in DNA methylation of at least 0.05 between the two groups. For some CpG sites, we observed age-dependent decrease or increase in methylation, which in some cases was specific for the responders and non-responders groups. Finally, we divided the cohort in two subgroups including younger (age < 50) and older (age ≥ 50) subjects and compared DNA methylation between responders and non-responders, correcting for sex and batch in each subgroup. We identified 142 differentially methylated CpG sites in the young subgroup and 305 in the old subgroup, suggesting a larger epigenetic remodeling at older ages. Interestingly, some of the differentially methylated probes mapped in genes involved in immunosenescence (CD40) and in innate immunity responses (CXCL16, ULK1, BCL11B, BTC). In conclusion, the analysis of epigenetic landscape can shed light on the biological basis of vaccine responsiveness during aging, possibly providing new appropriate biomarkers of this process.

Systemic Age-Associated DNA Hypermethylation of ELOVL2 Gene: In Vivo and In Vitro Evidences of a Cell Replication Process.

Epigenetic remodeling is one of the major features of the aging process. We recently demonstrated that DNA methylation of ELOVL2 and FHL2 CpG islands is highly correlated with age in whole blood. Here we investigated several aspects of age-associated hypermethylation of ELOVL2 and FHL2. We showed that ELOVL2 methylation is significantly different in primary dermal fibroblast cultures from donors of different ages. Using epigenomic data from public resources, we demonstrated that most of the tissues show ELOVL2 and FHL2 hypermethylation with age. Interestingly, ELOVL2 hypermethylation was not found in tissues with very low replication rate. We demonstrated that ELOVL2 hypermethylation is associated with in vitro cell replication rather than with senescence. We confirmed intra-individual hypermethylation of ELOVL2 and FHL2 in longitudinally assessed participants from the Doetinchem Cohort Study. Finally we showed that, although the methylation of the two loci is not associated with longevity/mortality in the Leiden Longevity Study, ELOVL2 methylation is associated with cytomegalovirus status in nonagenarians, which could be informative of a higher number of replication events in a fraction of whole-blood cells. Collectively, these results indicate that ELOVL2 methylation is a marker of cell divisions occurring during human aging.