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Purpose: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown.Experimental Design: The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches.Results: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax.Conclusions: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.

Supplementary Data from Intracellular Clusterin Inhibits Mitochondrial Apoptosis by Suppressing p53-Activating Stress Signals and Stabilizing the Cytosolic Ku70-Bax Protein Complex

Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological processes and is differentially expressed in many severe physiological disturbances, including tumor formation and in vivo cancer progression. Despite extensive efforts, clarification of CLU’s biological role has been exceptionally difficult and its precise function remains elusive. Short RNA duplexes, referred to as small interfering RNAs (siRNAs), provide a new approach for the elucidation of gene function in human cells. Here, we describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis. Moreover, CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H2O2, respectively. These effects were more pronounced in cell lines that express high endogenous steady-state levels of the CLU protein and occur through hyperactivation of the cellular apoptotic machinery. Overall, our results reveal that, in the distinct cellular contexts of the osteosarcoma and prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific siRNA oligonucleotides that can potently silence CLU gene expression may thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.

Supplementary Figure from Silencing Expression of the Clusterin/Apolipoprotein J Gene in Human Cancer Cells Using Small Interfering RNA Induces Spontaneous Apoptosis, Reduced Growth Ability, and Cell Sensitization to Genotoxic and Oxidative Stress

Circulating cell-free DNA (cf-DNA) has emerged as a promising biomarker of ageing, tissue damage and cellular stress. However, less is known about health behaviours, ageing phenotypes and metabolic processes that lead to elevated cf-DNA levels. We sought to analyse the relationship of circulating cf-DNA level to age, sex, smoking, physical activity, vegetable consumption, ageing phenotypes (physical functioning, the number of diseases, frailty) and an extensive panel of biomarkers including blood and urine metabolites and inflammatory markers in three human cohorts (N = 5385; 17–82 years). The relationships were assessed using correlation statistics, and linear and penalised regressions (the Lasso), also stratified by sex.cf-DNA levels were significantly higher in men than in women, and especially in middle-aged men and women who smoke, and in older more frail individuals. Correlation statistics of biomarker data showed that cf-DNA level was higher with elevated inflammation (C-reactive protein, interleukin-6), and higher levels of homocysteine, and proportion of red blood cells and lower levels of ascorbic acid. Inflammation (C-reactive protein, glycoprotein acetylation), amino acids (isoleucine, leucine, tyrosine), and ketogenesis (3-hydroxybutyrate) were included in the cf-DNA level-related biomarker profiles in at least two of the cohorts.In conclusion, circulating cf-DNA level is different by sex, and related to health behaviour, health decline and metabolic processes common in health and disease. These results can inform future studies where epidemiological and biological pathways of cf-DNA are to be analysed in details, and for studies evaluating cf-DNA as a potential clinical marker.

[Abstract] Introduction: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. Methods: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). Results: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p < 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p < 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. Conclusions: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals. Ministerio de Ciencia e Innovación (España); PID2020-113788RB-I00 Xunta de Galicia; ED431B 2022/16 Xunta de Galicia; ED431F 2017/09

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.

The tight regulation of proteostasis is essential for physiological cellular function. Mammalian cells possess a network of mechanisms that ensure proteome integrity under normal or stress conditions. The proteasome, being the major cellular proteolytic machinery, is central to proteostasis maintenance in response to distinct intracellular and extracellular conditions. The proteasomes are multisubunit protease complexes that selectively catalyze the degradation of short-lived regulatory proteins and damaged peptides. Different forms of the proteasome complexes comprising of different subunits and attached regulators directly affect the substrate selectivity and degradation. Thus, the proteasome participates in the turnover of a multitude of factors that control key processes that affect the cellular state, such as adaptation to environmental cues, growth, development, metabolism, signaling, senescence, pluripotency, differentiation, and immunity. Aberrations on its function are related to normal processes like aging and pathological conditions such as neurodegeneration and cancer. The past few years of research have highlighted that proteasome abundance, activity, assembly, and localization are subject to a dynamic transcriptional control that secures the continuous adaptation of the proteasome to internal or external stimuli. This review focuses on the factors and signaling pathways that are involved in the regulation of the mammalian proteasome at the transcriptional level. A comprehensive understanding of proteasome regulation has critical implications on disease prevention and treatment.

