Altered modulation of lamin A/C‐HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson–Gilford progeria, a severe LMNA‐linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C‐HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C‐HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.     

Review: a meta-analysis of GWAS and age-associated diseases

Genome‐Wide Association studies (GWAS) offer an unbiased means to understand the genetic basis of traits by identifying single nucleotide polymorphisms (SNPs) linked to causal variants of complex phenotypes. GWAS have identified a host of susceptibility SNPs associated with many important human diseases, including diseases associated with aging. In an effort to understand the genetics of broad resistance to age‐associated diseases (i.e., ‘wellness’), we performed a meta‐analysis of human GWAS. Toward that end, we compiled 372 GWAS that identified 1775 susceptibility SNPs to 105 unique diseases and used these SNPs to create a genomic landscape of disease susceptibility. This map was constructed by partitioning the genome into 200 kb ‘bins’ and mapping the 1775 susceptibility SNPs to bins based on their genomic location. Investigation of these data revealed significant heterogeneity of disease association within the genome, with 92% of bins devoid of disease‐associated SNPs. In contrast, 10 bins (0.06%) were significantly (P < 0.05) enriched for susceptibility to multiple diseases, 5 of which formed two highly significant peaks of disease association (P < 0.0001). These peaks mapped to the Major Histocompatibility (MHC) locus on 6p21 and the INK4/ARF (CDKN2a/b) tumor suppressor locus on 9p21.3. Provocatively, all 10 significantly enriched bins contained genes linked to either inflammation or cellular senescence pathways, and SNPs near regulators of senescence were particularly associated with disease of aging (e.g., cancer, atherosclerosis, type 2 diabetes, glaucoma). This analysis suggests that germline genetic heterogeneity in the regulation of immunity and cellular senescence influences the human healthspan.     

Structural organization, mapping, characterization and evolutionary relationships of CDKN2 gene family members in Xiphophorus fishes

Xiphophorus fishes and their hybrids are used as models for the study of melanoma and other diseases. The cyclin-dependent kinase inhibitor gene family in humans is comprised of four members, including CDKN2A (P16), and dysregulation of this gene is implicated in numerous neoplasms including melanomas. We have investigated the status of the gene family in the southern platyfish X. maculatus. Xiphophorus harbors at least two such loci, which we now term CDKN2A/B and CDKN2D. Both loci map to Xiphophorus linkage group 5, a genomic area that has long been known to harbor the DIFF tumor suppressor locus. Within this report, we report on the complete cloning, genomic exon/intron boundary delineation, linkage mapping and expressional characteristics of Xiphophorus CDKN2D. We also compare and contrast this expression to that of the previously isolated CDKN2AB locus in normal and neoplastic tissues derived from non-hybrid and hybrid fishes. The hypothetical evolutionary relationships of gene family members and their involvement in melanoma is evaluated. In comparison to CDKN2A/B, the RNA expression of Xiphophorus CDKN2D differs in normal tissues and is not associated with melanotic/pathologic tissues, confirming functional divergence between obvious homologues.     

An 11p15 imprinting centre region 2 deletion in a family with Beckwith Wiedemann syndrome provides insights into imprinting control at CDKN1C

We report a three generation family with Beckwith Wiedemann syndrome (BWS) in whom we have identified a 330 kb deletion within the KCNQ1 locus, encompassing the 11p15.5 Imprinting Centre II (IC2). The deletion arose on the paternal chromosome in the first generation and was only associated with BWS when transmitted maternally to subsequent generations. The deletion on the maternal chromosome was associated with a lower median level of CDKN1C expression in the peripheral blood of affected individuals when compared to a cohort of unaffected controls (p<0.05), however was not significantly different to the expression levels in BWS cases with loss of methylation (LOM) within IC2 (p<0.78). Moreover the individual with a deletion on the paternal chromosome did not show evidence of elevated CDKN1C expression or features of Russell Silver syndrome. These observations support a model invoking the deletion of enhancer elements required for CDKN1C expression lying within or close to the imprinting centre and importantly extend and validate a single observation from an earlier study. Analysis of 94 cases with IC2 loss of methylation revealed that KCNQ1 deletion is a rare cause of loss of maternal methylation, occurring in only 3% of cases, or in 1.5% of BWS overall.     

