Molecular mechanisms of ageing and related diseases.

Accepted Article

Received Date : 01-Jan-2014 Revised Date

: 07-Apr-2014

Accepted Date : 24-Apr-2014 Article type

: Original Article

Molecular mechanisms of aging and related diseases

Jun-Ping Liu *, Editor-in-Chief, CEPP

Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Zhejiang 311121, China; Department of Immunology, Monash University Central Clinical School, Prahran, Victoria 3181, Australia; Department of Genetics, Faculty of Science, University of Melbourne, Parkville, Victoria 3010, Australia.

Keywords: senescence, longevity, lifespan, telomeres, telomerase, mitochondria, endoplasmic reticulum, lysosome, aging, Parkinson’s disease, diabetes, hypertension, dyskeratosis congenita.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.12247 This article is protected by copyright. All rights reserved.

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* Corresponding author:

Email: [email protected]

Email: [email protected]

Abstract

Human and other multi-cellular life species age and the aging processes become dominant in a late phase of lives. However, recent studies challenged the dogma suggesting that ageing in some animal species may never occur, but mammals undertake cell replicative senescence as early as before birth in embryos under physiological conditions. How the molecular machineries operate, and why aging cells dominate under some circumstances, are intriguing. Recent studies show that cell aging involves extensive cellular remodeling, including telomere attrition, heterochromatin formation, endoplasmic reticulum stress, mitochondrial disorders, lysosome processing organelles and chromatins. This article provides an update in the molecular mechanisms underlying aging of various cell types, the newly described developmental and programmed replicative senescence, and the critical roles of cellular organelles and effectors in Parkinson’s disease, diabetes, hypertension and dyskeratosis congenita.

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Introduction

Aging is a risk factor of many diseases under physiological and pathophysiological conditions. Many age-related diseases stem from a single cell or a group of cells with age-related injures (1-7). The aging cells initiate inflammation by releasing detrimental substances to inflame surrounding tissues to cause further aging, known as the senescence-associated secretary phenotype (SASP) (8-14). Senescence occurs as a degenerative phenotype with the term most often used in studies of cellular senescence. The term senescence is also used occasionally in organismal senescence (15), organ senescence (16, 17) and organelle senescence (18). Cellular senescence is a status of mitotic arrest of dividing cells, commonly known as replicative senescence or the Hayflick limit (19). In contrast to quiescent cells that temporarily and reversibly exit to the G0 status of the cell cycle, senescent cells are produced from dividing cells to the irreversible exit of the cell cycle cells, by significant impacts including unresolvable DNA replication stress (20-23), metabolic pressures of certain oncogenes (24-26) or tumor suppressors (27). In addition to undergoing mitotic slippage to enter G0 or permanent growth arrest in G1/G0 checkpoint, recent studies showed that human uveal melanoma cell line 92-1 cells undergo G2 slippage to become senescent in association with down-regulations of G2-M transition genes and S/G2-specific markers (Cyclin B1 and Aurora A) and up-regulations of the G1-specific markers (Cyclin D1 and Caveolin-1) (28). As a mechanism alternative to apoptosis for cells, cell senescence prevents cell replication with serious internal errors or mutations, preventing tissue overgrowth and tumor formation (29-31). Cell senescence also instigates organic and This article is protected by copyright. All rights reserved.

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organismal aging, and removal of senescent cells from the tissues delays tissue dysfunction and extend health span of the animals (32).

Recent findings that cell senescence is programed to occur physiologically in mammal’s embryos (29, 30) and may not occur in certain reptiles (33) challenged the notion that cellular senescence is age-dependent during organismal aging and raised the new issues of how cellular senescence is genetically programmed and post-genetically regulated to switch functions from precipitating organic aging to fostering embryonic development. Further analysis is required to delineate not only the diversities of cell aging, but also the apparently different mechanisms underpinning various cell aging-related diseases for recognition and management.

Definition and types of cell aging

Aging is defined by biologists as an age-dependent or age-progressive decline in intrinsic physiological function, leading to an increase in age-specific mortality rate and a decrease in age-specific reproductive rate (34). By analogy, cell aging is an age-dependent loss of cellular intrinsic function, e.g. cell division, communication, transport and delivery, leading to an increase in the susceptibility to cell death and removal. As aging occurs to all types of cells including germ line and stem cells (35-37), the characteristics and consequences of different types of cell aging are different with dividing cells displaying cessation of cell division which is called cell This article is protected by copyright. All rights reserved.