Stress accelerates aging by affecting relevant cellular pathways including, among others, leucocyte telomere length (LTL) and proteasome levels. Their impaired function underlies several age-related and non-communicable conditions, such as type 2 diabetes mellitus. The aim of the present study was to investigate, for the first time, the dynamics of stress-related aging factors in the frame of a novel stress-management technique, the Pythagorean Self Awareness Intervention (PSAI), in healthy volunteers and adults with type 2 diabetes. To this end a cohort of 311 healthy volunteers was initially studied and LTL and proteasome levels were analysed in a subgroup of healthy volunteers and adults with type 2 diabetes who were enrolled in the PSAI, with regards to specific physio- and psychometric characteristics of the participants (baseline and post-intervention). We have found a significant improvement of aging biomarkers and of psycho-/bio-factors in all participants. More specifically, post-intervention, both healthy adults and patients with type 2 diabetes demonstrated improved LTL and proteasome levels. Significant improvements were also observed in psychometric, anthropometric and key metabolic features as well as in hair cortisol. In conclusion our results highlighted potential key targets of such interventions and prognostic tools for the assessment of aging pace in clinical practice.

Ageing leaves characteristic traces in the DNA methylation make-up of the genome. However, the importance of DNA methylation in ageing remains unclear. The study of subtelomeric regions could give promising insights into this issue. Previously reported associations between susceptibility to age-related diseases and epigenetic instability at subtelomeres suggest that the DNA methylation profile of subtelomeres undergoes remodelling during ageing. In the present work, this hypothesis has been tested in the context of the European large-scale project MARK-AGE. In this cross-sectional study, we profiled the DNA methylation of chromosomes 5 and 21 subtelomeres, in more than 2000 age-stratified women and men recruited in eight European countries. The study included individuals from the general population as well as the offspring of nonagenarians and Down syndrome subjects, who served as putative models of delayed and accelerated ageing, respectively. Significant linear changes of subtelomeric DNA methylation with increasing age were detected in the general population, indicating that subtelomeric DNA methylation changes are typical signs of ageing. Data also show that, compared to the general population, the dynamics of age-related DNA methylation changes are attenuated in the offspring of centenarian, while they accelerate in Down syndrome individuals. This result suggests that subtelomeric DNA methylation changes reflect the rate of ageing progression. We next attempted to trace the age-related changes of subtelomeric methylation back to the influence of diverse variables associated with methylation variations in the population, including demographics, dietary/health habits and clinical parameters. Results indicate that the effects of age on subtelomeric DNA methylation are mostly independent of all other variables evaluated. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00347-9.

Frailty among elderly people leads to an increased risk for negative health outcomes. To prevent frailty, we need a better understanding of the underlying mechanisms and early detection of individuals at risk. Both may be served by identifying candidate (bio)markers, i.e. biomarkers and markers, for the physical, cognitive, and psychological frailty domains. We used univariate (Rank-ANOVA) and multivariate (elastic net) approaches on the RASIG study population (age range: 35–74 years, n = 2220) of the MARK-AGE study to study up to 331 (bio)markers between individuals with and without frailty for each domain. Biomarkers and markers identified by both approaches were studied further regarding their association with frailty using logistic regression. Univariately, we found lower levels of antioxidants, including β-cryptoxanthin and zeaxanthin, in those who were physically, cognitively or psychologically frail. Additionally, self-reported health was worse in these three frail groups. Multivariately, we observed lower levels of β-cryptoxanthin and zeaxanthin in the cognitively frail. Levels of these carotenoids were inversely associated with the risk of being cognitively frail after adjusting for confounders. Antioxidants and self-reported health are potential (bio)markers to detect persons at risk of becoming frail. The biomarkers identified may indicate the involvement of inflammation in frailty, especially for physical and cognitive frailty.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the “identify (OS) and treat (by low molecular weight antioxidants, LMWA)” approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different “antioxidants” are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the “identify and treat” approach is questionable.