Microdeletions in 9p21.3 induce false negative results in CDKN2A FISH analysis of Ewing sarcoma

Deletion of the CDKN2A locus at 9p21.3 has been reported to be a poor prognostic sign in the Ewing sarcoma family of tumours. In clinical applications CDKN2A deletion is primarily detected using fluorescent in situ hybridisation (FISH) with a commercial probe, size approximately 190 kb. Due to limitations in resolution, FISH analysis may fail to detect microdeletions smaller than 190 kb. In the present study, we performed 44K array comparative genomic hybridisation (CGH) on eleven Ewing sarcoma cell lines and 26 tissue samples in order to define the sizes of 9p21.3 deletions. Microarray CGH analysis revealed 9p21.3 deletions encompassing the CDKN2A locus in eight cell lines (73%) and in six tumours (23%). In four cases (two cell lines and two tissue samples) the deletion was less than 190 kb in size. In one cell line sample, we detected a microdeletion of approximately 58 kb in 9p21.3 harbouring the CDKN2A locus. We confirmed this result using 244K microarray CGH and TaqMan quantitative RT-PCR analysis and further performed FISH analysis on this cell line sample. Here, we show that CDKN2A FISH analysis can give false negative results in cases with small microdeletions. Our results suggest that new and more accurate FISH methods should be developed for detection of deletions in the CDKN2A locus.     

The Piwi‐piRNA pathway: road to immortality

Despite its medical, social, and economic significance, understanding what primarily causes aging, that is, the mechanisms of the aging process, remains a fundamental and fascinating problem in biology. Accumulating evidence indicates that a small RNA‐based gene regulatory machinery, the Piwi‐piRNA pathway, represents a shared feature of nonaging (potentially immortal) biological systems, including the germline, somatic cancer stem cells, and certain ‘lower’ eukaryotic organisms like the planarian flatworm and freshwater hydra. The pathway primarily functions to repress the activity of mobile genetic elements, also called transposable elements (TEs) or ‘jumping genes’, which are capable of moving from one genomic locus to another, thereby causing insertional mutations. TEs become increasingly active and multiply in the genomes of somatic cells as the organism ages. These characteristics of TEs highlight their decisive mutagenic role in the progressive disintegration of genetic information, a molecular hallmark associated with aging. Hence, TE‐mediated genomic instability may substantially contribute to the aging process.     

Long noncoding RNA FAM83H‐AS1 exerts an oncogenic role in glioma through epigenetically silencing CDKN1A (p21)

Gliomas are the commonest and most aggressive primary malignant tumor in the central nervous system. Long noncoding RNAs (lncRNAs) have been identified to act as crucial regulators in multiple biological processes, including tumorigenesis. FAM83H antisense RNA1 (FAM83H‐AS1) has been uncovered to be dysregulated in several cancers. However, the biological role of FAM83H‐AS1 in glioma still needs to be investigated. Currently, our findings indicated that FAM83H‐AS1 was upregulated in glioma tissues and cell lines and high level of FAM83H‐AS1 was associated with poor prognosis of glioma. Loss‐of‐function assays demonstrated that silenced FAM83H‐AS1 obviously suppressed cell proliferation via regulating the cell‐cycle distribution and cell apoptosis rate, and mechanistic experiments revealed that FAM83H‐AS1 could epidemically silence CDKN1A expression through recruiting EZH2 to the promoter of CDKN1A, thereby influencing the cell cycle and proliferation. Collectively, our findings suggested that FAM83H‐AS1 participated in the progression of glioma and might act as a potential therapeutic target and prognosis biomarker for human glioma.     

Hydrochar enhances growth of poplar for bioenergy while marginally contributing to direct soil carbon sequestration

Hydrothermal carbonization (HTC) has been proposed as an alternative method to pyrolysis for producing C‐rich amendments for soil C sequestration. However, the use of hydrochar (HC) as soil amendment is still controversial due to the limited information on the potential benefits and trade‐offs that may follow its application into soil. This study investigated the effects of HC starting from maize silage on plant growth in a 2‐year controlled experiment on poplar for bioenergy and evaluated HC stability in soil by periodic soil respiration and isotopic (δ¹³C) measurements. HC application caused a substantial and significant increase in plant biomass after one and two years after planting, and no evident signs of plant diseases were evident. Isotopic analysis on soil and CO₂ efflux showed that slightly less than half of the C applied was re‐emitted as CO₂ within 12 months. On the contrary, considering that the difference in the amount of N fixed in wood biomass in treated and not‐treated poplars was 16.6 ± 4.8 g N m^?2 and that the soil N stocks after one year since application did not significantly change, we estimated that approximately 85% of the N applied with HC could have been potentially lost as leachate or volatilized into the atmosphere as N₂O, in response to nitrification/denitrification processes in the soil. Thus, the permanence, additionality and leakage of C sequestration strategy using HC are deeply discussed.     