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senescence. Since many terminally differentiated cells do not divide, exhibiting physiologically post-mitotic features, such as neurons, pancreatic β cells, cardiac and skeletal muscle cells, cellular aging of the terminally differentiated cells manifests the loss of their specific function such as neurotransmission, stimulus-coupled exocytosis and depolarization-triggered contraction respectively. Differing from mitotic cells, such as epithelial cells and stem cells, aging of which is loss of cell divisions leading to compromised tissue growth and repair, terminally differentiated cells undergo cellular aging with loss of cell type specific functions leading to particular diseases such as neurodegenerative diseases, diabetes and muscle atrophy. Describing terminally differentiated cell aging as senescence may be confounding (38-41), noting the mechanisms of the different kinds of cell aging are apparently deeply different with the predominant deficits being primarily in the cytoplasm for terminally differentiated cell aging and in the nucleus for proliferative cell aging or cell senescence.

For convenience of studying the cellular and molecular mechanisms of the broadly two types of cell aging, I discuss the terminally differentiated cell aging and aging in cells that fail to proliferate senescence aging separately. Multiple lines of evidence indicate that the differentiated cell aging is chiefly triggered by accumulative age-related damages in cellular organelle structure and function. For example, protein misfolding and endoplasmic reticulum (ER) stress has been demonstrated to underpin brain aging-related diseases (42), mutations in mitochondrial DNA transmitted maternally aggravate ageing and brain lesions in mice (43, 44), and increased defective This article is protected by copyright. All rights reserved.

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mitochondrial DNA polymerase precipitates aging with cardiac enlargement (45). The muscle cell aging induced by protein aggregates can be significantly alleviated by the transcription factor FOXO and its target 4E-BP through the autophagy/lysosome system (46). Consistently, functionally demanding of specific stresses puts pressures on cellular organelles such as the ER, mitochondria and lysosomes resulting in stress-induced injuries of the organelles, thereby causing some cells to selectively lose their intrinsic function undergoing aging. Although accumulative damaging substances in terminally differentiated cells may insult the genomes causing genetic and epigenetic alterations of gene expressions, the scales and spectrums of DNA damage response (DDR) seen in non-dividing cells may not be as intensive as that incurred from constant DNA replication mitotic stresses in proliferating cells to cause cell aging.

In contrast, the cell senescence appears to be triggered principally by DDR. At least two types of cell senescence have been defined: replicative senescence and stress- or oncogene-induced premature senescence (SIPS/OIS) that are induced by replicative limits/exhaustions and oncogenic stresses/ insults respectively. A number of lines of evidence indicate that progressive losses of telomeres (ends of chromosomes) operate as a biological clock in inducing replicative senescence of various types of cells including yeast, ciliates, hematopoietic stem cells and cancerous cells (47-55). Upon telomere attrition, the DDR kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) are recruited to the damaged foci of double-stranded and single-stranded DNA respectively, initiating DDR and activating This article is protected by copyright. All rights reserved.

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p53 and p21CIP1 to trigger cell senescence (Fig. 1) (56). Acting as a break, OIS withdraws the dividing cells from the proliferative pool thereby crafting a crucial pathophysiological barrier to prevent tumorigenesis (24, 57). Recently, studies show that OIS is also mediated by a DDR (25, 26). While normal human cells undergo cellular senescence upon an activated oncogene (H-RasV12), inhibition of DDR abrogates OIS (25). DDR and OIS are initiated by oncogene-induced DNA replication stress with augmented numbers of active replicons and alterations in DNA replication fork progression (25). Prematurely terminated DNA replication forks and DNA double-strand breaks have been observed in OIS, suppression of which by targeting ATM overpowers OIS leading to increased tumorigenesis in mice (26). Senescent cells display increased senescence-associated β-galactosidase (SA-βGal) activity, senescence-associated heterochromatin foci (SAHF) containing H3K9me3 and heterochromatin protein 1γ (HP1γ), increased cyclin-dependent protein kinase inhibitors p16INK4a and p21CIP1.