Human longevity may be found in single individuals as well as in the population as a whole ("population longevity"). Longevity Blue Zones (LBZs), which are areas with an unusually high number of oldest old, have been identified in Sardinia and the Greek island of Ikaria. We compared the lifestyle, health status and some genetic markers of the LBZ populations with those of reference populations from Italy and Greece; the data were extracted from the GEHA database. In the LBZs, the proportion of individuals who never married or were married and still living with their spouse was significantly greater. Nonagenarians males and females with a high self‒perception of optimism and/or a high score for self-rated health were also found in larger proportions in LBZs. Among the variables with lower frequency were the proportion of the widowed, the percentage of subjects who had suffered a stroke and the frequency of Apoe4 and Apoe2 and the TT genotype of FOXO3A gene. Compared to behavioral and health indicators, the impact of genetic factors might be relatively less important in the LBZs. Nevertheless, further research is needed to identify potential epigenetic traits that might play a predominant role due to the interaction between genetics and the human and physical environments.

Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic β5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

The analysis of copper (Cu) and zinc (Zn) along with their major serum carriers, albumin (Alb) and ceruloplasmin (Cp), could provide information on the capacity of humans to maintain homeostasis of metals (metallostasis). However, their relationship with aging, sex, body mass index, as well as with nutritional and inflammatory markers was never investigated in a large-scale study. Here, we report results from the European large-scale cross-sectional study MARK-AGE in which Cu, Zn, Alb, Cp, as well as nutritional and inflammatory parameters were determined in 2424 age-stratified participants (35–75 years), including the general population (RASIG), nonagenarian offspring (GO), a well-studied genetic model of longevity, and spouses of GO (SGO). In RASIG, Cu to Zn ratio and Cp to Alb ratio were higher in women than in men. Both ratios increased with aging because Cu and Cp increased and Alb and Zn decreased. Cu, Zn, Alb, and Cp were found associated with several inflammatory as well as nutritional biomarkers. GO showed higher Zn levels and higher Zn to Alb ratio compared to RASIG, but we did not observe significant differences with SGO, likely as a consequence of the low sample size of SGO and the shared environment. Our results show that aging, sex, body mass index, and GO status are characterized by different levels of Cu, Zn, and their serum carrier proteins. These data and their relationship with inflammatory biomarkers support the concept that loss of metallostasis is a characteristic of inflammaging.

At a recent symposium on aging biology, a debate was held as to whether or not we know what biological aging is. Most of the participants were struck not only by the lack of consensus on this core question, but also on many basic tenets of the field. Accordingly, we undertook a systematic survey of our 71 participants on key questions that were raised during the debate and symposium, eliciting 37 responses. The results confirmed the impression from the symposium: there is marked disagreement on the most fundamental questions in the field, and little consensus on anything other than the heterogeneous nature of aging processes. Areas of major disagreement included what participants viewed as the essence of aging, when it begins, whether aging is programmed or not, whether we currently have a good understanding of aging mechanisms, whether aging is or will be quantifiable, whether aging will be treatable, and whether many non-aging species exist. These disagreements lay bare the urgent need for a more unified and cross-disciplinary paradigm in the biology of aging that will clarify both areas of agreement and disagreement, allowing research to proceed more efficiently. We suggest directions to encourage the emergence of such a paradigm.

Torquetenovirus (TTV) viremia has been associated with increased mortality risk in the elderly population. This work aims to investigate TTV viremia as a potential biomarker of immunosenescence. We compared levels of circulating TTV in 1813 participants of the MARK-AGE project, including human models of delayed (offspring of centenarians [GO]) and premature (Down syndrome [DS]) immunosenescence. The TTV load was positively associated with age, cytomegalovirus (CMV) antibody levels, and the Cu/Zn ratio and negatively associated with platelets, total cholesterol, and total IgM. TTV viremia was highest in DS and lowest in GO, with intermediate levels in the SGO (spouses of GO) and RASIG (Randomly Recruited Age-Stratified Individuals From The General Population) populations. In the RASIG population, TTV DNA loads showed a slight negative association with CD3+T-cells and CD4+T-cells. Finally, males with ≥4log TTV copies/mL had a higher risk of having a CD4/CD8 ratio

