Dina Bellizzi

Variability of the SIRT3 gene, human silent information regulator Sir2 homologue, and survivorship in the elderly

The human sirtuin 3 (SIRT3) gene encodes a putative mitochondrial NAD-dependent deacetylase (SIRT3) which belongs to the evolutionary conserved family of sirtuin 2 proteins. Studies in model organisms have demonstrated that SIR2 genes control lifespan, while no data are available regarding a possible role of SIRT3 in human longevity. By analysing the genotype-specific survival function relevant to the G477T marker of SIRT3, we found that in males the TT genotype increases (p=0.0272), while the GT genotype decreases (p=0.0391) survival in the elderly. Since SIRT3 lies in a chromosomal region (11p15.5) where four genes potentially associated with longevity are located (HRAS1, Insulin-like Growth Factor 2, Proinsulin, and Tyrosine Hydroxylase) we tested for linkage-disequilibrium between G477T alleles and alleles of the above genes. The disequilibrium was not significant in any case, thus suggesting that SIRT3 itself, or a gene strictly linked to SIRT3, may have a role in human longevity.

The control region of mitochondrial DNA shows an unusual CpG and non-CpG methylation pattern.

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.

Association of the mitochondrial DNA haplogroup J with longevity is population specific

Evidences are accumulating on the effects of the variability of mitochondrial DNA (mtDNA) on many complex traits. In particular, mtDNA haplogroup J has been reported to increase the individual chance to attain longevity in northern Italians, Northern Irish and Finns. However, since the genetic contribution to longevity may be population specific, we wanted to verify if haplogroup J does affect longevity also in a southern European population having a different genetic and environmental history. We analysed a population sample (883 subjects, 371 males and 521 females; age range 18–108 years) from southern Italy for the presence of haplogroup J. No frequency increase of this mtDNA haplogroup was found in the older cohorts, suggesting that, in this population, haplogroup J does not play a significant role in longevity. This finding shows that, as for other genetic factors, the association of mtDNA inherited variability with longevity is population specific.

Global DNA methylation levels are modulated by mitochondrial DNA variants

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.

miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.

Global DNA methylation in old subjects is correlated with frailty

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.

The allele (A)(-110) in the promoter region of the HSP70-1 gene is unfavorable to longevity in women.

Heat shock proteins (HSPs) are crucial for maintenance of cell homeostasis and survival both during and after various stresses. The capability to cope with stress is believed to affect the chance of health and survival at organismal level. We have investigated whether the gene pool relevant to the (A/C)-110 polymorphism in the promoter region of the HSP70-1 gene changes as the population ages and survival selection occurs. A total of 591 southern Italian subjects were enrolled in the study (263 males and 328 females; age range 18–109 years), free of clinically manifest diseases and with normal haemato-chemical parameters. A significant age-related decrease of the frequency of allele (A)-110 was observed in females. The probability ratio of 0.403 (95% confidence interval [0.163, 0.910]) computed by considering female centenarians as cases and young women (18–49 years old) as controls showed that the (A)-110 allele is unfavorable to longevity in females.

Exploring the Role of Genetic Variability and Lifestyle in Oxidative Stress Response for Healthy Aging and Longevity

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.

The mitochondrial DNA control region shows genetically correlated levels of heteroplasmy in leukocytes of centenarians and their offspring

BackgroundStudies on heteroplasmy occurring in the mitochondrial DNA (mtDNA) control region (CR) in leukocytes of centenarians and younger subjects have shown that the C150T somatic transition is over-represented in centenarians. However, whether the occurrence/accumulation of heteroplasmy is a phenotypic consequence of extreme ageing or a genetically controlled event that may favor longevity is a question that deserves further attention. To clarify this point, we set up a Denaturing High Performance Liquid Chromatography (DHPLC) protocol to quantify mtDNA CR heteroplasmy. We then analyzed heteroplasmy in leukocytes of centenarians (100 subjects), their offspring and nieces/nephews (200 subjects, age-range 65–80 years, median age 70 years), and in leukocytes of 114 control subjects sex- and age-matched with the relatives of centenarians.ResultsThe centenarians and their descendants, despite the different ages, showed similar levels of heteroplasmy which were significantly higher than levels in controls. In addition we found that heteroplasmy levels were significantly correlated in parent-offspring pairs (r = 0.263; p = 0.009), but were independent of mtDNA inherited variability (haplogroup and sequence analyses).ConclusionOur findings suggest that the high degree of heteroplasmy observed in centenarians is genetically controlled, and that such genetic control is independent of mtDNA variability and likely due to the nuclear genome.

Gene expression of cytokines and cytokine receptors is modulated by the common variability of the mitochondrial DNA in cybrid cell lines

Some lines of evidence indicate that common polymorphisms of mitochondrial DNA (mtDNA) act as susceptibility factors in complex traits, such as age‐related common diseases. There is also evidence that the cell capability to compensate ravages caused by intrinsic or extrinsic stress factors could contribute to some of these diseases. The cross‐talk between nuclear and mitochondrial genome may link the above observations if we assume that the transcription of stress‐responder nuclear genes is modulated according to the mtDNA common variability. Cytokines and cytokine receptors are key molecules in stress response. We could, therefore, check the above hypothesis by analyzing expression patterns of cytokine and cytokine receptor genes in response to stress in cell lines sharing the same nuclear genome but different mtDNA. By using a cybrid model (143B.TK− osteosarcoma cells depleted of their own mtDNA and repopulated with foreign mitochondria) we show that the transcription patterns of some of such genes are specifically modulated by the variability of the mitochondrial genome not only under stress conditions (interleukin‐6) but also at basal conditions (interleukin‐1β and tumor necrosis factor receptor 2). These findings provide a first experimental evidence of a relationship between mtDNA common variability and expression pattern of stress responder nuclear genes in human cells.