Developmental and programmed senescence

This recently described physiological form of cell senescence in the developing embryo tissues of mice features detected positivity of SA-βGal, H3K9me3, HP1γ and p21CIP1, together with the negativity of cell proliferation marker Ki67 or BrdU incorporation in some tissue areas of embryos (30, 58). Mechanistic specificity studies show that Developmental and programmed senescence (DPS) differs from OIS and replicative


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senescence both in the lack of oncogene-p38-p16INK4a or DDR-ATM-p53 mechanisms and in the significant activation of Smad signaling and p21CIP1 gene in DPS (30, 58). In addition, the most striking feature of DPS may be the inducible mechanisms of DPS under physiological conditions involving transforming growth factor-β (TGF-β) as well as other cytokines from the paracrine and autocrine milieu interieur (30, 58). Furthermore, DPS may share some features with OIS including the involvement of gene expression of p15INK4b and mediators of SASP including mitogen-activated protein kinases and Rel/NF-kB singling (58). Recently, the SASP induces paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo, with multiple components mediating paracrine senescence including TGF-β family ligands, VEGF, CCL2 and CCL20 where TGF-β ligands regulate p15INK4b and p21CIP1 (59).

Currently it remains to be investigated how DPS is fully induced and regulated by the developmental programs. It is well documented that TGF-β plays dominant roles in a number of scenarios including tissue remodeling, repercussions of transplantation and inflammatory processes. Our previous work showed that TGF-β family cytokines play a fundamental role in regulating telomerase activity required in the maintenance of telomeres during developmental and oncogenic processes, with Smad3 being a repressor of the telomerase reverse transcriptase (TERT) gene (60-64). Indeed, telomerase is under the regulations of a number of cytokines (for review, see (65)). Recently both p16INK4A and p15INK4B are involved in telomere DNA damage response This article is protected by copyright. All rights reserved.

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checkpoints (66, 67). So, the putative mechanism of cytokine-induced telomerase inhibition and telomeric DDR-induced p15INK4B activation may require to be investigated in DPS. Moreover, it is also unclear if DPS is a physiologically developmental process confined in embryonic development, and if DPS exists in other developmental and physiological processes such as in immune response, wound healing, tissue repair and organ generation.

The function of senescence in physiological conditions is unknown. It is noteworthy that DPS is frequently detected in the tissue regions that are among the most frequently disrupted areas affected by developmental birth defects and that the ultimate fate of the senescent cells is apoptosis and macrophage-mediated clearance (58). Recently, the human placental syncytiotrophoblast has been shown to exhibit the phenotype and expressed molecular markers of cellular senescence (68). During embryonic development, ERVWE1 (an endogenous retroviral locus involved in hominoid placental physiology) mediates cell fusion leading to formation of the syncytiotrophoblast to serve as the maternal/fetal interface at the placenta (68). However, expression of ERVWE1 causes normal and cancer cell fusion exhibiting hyperploid syncytia and features of cellular senescence by a mechanism of the p53- and p16-pRb-dependent pathways (68).


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Major molecular mechanisms underlying cellular senescence

Telomeres, TERRA and telomerase

Molecular defects that cause aging are often biomarkers important in predicting aging-related diseases. Telomere shortness in human beings is a prognostic marker of ageing, disease, and premature morbidity. As the DNA sequences of TTAGGG repeats and associated binding molecules at the ends of each chromosome in all nucleated cells, telomeres vary in length from 4–14 kb and undergo cell chromosome replication-dependent shortening, so telomere length in mitotic cells reflect faithfully of the numbers of cell replications as a biological clock (69-71). When reaching to a critical length, the shortest telomere triggers DNA damage response and signals to cell senescence (47, 50, 51, 72-78). In addition, chronic stress including cardiovascular disease, poor immunity and psychological pressures modulates telomere length homeostasis, leading to telomere shortening in post-mitotic differentiated cells as often determined in peripheral leukocytes. In healthy premenopausal women, for instance, psychological stress (both perceived stress and chronicity of stress) was associated with increased mitochondrial oxidative stress and decreased telomere length in blood leukocytes, and women with the highest levels of stress showed such shorter telomeres as having an additional decade of aging compared to low stress women (79). Comprehensive lifestyle changes (diet, activity, stress management, and social support) for 5-years in ten men with 25 external controls showed increased telomere length (80).