The regular use of medication may interfere with micronutrient metabolism on several levels, such as absorption, turnover rate, and tissue distribution, and this might be amplified during aging. This study evaluates the impact of self-reported medication intake on plasma micronutrients in the MARK-AGE Project, a cross-sectional observational study in 2217 subjects (age- and sex-stratified) aged 35&ndash, 75 years from six European countries that were grouped according to age. Polypharmacy as possible determinant of micronutrient concentrations was assessed using multiple linear regression models adjusted for age-group, dietary fruit, vegetables, and juice intake, and other confounders. Younger participants reported taking fewer drugs than older participants. Inverse associations between medication intake and lutein (&minus, 3.31% difference per increase in medication group), &beta, carotene (&minus, 11.44%), &alpha, 8.50%) and positive associations with retinol (+2.26%), &alpha, tocopherol/cholesterol (+2.89%) and &gamma, tocopherol/cholesterol (+1.36%) occurred in multiple adjusted regression models. Combined usage of a higher number of medical drugs was associated with poorer status of carotenoids on the one hand and higher plasma concentrations of retinol, &alpha, and &gamma, tocopherol on the other hand. Our results raise concerns regarding the safety of drug combinations via the significant and surprisingly multifaceted disturbance of the concentrations of relevant micronutrients.

Significance: It is well established that lifestyle and dietary habits have a tremendous impact on life span, the rate of aging, and the onset/progression of age-related diseases. Specifically, die...

Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases.

Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer’s disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer’s disease-associated amyloid β peptide. Biochemical, biophysical and biological assays using isolated amyloid β peptide, primary neurons and various established Alzheimer’s disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid β peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase—an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides. A fluorescence-based assay is used to screen cyclic peptides for their activity in preventing protein misfolding, an event that can generate pathogenic aggregates that lead to diseases such as Alzheimer’s disease or amyotrophic lateral sclerosis..

Background Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Replicative senescence has been linked to macromolecular damage, and especially oxidative stress-induced DNA damage. Recent studies on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has a telomere-length independent function that promotes survival. Methods Here, we studied the DNA damage accumulation and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that overexpress the catalytic subunit of telomerase (hTERT MSCs). Results We showed that hTERT MSCs at high passages have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation of such defects. Finally, hTERT MSCs were found to possess higher activities of the basic antioxidant enzymes, superoxide dismutase and catalase, than control MSCs. Discussion On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cell's basic antioxidant machinery.

The age-associated decline of adult stem cell function contributes to the physiological failure of homeostasis during aging. The proteasome plays a key role in the maintenance of proteostasis and its failure is associated with various biological phenomena including senescence and aging. Although stem cell biology has attracted intense attention, the role of proteasome in stemness and its age-dependent deterioration remains largely unclear. By employing both Wharton's-Jelly- and Adipose-derived human adult mesenchymal stem cells (hMSCs), we reveal a significant age-related decline in proteasome content and peptidase activities, accompanied by alterations of proteasomal complexes. Additionally, we show that senescence and the concomitant failure of proteostasis negatively affects stemness. Remarkably, the loss of proliferative capacity and stemness of hMSCs can be counteracted through proteasome activation. At the mechanistic level, we demonstrate for the first time that Oct4 binds at the promoter region of β2 and β5 proteasome subunits and thus possibly regulates their expression. A firm understanding of the mechanisms regulating proteostasis in stem cells will pave the way to innovative stem cell-based interventions to improve healthspan and lifespan.

Oxidative stress and antioxidants play a role in age-related diseases and in the aging process. We here present data on protein carbonyls, 3-nitrotyrosine, malondialdehyde, and cellular and plasma antioxidants (glutathione, cysteine, ascorbic acid, uric acid,α-tocopherol, and lycopene) and their relation with age in the European multicenter study MARK-AGE. To avoid confounding, only data from countries which recruited subjects from all three study groups (five of eight centers) and only participants aged ≥55 years were selected resulting in data from 1559 participants. These included subjects from (1) the general population, (2) members from long-living families, and (3) their spouses. In addition, 683 middle-aged reference participants (35–54 years) served as a control. After adjustment for age, BMI, smoking status, gender, and country, there were differences in protein carbonyls, malondialdehyde, 3-nitrotyrosine,α-tocopherol, cysteine, and glutathione between the 3 study groups. Protein carbonyls and 3-nitrotyrosine as well as cysteine, uric acid, and lycopene were identified as independent biomarkers with the highest correlation with age. Interestingly, from all antioxidants measured, only lycopene was lower in all aged groups and from the oxidative stress biomarkers, only 3-nitrotyrosine was increased in the descendants from long-living families compared to the middle-aged control group. We conclude that both lifestyle and genetics may be important contributors to redox biomarkers in an aging population.