Corneal Endothelial Cells Provide Evidence of Accelerated Cellular Senescence Associated with HIV Infection: A Case-Control Study

Background

Cellular senescence may be a key factor in HIV-related premature biological aging. We assessed features of the corneal endothelium that are known to be associated with biological aging, and cellular senescence markers in HIV-infected adults.

Methods

Case-control study of 242 HIV-infected adults and 249 matched controls. Using specular microscopy, the corneal endothelium was assessed for features of aging (low endothelial cell density [ECD], high variation in cell size, and low hexagonality index). Data were analysed by multivariable regression. CDKN2A expression (a cell senescence mediator) was measured in peripheral blood leukocytes and 8-hydroxy-2′-deoxyguanosine (8-OHDG; an oxidative DNA damage marker) levels were measured in plasma.

Results

The median age of both groups was 40 years. Among HIV-infected adults, 88% were receiving antiretroviral therapy (ART); their median CD4 count was 468 cells/µL. HIV infection was associated with increased odds of variation in cell size (OR = 1.67; 95% CI: 1.00–2.78, p = 0.04). Among HIV-infected participants, low ECD was independently associated with current CD4 count <200 cells/µL (OR = 2.77; 95%CI: 1.12–6.81, p = 0.03). In participants on ART with undetectable viral load, CDKN2A expression and 8-OHDG levels were higher in those with accelerated aging, as reflected by lower ECD.

Conclusions

The corneal endothelium shows features consistent with HIV-related accelerated senescence, especially among those with poor immune recovery.     

Fitness penalty in susceptible host is associated with virulence of Soybean mosaic virus on Rsv1‐genotype soybean: a consequence of perturbation of HC‐Pro and not P3

The multigenic Rsv1 locus in the soybean plant introduction (PI) ‘PI96983’ confers extreme resistance against the majority of Soybean mosaic virus (SMV) strains, including SMV‐N, but not SMV‐G7 and SMV‐G7d. In contrast, in susceptible soybean cultivars lacking a functional Rsv1 locus, such as ‘Williams82’ (rsv1), SMV‐N induces severe disease symptoms and accumulates to a high level, whereas both SMV‐G7 and SMV‐G7d induce mild symptoms and accumulate to a significantly lower level. Gain of virulence by SMV‐N on Rsv1‐genotype soybean requires concurrent mutations in both the helper‐component proteinase (HC‐Pro) and P3 cistrons. This is because of the presence of at least two resistance (R) genes, probably belonging to the nucleotide‐binding leucine‐rich repeat (NB‐LRR) class, within the Rsv1 locus, independently mediating the recognition of HC‐Pro or P3. In this study, we show that the majority of experimentally evolved mutational pathways that disrupt the avirulence functions of SMV‐N on Rsv1‐genotype soybean also result in mild symptoms and reduced accumulation, relative to parental SMV‐N, in Williams82 (rsv1). Furthermore, the evaluation of SMV‐N‐derived HC‐Pro and P3 chimeras, containing homologous sequences from virulent SMV‐G7 or SMV‐G7d strains, as well as SMV‐N‐derived variants containing HC‐Pro or P3 point mutation(s) associated with gain of virulence, reveals a direct correlation between the perturbation of HC‐Pro and a fitness penalty in Williams82 (rsv1). Collectively, these data demonstrate that gain of virulence by SMV on Rsv1‐genotype soybean results in fitness loss in a previously susceptible soybean genotype, this being a consequence of mutations in HC‐Pro, but not in P3.     