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Among a cohort of healthy 18- to 55-year-olds, shorter telomere length in a T cell population was associated with increased risk for experimentally induced acute upper respiratory infection and clinical illness (81). When 152 healthy American residents of Pittsburgh, Pennsylvania were quarantined (single rooms), administered nasal drops containing a common cold virus (rhinovirus 39) and monitored for 5 days for development of infection and clinical illness, it was found that the rates of infections and clinical illness were 69% (n = 105) and 22% (n = 33), respectively, and shorter telomeres in the CD8 T cells positive for the T cell receptor CD28 were associated with greater odds of infection without dependence on prechallenge virus-specific antibody, demographics, contraceptive use, season, or body mass index (81). Previous studies show that cigarette smoking is associated with telomere shortening in blood mononuclear cells (82-85). This might implicate telomere damage in the context as a possible intermediate mechanism involved in smoking-associated immune compromise and viral infection. However, recent studies suggest that the dynamics of leukocyte telomere length (LTL) are defined at birth and age-dependent shortening occurs remarkably at rapid rates of attritions during the first 20 years of life (86-88). Comparing telomere shortening in somatic tissues of leukocytes, skeletal muscle, skin and subcutaneous fat of 87 adults (aged 19-77 years), longest telomeres in muscle and shortest telomeres in leukocytes assume similar rates in telomere shortening in the four tissues, so differences in telomere length between proliferative (blood and skin) and minimally proliferative tissues (muscle and fat) are established during early life, and that in adulthood, stem cells replicate and telomeres shorten at similar rates (89).


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Recently, telomere shortening has been shown to induce the expression of noncoding telomere repeat-containing RNA (TERRA), leading to the accumulation of TERRA molecules into a nuclear focus (90). Human TERRA sequences of UUAGGG repeats form parallel-stranded G-quadruplexes (91), and form RNA:DNA hybrid structures or R-loops that involve the long-noncoding RNA TERRA at telomeres with the RNA moity being removed by RNase H enzymes in eukaryotes (92-94). TERRA promotes telomere shortening through exonuclease 1-mediated resection of chromosome ends, with replication stress further enhancing the short telomere phenotype (92, 93). Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters, suggesting that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres (90). TERRA promotes POT1 binding to telomeric ssDNA by removing hnRNPA1 (95). In the budding yeast Saccharomyces cerevisiae, telomeric RNA-DNA hybrids promote recombination-mediated elongation events that delay the onset of cellular senescence, but in the absence of both telomerase and homologous recombination, accumulation of telomeric RNA-DNA hybrids leads to telomere loss and accelerated rates of cellular senescence (94). TERRA antagonizes the functions of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) to promote single telomere DNA replication by replication protein A (RPA) (95) and perhaps also regulate telomerase (96). Consistent with telomere shortening regulation of TERRA expression and TEERA regulation of telomere length, TERRA accumulates in highly proliferating


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normal and cancer cells, and forms large nuclear foci distinct from gammaH2AX foci, promyelocytic leukemia (PML) or Cajal bodies of rapidly proliferating tumor cells and progenitor neurons in highly proliferating zones of normal developing cerebellum, suggesting that elevated TERRA levels reflect a novel early form of telomere regulation during replication stress and cancer cell evolution (97).

With variations in telomere length and their associations with age-associated diseases, rare monogenic diseases such as dyskeratosis congenita and more common diseases like pulmonary fibrosis and some forms of aplastic anemia have been identified to stem from catastrophic mutations in telomere-regulatory genes (98, 99). A genome-wide meta-analysis of 37,684 individuals with replication of selected variants in an additional 10,739 individuals identified seven loci with mean LTL (P < 5 x 10(-8)) (100). Five of the loci contain candidate genes of TERC, TERT, NAF1, OBFC1 and RTEL1 with leading SNPs at two loci of TERC and TERT (100). Genetic risk score analysis combining lead variants at all seven loci in 22,233 coronary artery disease cases and 64,762 controls showed a significant association of the shorter LTL alleles with increased risk of coronary artery disease, supporting a causal role of telomere length variation in some age-related diseases (100). Mice lacking dystrophin and TERC (mdx/mTERC(KO)) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality, with the cardiac defects accompanied by telomere erosion, mitochondrial fragmentation and increased oxidative stress, supporting for a link between telomere length and This article is protected by copyright. All rights reserved.

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dystrophin deficiency in the etiology of dilated cardiomyopathy in Duchenne muscular dystrophy (101). TERT (telomerase reverse transcriptase) and TERC (telomerase RNA component) form the minimal core structure of telomerase that is a large ribonucleoprotein complex operating as a homodimer by binding to two telomeric DNA substrates (102, 103). Telomerase repetitively reverse transcribes its short RNA template to add multiple telomeric repeats onto telomere DNA substrate. Telomerase activity requires the telomere end binding protein complex Pot1-TPP1, and is regulated by multiple factors (65, 104). Pot1-TPP1 complex stimulates markedly telomerase processivity by binding telomerase and the telomere DNA products to promote dissociation (105, 106). Germline mutations in TERT produce an allele of two non-synonymous substitution variations of V791I and V867M (107) and in the TERT T motif (T567M) (108) resulting in decreased telomerase processivity and telomere shortening, but more interestingly, mutations of Tetrahymena TERT (L813Y) (109), human TERT (V658A and D684A) (110) and human TERT (L866Y) result in increased processivity (110). Cells expressing human TERT-L866Y display heterogenous telomere lengths, telomere elongation, multiple telomeric signals indicative of fragile sites and replicative stress, and an increase in short telomeres (111). TERT-locus SNPs have recently been reported to be associated with risks of multiple cancers. Analyzing approximately 480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls, and measuring LTL in 53,724 individuals, Bojesen et al reported that most associations This article is protected by copyright. All rights reserved.