Recently, Weber et al. published a thorough investigation of the age-dependency of oxidative stress (OS) determined by the steady state concentrations of different compounds - oxidation products and antioxidants - that are in common use as biomarkers of OS in 2207 healthy individuals of the cross-sectional MARK-AGE Project. The correlations among biomarkers were significant but weak. These findings may indicate different manifestations of OS and must further be evaluated. Here, we report a refined analysis of OS based on the above-mentioned original data. We show that malondialdehyde (MDA) appears to be sensitive to both gender and age. It is significantly lower and shows a greater age-dependence in women than in men. The age-dependency of MDA in women arises in a stepwise fashion. The age-dependent slope of the steady state concentration is maximal at the age between 50 and 55 years, indicating that it may be attributed to the change of metabolism in the post-menopause. Interestingly, total glutathione (GSH) decreased with age simultaneously with the increase in MDA.Different biomarkers yield different gender- and age-dependencies. Unlike the concentration of MDA, the concentrations of the other two oxidation products, i.e. protein carbonyls and 3-nitrotyrosine were similar in men and women and appeared to be independent of age in the healthy study population. The analyzed antioxidants exhibited different gender- and age-dependencies. In conclusion, it appears that all the biomarkers assessed here reflect different types of OS and that MDA and GSH reflect the same type of OS. Keywords: Oxidative stress, Age-dependency, Gender-associated differences, Biomarkers

Protein synthesis and degradation balance have a crucial role in maintenance of cellular homeostasis and function. The ubiquitin-proteasome system is one of the major cellular proteolytic machineries responsible for the removal of normal, abnormal, denatured or in general damaged proteins. Proteasome is a multisubunit enzyme that consists of the 20S core and the 19S regulatory complexes giving rise to multiple active forms. In the present study we investigated the crosstalk between protein synthesis and proteasome-mediated protein degradation. Pharmacological protein synthesis inhibition led to increased proteasome function and assembly of 30S/26S proteasome complexes, in human primary embryonic fibroblasts. The enhancement in proteasome function counted for the degradation of ubiquitinated, misfolded and oxidized proteins. Additionally, it was found that heat shock proteins 70 and 90 are probably involved in the elevated proteasome assembly. Our results provide an insight on how the mechanisms of protein synthesis, protein degradation and heat shock protein chaperones machinery interact under various cellular conditions.

This is a PDF file of an unedited manuscript that has been accepted for publication. The final version of this article is available at [https://doi.org/10.1016/j.mad.2018.12.002].

Several laboratory animal models have shown that dietary energy restriction (ER) can promote longevity and improve various health aspects in old age. However, whether the entire spectrum of ER-induced short- and long-term physiological and metabolic adaptions is translatable to humans remains to be determined. In this review article, we present recent evidence towards the elucidation of the impact of ER on brain physiology and in age-related neurodegenerative diseases. We also discuss modulatory influences of ER on metabolism and overall on human health, limitations of current experimental designs as well as future perspectives for ER trials in humans. Finally, we summarize signaling pathways and processes known to be affected by both aging and ER with a special emphasis on the link between ER and cellular proteostasis.