Circulating levels of monocyte chemoattractant protein‐1 as a potential measure of biological age in mice and frailty in humans

A serum biomarker of biological versus chronological age would have significant impact on clinical care. It could be used to identify individuals at risk of early‐onset frailty or the multimorbidities associated with old age. It may also serve as a surrogate endpoint in clinical trials targeting mechanisms of aging. Here, we identified MCP‐1/CCL2, a chemokine responsible for recruiting monocytes, as a potential biomarker of biological age. Circulating monocyte chemoattractant protein‐1 (MCP‐1) levels increased in an age‐dependent manner in wild‐type (WT) mice. That age‐dependent increase was accelerated in Ercc1^?/Δ and Bubr1^H/H mouse models of progeria. Genetic and pharmacologic interventions that slow aging of Ercc1^?/Δ and WT mice lowered serum MCP‐1 levels significantly. Finally, in elderly humans with aortic stenosis, MCP‐1 levels were significantly higher in frail individuals compared to nonfrail. These data support the conclusion that MCP‐1 can be used as a measure of mammalian biological age that is responsive to interventions that extend healthy aging.     

CDKN2B expression and subcutaneous adipose tissue expandability: Possible influence of the 9p21 atherosclerosis locus

Risk alleles within a gene desert at the 9p21 locus constitute the most prevalent genetic determinant of cardiovascular disease. Previous research has demonstrated that 9p21 risk variants influence gene expression in vascular tissues, yet the biological mechanisms by which this would mediate atherosclerosis merits further investigation. To investigate possible influences of this locus on other tissues, we explored expression patterns of 9p21-regulated genes in a panel of multiple human tissues and found that the tumor suppressor CDKN2B was highly expressed in subcutaneous adipose tissue (SAT). CDKN2B expression was regulated by obesity status, and this effect was stronger in carriers of 9p21 risk alleles. Covariation between expression of CDKN2B and genes implemented in adipogenesis was consistent with an inhibitory effect of CDKN2B on SAT proliferation. Moreover, studies of postprandial triacylglycerol clearance indicated that CDKN2B is involved in down-regulation of SAT fatty acid trafficking. CDKN2B expression in SAT correlated with indicators of ectopic fat accumulation, including markers of hepatic steatosis. Among genes regulated by 9p21 risk variants, CDKN2B appears to play a significant role in the regulation of SAT expandability, which is a strong determinant of lipotoxicity and therefore might contribute to the development of atherosclerosis.     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

Analysis of somatic mutations identifies signs of selection during in vitro aging of primary dermal fibroblasts

Somatic mutations are critical for cancer development and may play a role in age‐related functional decline. Here, we used deep sequencing to analyze the prevalence of somatic mutations during in vitro cell aging. Primary dermal fibroblasts from healthy subjects of young and advanced age, from Hutchinson–Gilford progeria syndrome and from xeroderma pigmentosum complementation groups A and C, were first restricted in number and then expanded in vitro. DNA was obtained from cells pre‐ and post‐expansion and sequenced at high depth (1656× mean coverage), over a cumulative 290 kb target region, including the exons of 44 aging‐related genes. Allele frequencies of 58 somatic mutations differed between the pre‐ and post‐cell culture expansion passages. Mathematical modeling revealed that the frequency change of three of the 58 mutations was unlikely to be explained by genetic drift alone, indicative of positive selection. Two of these three mutations, CDKN2A c.53C>T (T18M) and ERCC8 c.*772T>A, were identified in cells from a patient with XPA. The allele frequency of the CDKN2A mutation increased from 0% to 55.3% with increasing cell culture passage. The third mutation, BRCA2 c.6222C>T (H2074H), was identified in a sample from a healthy individual of advanced age. However, further validation of the three mutations suggests that other unmeasured variants probably provide the selective advantage in these cells. Our results reinforce the notions that somatic mutations occur during aging and that some are under positive selection, supporting the model of increased tissue heterogeneity with increased age.     

Markers of cellular senescence in zero hour biopsies predict outcome in renal transplantation

Although chronological donor age is the most potent predictor of long-term outcome after renal transplantation, it does not incorporate individual differences of the aging-process itself. We therefore hypothesized that an estimate of biological organ age as derived from markers of cellular senescence in zero hour biopsies would be of higher predictive value. Telomere length and mRNA expression levels of the cell cycle inhibitors CDKN2A (p16INK4a) and CDKN1A (p21WAF1) were assessed in pre-implantation biopsies of 54 patients and the association of these and various other clinical parameters with serum creatinine after 1 year was determined. In a linear regression analysis, CDKN2A turned out to be the best single predictor followed by donor age and telomere length. A multiple linear regression analysis revealed that the combination of CDKN2A values and donor age yielded even higher predictive values for serum creatinine 1 year after transplantation. We conclude that the molecular aging marker CDKN2A in combination with chronological donor age predict renal allograft function after 1 year significantly better than chronological donor age alone.