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cluster into three independent peaks: the allele at the peak 1 SNP rs2736108 associates with longer telomeres, lower risks for estrogen receptor-negative and BRCA1 mutation carrier breast cancers; the allele at the peak 2 SNP rs7705526 associates with longer telomeres, higher risk of low-malignant-potential ovarian cancer; and the alleles at the peak 3 SNPs rs10069690 and rs2242652 increase estrogen receptor-negative and BRCA1 mutation carrier breast and invasive ovarian cancer risks but not via altered telomere length (112). The peak 1 and peak 2 SNP associate with increased TERT gene promoter activity, whereas the peak 3 SNP associates with truncated TERT (112).

Metabolic stress-triggered cell cycle arrest

B-Raf (V600E)-induced senescence requires simultaneous suppression of the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) inhibitory enzyme pyruvate dehydrogenase kinase 1 (PDK1) and induction of the PDH-activating enzyme pyruvate dehydrogenase phosphatase 2 (PDP2) (24) (Fig. 1). The resultant active form of PDH without phosphorylation increases the use of pyruvate in the tricarboxylic acid cycle (TCA cycle or the Krebs cycle) causing elevated respiration and redox stress and thus cell senescence (24). Enforced normalization of either PDK1 or PDP2 expression levels inhibits PDH and abrogates OIS, thereby permitting B-Raf (V600E)-driven melanoma development (24). Currently it is unclear if PDH activation and OIS involve DDR and subsequent activation of p53 and p21CIP1. Consistent with the role of increased metabolic stress in facilitating OIS, down-regulation of the TCA


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cycle-associated malate enzymes ME1 and ME2 induces robust OIS in human and mouse cells (27) (Fig. 1). The specific causality of ME1 and ME2 down-regulation to OIS is further bolstered by the interconnection of p53 tumor suppressor whereby both malic enzymes inhibit p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms and cellular senescence (27) (Fig. 1). Further studies are required to elucidate the roles of ME1, ME2 and PDH from the metabolic pathways in the convergence of replicative senescence and OIS.

Signalling to p53, p21CIP1 and p16INK4a

Cellular senescence may be controlled by multiple mechanisms. A pro OIS posttranslational modification mechanism has recently been identified for the multifunctional acetyltransferase Tip61 and protein kinase PRAK (113). Activation by ras, p38 induces Tip60 acetyltransferase activity through phosphorylation (on Thr158, Tip60), then directly interacts with and induces the protein kinase activity of PRAK through acetylation (on K364), to induce OIS (113). Like Tip60, MOZ (monocytic leukemia zinc finger)/KAT6A is a histone acetyltransferase that also functions as an acetyltransferase of p53 at K120 and K382 and co-localizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress (114). The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and the ternary complex enhances p53-dependent p21CIP1 expression and cell senescence (114). Moreover, Akt-mediated phosphorylation of MOZ at T369 inhibits PML and MOZ complex


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formation to prevent cell senescence, whereas PML-mediated suppression of Akt increases PML-MOZ interaction enhancing p53- and p21CIP1-dependent cell senescence upon forced PML expression (114). These post-translational acetylation modifications by Tip60 and MOZ are required for the signaling pathway mediating senescence.

Peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) inhibits skin tumorigenesis through promoting p53-dependent OIS. PPARbeta/delta expression increases p-ERK and decreases p-AKT activity, and decreased p-AKT activity in turn promotes cellular senescence through up-regulation of p53 and p27 expression (115). In addition, the tumor suppressor ASPP2 (53BP2L) that functions to enhance p53-dependent apoptosis with its C-terminal p53-binding domain has been shown to have a Ras-association domain in the N terminus, and thus by binding Ras-GTP to stimulate Ras-induced senescence in nontransformed human cells (116). The transcription coactivator Yes-associated protein (YAP) plays a key role in preventing cellular senescence, so YAP deficiency induces cellular replicative senescence by p53/p16/Rb-mediated and TEAD-Cdk6-dependent mechanisms (117). Recently, oncogene-induced tumor suppressor protein BRCA1 dissociation from chromatin and down-regulation can drive senescence by promoting SAHF formation. As a chromatin-remodeling factor that interacts with BRCA1 and pRB, BRG1 is required for SAHF formation and cell senescence induced by oncogenic RAS or