Highlights • Biomarkers of human immunosenescence are discussed. • Longitudinal studies are essential. • Associations of immune markers in older adults with clinical outcome are context-dependent. • There are no universal biomarkers of human immunosenescence. • There are common age-associated changes to peripheral immune markers in humans., There is a great deal of debate on the question of whether or not we know what ageing is (Ref. Cohen et al., 2020). Here, we consider what we believe to be the especially confused and confusing case of the ageing of the human immune system, commonly referred to as “immunosenescence”. But what exactly is meant by this term? It has been used loosely in the literature, resulting in a certain degree of confusion as to its definition and implications. Here, we argue that only those differences in immune parameters between younger and older adults that are associated in some definitive manner with detrimental health outcomes and/or impaired survival prospects should be classed as indicators of immunosenescence in the strictest sense of the word, and that in humans we know remarkably little about their identity. Such biomarkers of immunosenescence may nonetheless indicate beneficial effects in other contexts, consistent with the notion of antagonistic pleiotropy. Identifying what could be true immunosenescence in this respect requires examining: (1) what appears to correlate with age, though generality across human populations is not yet confirmed; (2) what clearly is part of a suite of canonical changes in the immune system that happen with age; (3) which subset of those changes accelerates rather than slows aging; and (4) all changes, potentially population-specific, that accelerate agig. This remains an immense challenge. These questions acquire an added urgency in the current SARS-CoV-2 pandemic, given the clearly greater susceptibility of older adults to COVID-19.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35–75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan. published

Objective: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel.Methods: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers.Results: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified.Conclusion: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) alpha-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin alpha, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGF beta (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (REIN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Nanobionic ® are mineral based and bio-ceramic coating on microfibers clothes based on newly developed high-tech textile. Previous studies have shown that use of these vests has positive effects on athletic performance; however, the mechanism of function remains unknown. In the current report we provide evidence that these clothes have a high energy emission up to 99%, giving it the most positive results of emitting infrared rays. Moreover, we demonstrate that mild use of Nanobionic ® clothing (2 hours/day for 6 days) reduce free radical levels in peripheral blood mononuclear cells (PBMCs) in two-thirds of healthy volunteers ranging from 9.13 to 95.38%. Given these findings we have also examined Nanobionic ® clothing effects on a pathophysiological condition that is linked to oxidative stress and free radicals, namely, Raynaud syndrome which is a rare blood disease. Importantly, we have recorded a significant increase in hands’ temperature in these patients. In conclusion, this report demonstrates the beneficial effects of Nanobionic® vests in reducing free radical levels in healthy individuals as well as in patients suffering from free-radical-related disorders.

Several studies have employed DNA microarrays to identify gene expression signatures that mark human ageing; yet the features underlying this complicated phenomenon remain elusive. We thus conducted a bioinformatics meta-analysis on transcriptomics data from human cell- and biopsy-based microarrays experiments studying cellular senescence orin vivotissue ageing, respectively. We report that coregulated genes in the postmitotic muscle and nervous tissues are classified into pathways involved in cancer, focal adhesion, actin cytoskeleton, MAPK signalling, and metabolism regulation. Genes that are differentially regulated during cellular senescence refer to pathways involved in neurodegeneration, focal adhesion, actin cytoskeleton, proteasome, cell cycle, DNA replication, and oxidative phosphorylation. Finally, we revealed genes and pathways (referring to cancer, Huntington’s disease, MAPK signalling, focal adhesion, actin cytoskeleton, oxidative phosphorylation, and metabolic signalling) that are coregulated during cellular senescence andin vivotissue ageing. The molecular commonalities between cellular senescence and tissue ageing are also highlighted by the fact that pathways that were overrepresented exclusively in the biopsy- or cell-based datasets are modules either of the same reference pathway (e.g., metabolism) or of closely interrelated pathways (e.g., thyroid cancer and melanoma). Our reported meta-analysis has revealed novel age-related genes, setting thus the basis for more detailed future functional studies.

Protein homeostasis (proteostasis) is one of the nodal points that need to be preserved to retain physiologic cellular/organismal balance. The ubiquitin-proteasome system (UPS) is responsible for the removal of both normal and damaged proteins, with the proteasome being the downstream effector. The proteasome is the major cellular protease with progressive impairment of function during aging and senescence. Despite the documented age-retarding properties of proteasome activation in various cellular models, simultaneous enhancement of the 20S core proteasome content, assembly, and function have never been reported in any multicellular organism. Consequently, the possible effects of the core proteasome modulation on organismal life span are elusive. In this study, we have achieved activation of the 20S proteasome at organismal level. We demonstrate enhancement of proteasome levels, assembly, and activity in the nematode Caenorhabditis elegans, resulting in life span extension and increased resistance to stress. We also provide evidence that the observed life span extension is dependent on the transcriptional activity of Dauer formation abnormal/Forkhead box class O (DAF-16/FOXO), skinhead-1 (SKN-1), and heat shock factor-1 (HSF-1) factors through regulation of downstream longevity genes. We further show that the reported beneficial effects are not ubiquitous but they are dependent on the genetic context. Finally, we provide evidence that proteasome core activation might be a potential strategy to minimize protein homeostasis deficiencies underlying aggregation-related diseases, such as Alzheimer’s disease (AD) or Huntington’s disease (HD). In summary, this is the first report demonstrating that 20S core proteasome up-regulation in terms of both content and activity is feasible in a multicellular eukaryotic organism and that in turn this modulation promotes extension of organismal health span and life span.—Chondrogianni, N., Georgila, K., Kourtis, N., Tavernarakis, N., Gonos, E. S. 20S proteasome activation promotes life span extension andresistance to proteotoxicity in Caenorhabditis elegans.