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BRCA1 loss (118). Over-expression of BRG1 induces SAHF and cell senescence by a mechanism dependent on BRG1 chromatin-remodeling activity and interaction with pRB without requiring DNA damage, whereas knockdown of BRG1 inhibits BRCA1 knockdown-induced SAHF and cell senescence (118). Furthermore, SAHF induced by BRG1 is inhibited by knockdown of pRB, p21CIP1, and p16INK4a, but not p53 (118). Therefore, BRG1 acts downstream of BRCA1 down-regulation and Ras activation, and upstream of pRB, p21CIP1, and p16INK4a, to promote SAHF and senescence (118).

Mechanisms governing the transcription of p16INK4a as one of the major regulators of cellular senescence have been extensively studied. The p16INK4 locus is epigenetically repressed by Polycomb proteins in healthy cells and reactivated as a hallmark of senescence (Fig. 2). Recently, the oncogenes homeobox proteins HLX1 (H2.0-like homeobox 1) and HOXA9 (Homeobox A9) have been shown to recruit polycomb protein complexes to repress INK4a locus (119). A mechanism of reactivating Polycomb-silenced genes including INK4a is mediated by the epigenetic factor ZRF1 that is increased in senescent cells following Ras expression and bound to the p15INK4b, ARF and p16INK4a promoters (120). Depletion of ZRF1 in oncogenic Ras-expressing cells restores proliferation by preventing Arf and p16Ink4a expression, so ZRF1 activates the INK4-ARF locus during cellular senescence (120). In addition, Forkhead box A1 protein (FOXA1) that is a direct target of Polycomb-mediated repression and up-regulated in both replicative and oncogene-induced senescence activates p16(INK4a) transcription both as a classic sequence-specific transcriptional activator This article is protected by copyright. All rights reserved.

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and as an epigenetic regulator in decreasing nucleosome density at the p16(INK4a) promoter in senescent fibroblasts (121) (Fig. 2).

Cell senescence and related diseases

Cell senescence represents the loss of cell intrinsic function of mitosis. An increasing body of evidence has pointed the main cellular and molecular pathologies of cell senescence to the nuclear structure, function and genome replication failures. Thus, DDR and continuous activations of the cell cycle check-points serve as the primary key contributors to the cell senescent relevant diseases. Typical examples of cell senescent diseases induced by gene mutations of telomerase or shelterin components include sporadic and familial aplastic anemia (122), myelodysplastic syndrome (preleukemia) (123), autosomal dominant dyskeratosis congenita (124, 125), X-linked dyskeratosis congenita (126), idiopathic pulmonary fibrosis (sporadic and familial) (127-129).

Although there are debates about whether or not cancers are aging diseases, increased cancer morbidity and mortality with age show a significant age-dependent decline in the physiologically regulated function of cell divisions. So, cell senescent diseases may include senescence failure-induced cell proliferative diseases with benign or malignant cell growth and proliferation. It is noteworthy that the senescence failure-induced phenotypes in the disease tissues may be in contrast to the general senescent phenotypes of the biological bodies. For example, whereas telomeres are lengthened in This article is protected by copyright. All rights reserved.

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some cancers and the vascular smooth muscle cells of genetic hypertensive animals (130), telomeres are shortened in circulating white blood cells of a number of aging related diseases including dyskeratosis congenita, Hoyeraal Hreidarsson syndrome (HHS) and hypertensive patients (131-134).

Dyskeratosis congenita (DC)

Dyskeratosis congenita (DC) is a rare progressive congenital disorder resembling premature aging (similar to progeria somehow). The disease mainly affects the integumentary system characterized by cutaneous pigmentation, premature graying, dystrophy of the nails, leukoplakia of the oral mucosa, continuous lacrimation, anemia, thrombocytopenia, testicular atrophy in the male carriers, and predisposition to cancer with significantly shortened lifespans (125, 126, 135-137). About one half of known DC families are from germline mutations in telomere-regulatory genes, and currently 34 disease-associated gene variants have been described within TERT (108, 138). Dominant mutations that cause DC and its particularly severe form HHS have also been observed in other telomere-regulating genes including TERC, DKC1, TIN2, Apollo, RTEL1 and CTC1 (139-143). HHS is characterized by bone marrow failure, intrauterine growth retardation, developmental delay, microcephaly, cerebellar hypoplasia, immunodeficiency, and extremely short telomeres (138, 143). Recent studies show 2 sibling HHS cases caused by a homozygous mutation (T567M) within


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the TERT T motif, resulting in a markedly reduced telomerase processivity and early-onset multisystem disease (108). Dyskerin that in humans is encoded by the DKC1 gene is a highly conserved nucleolar protein, and as a component of telomerase complex it is essential for TERC maturation and stability, however, recent studies show that dyskerin is modified by small ubiquitin-like modifiers (SUMOs) and mutations in highly conserved dyskerin SUMOylation consensus sites lead to dyskerin instability and altered TERC accumulation, telomerase activity and telomere maintenance characteristic of DC (143). Recent exome sequencing identified mutations in RTEL1, a helicase recruited to telomeres by TRF1 for critical telomeric maintenance and homologous recombination, in HHS (144). In two families with HHS, with the first family of two siblings having very short telomeres inherited a premature stop codon from their mother who has short telomeres and the second family inheriing a premature stop codon in RTEL1 from his father and a missense mutation from his mother who also has short telomeres suggesting a new telomere regulatory gene RTEL1 in the etiology of DC (138). Whole-genome exome sequencing has recently revealed compound heterozygous mutations in four siblings affected with HHS in the gene of RTEL1, with the cell lines established from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displaying telomere shortening, fragility and fusion, and growth defects (144). Using whole-exome sequencing, Walne et al identified biallelic mutations in RTEL1 in an individual with familial HHS, which has been further demonstrated by additional screening in 6/23 index cases with HHS but none in 102 DC (145). All 11 This article is protected by copyright. All rights reserved.

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mutations in RTEL1 from ten HHS individuals of seven families showed an autosomal-recessive disorder with significantly shortened telomeres and high levels of telomeric circles, responsible for a major subgroup (approximately 29%) of HHS (145). Thus, not only telomerase holoenzyme components including dyskerin, but also RTEL1, are required for telomere maintenance with mutations to cause DC.

Hypertension

In 1997, Aviv and Aviv hypothesized that telomeres may be involved in the pathophysiology of essential hypertension (146). Using the animal model of spontaneously hypertensive rats (SHR), we compared cell proliferation, telomerase activity responsible for telomere lengthening and the telomere length in primary vascular smooth muscle cells (VSMC) with that in the controls (130). VSMC proliferate in both SHR and controls continuously, but SHR VSMC show increased proliferative potentials (130). Consistently, SHR VSMC show significant increases in telomerase activity and telomere length. Gene analysis suggested that TERT gene expression is increased. Thus, the genetic hypertensive rats have increased telomerase gene, activity and sizes of telomeres in their vascular smooth muscle cells licensing the cells with increased proliferative potentials (130). The mechanism of increased telomerase gene expression remains to be determined. To investigate if telomerase activation plays a key role in increased proliferation of SHR VSMC, we inhibited telomerase activity using specific gene silencing of TERT and found that inhibition of


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telomerase activity leads to VSMC apoptosis and reduced proliferation, demonstrating that telomerase activation in the vascular walls of SHR is critical in VSMC biology and probably the development of hypertension (56).

To investigate whether cell senescence contributes to pulmonary vessel remodeling and pulmonary hypertension in chronic obstructive pulmonary disease (COPD), Noureddine et al. examined 124 patients with COPD by right heart catheterization and found a negative correlation between leukocyte telomere length and pulmonary hypertension severity (131). Greater severity of lung vascular remodeling and increases in p16INK4a, p21CIP1 and Ki67-staining in VSMC of 14 patients with COPD have been evidence compared to 13 age- and sex-matched control subjects (131). Interestingly senescent VSMC stained for p16INK4a and p21CIP1 were virtually confined to the media, whereas the Ki67-positive cells predominated in the neointima and hypertrophied media. In addition, it was found that senescent VSMC stimulates the growth and migration of non-senescent VSMC through the production and release of paracrine soluble and insoluble factors (131). Thus, complex cellular interactions between senescent and proliferative VSMC have been described in the vascular walls underpinning the pathogenesis of chronic obstructive pulmonary disease. Measurements of telomere length in 686 male US World War II and Korean War veteran monozygotic (MZ) and dizygotic (DZ) twins (including 181 MZ and 125 DZ complete pairs) with a mean age of 77.5 years (range 73-85 years) by double-blinded


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methods showed that the white blood cell mean telomere length shortened in this elderly population by 71 base pairs per year (P < 0.0001), there was no evidence of heritable effects on telomere length maintenance in this elderly population, but telomere length was largely associated with shared environmental factors (P < 0.0001). Individuals with hypertension and cardiovascular disease had significantly shorter telomeres (133).