Due to the successful implementation of vaccination strategies early-life morbidity and mortality due to infectious disease has been reduced dramatically. Vaccines against tetanus and diphtheria are among the most frequently used vaccines worldwide, but various studies in different European countries have shown that protection against tetanus and particularly against diphtheria is unsatisfactory in adults and older persons. In this study we analyzed tetanus- and diphtheria-specific antibody concentrations in 2100 adults of different age from 6 selected European countries (Austria, Belgium, Germany, Greece, Italy, Poland) in order to investigate differences in the level of protection against tetanus and diphtheria across Europe. Our data reveal that tetanus- and diphtheria-specific antibody concentrations vary greatly between countries, which is also reflected in the percentage of persons with antibody concentrations below the protective level (0.1 IU/ml), which ranged from 2 to 31% percent for tetanus and 28–63% for diphtheria. In most countries, tetanus- and diphtheria-specific antibody concentrations decrease with age. This phenomenon is more pronounced in countries with generally low antibody levels, such as Italy, Poland and Greece. Interestingly, tetanus-specific antibody concentrations are generally higher in males than in females, which is probably due to vaccination during their military service or more frequent booster vaccinations after injuries, whereas no gender-related differences were found for diphtheria-specific antibodies. In conclusion, our study demonstrates that the European population is not fully protected against tetanus and diphtheria. Measures to improve protection should include a life-long perspective on vaccination, more education to increase awareness of and compliance with vaccination guidelines, and a harmonization of recommendations and incentives across Europe.

Aging is a natural biological process that is characterized by a progressive accumulation of macromolecular damage. In the proteome, aging is accompanied by decreased protein homeostasis and function of the major cellular proteolytic systems, leading to the accumulation of unfolded, misfolded, or aggregated proteins. In particular, the proteasome is responsible for the removal of normal as well as damaged or misfolded proteins. Extensive work during the past several years has clearly demonstrated that proteasome activation by either genetic means or use of compounds significantly retards aging. Importantly, this represents a common feature across evolution, thereby suggesting proteasome activation to be an evolutionarily conserved mechanism of aging and longevity regulation. This review article reports on the means of function of these proteasome activators and how they regulate aging in various species.

Recent longitudinal studies in dietary daily intake in human centenarians have shown that a satisfactory content of some micronutrients within the cells maintain several immune functions, a low grade of inflammation and preserve antioxidant activity. Micronutrients (zinc, copper, selenium) play a pivotal role in maintaining and reinforcing the performances of the immune and antioxidant systems as well as in affecting the complex network of the genes (nutrigenomic) with anti- and pro-inflammatory tasks. Genes of pro- and anti-inflammatory cytokines and some key regulators of trace elements homeostasis, such as Metallothioneins (MT), are involved in the susceptibility to major geriatric disease/disorders. Moreover, the genetic inter-individual variability may affect the nutrients’ absorption (nutrigenetic) with altered effects on inflammatory/immune response and antioxidant activity. The interaction between genetic factors and micronutrients (nutrigenomic and nutrigenetic approaches) may influence ageing and longevity because the micronutrients may become also toxic. This review reports the micronutrient–gene interactions in ageing and their impact on the healthy state with a focus on the method of protein–metal speciation analysis. The association between micronutrient–gene interactions and the protein–metal speciation analysis can give a complete picture for a personalized nutrient supplementation or chelation in order to reach healthy ageing and longevity.