Since some hypertensive patients are more prone than others to atherosclerotic lesions, Benetos et al. determined the relationship between telomere length in the white blood cells and carotid artery atherosclerotic plaques in hypertensive males, and found that among 163 treated hypertensive men, telomere length was shorter in hypertensive men with extracranial carotid artery plaques assessed with B-mode ultrasound, compared with that of hypertensive men without plaques (132). Furthermore, Ju and colleagues measured leukocyte telomere length as a risk factor of hypertension in a Chinese population of 379 healthy controls and 388 hypertensive patients, aged 30 to 80 years, and found that the median telomere length ratio was shorter in hypertensive than in healthy normotensive subjects (134). After 5 years of follow-up, subjects with shorter telomeres were at a higher risk of developing coronary artery disease than individuals with longer telomeres (134). Thus, in hypertensive vessel walls, there are senescent and proliferative vascular smooth muscle cells with shorter and longer telomeres respectively. Telomeres are shorter in circulating white blood cells of hypertensive patients than normotensive controls, with shorter telomeres correlated with increased risks of developing atherosclerotic lesions and coronary artery disease. This article is protected by copyright. All rights reserved.

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Differentiated cell aging and related diseases

Differentiated cell aging occurs with susceptibility to cellular organelle degenerative injuries. Failure of organelle regeneration (organelle senescence) or failure of injured organelle removal and disposal causes loss of organelle intrinsic function and accumulation of organelle injuries leading to the differentiated cell aging and related diseases. Currently there are no universal cellular markers or no longitudinal cellular demarcations in the cellular developments between normal, aging and demise statues for the differentiated cell aging from the aged tissues. The differentiated cell aging related diseases comprise neurodegenerative diseases and the diseases arisen from other non-dividing cells such as cardiac muscle cells, skeletal muscle cells, adipocytes and pancreatic β cells. Recently, age-progressive damages to such organelles as mitochondria and endoplasmic reticulum (ER) that require lysosome and proteasomes to selectively eliminate and recycle have been the core development as the primary pathologies in the differentiated cell aging diseases: Parkinson’s disease and diabetes.

Parkinson's disease

Parkinson's disease (PD) is a prevalent neurodegenerative disease characteristic progressive loss of midbrain dopaminergic neurons resulting in motor dysfunction. While most PD is sporadic in nature and aging neurons with losing intrinsic neurotransmission function are poorly characterized, a subset can be linked to either dominant or recessive germ line mutations. Studies of the PD related genes indicate that This article is protected by copyright. All rights reserved.

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two mitochondrial proteins play fundamental roles in eliminating degenerative or senescent mitochondria, which are PINK1 (the mitochondrial PTEN-induced putative kinase 1 encoded by the PARK6 gene) and parkin (a RING-between-RING-type E3 ubiquitin ligase encoded by the most frequently mutated PARK2 gene). The two mitochondrial proteins cooperate in a quality control pathway preventing the accumulation of dysfunctional mitochondria by mediating the autophagic clearance of damaged mitochondria (mitophagy) (147, 148).

Accumulation and auto-phosphorylation of PINK1 in damaged mitochondria results in the recruitment of parkin to trigger the degradation of the damaged mitochondria by the proteasome and lysosomes. The PINK1 forms a dimer complex on mitochondria following a decrease in the mitochondrial membrane potential with the dimer correlating with intermolecular phosphorylation of PINK1 and being inhibited by the most disease-associated PINK1 mutations with compromised kinase activity (149). Disruption of PINK1 complex formation by the PINK1 S402A mutation has been shown to have reduced parkin recruitment onto depolarized mitochondria, suggesting that phosphorylation-dependent formation of the dyadic PINK1 complex is an important step for parkin recruitment to damaged mitochondria (149). Once recruited to the mitochondrial outer membrane (MOM) upon depolarization, parkin ubiquitylates the β barrel transmembrane protein porin, GTPase mitofusin (Mfn) and Ras family member Miro, as well as numerous MOM targets, autophagy receptors,


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and the proteasome (150). Mutation of the parkin active site residue C431, which has been found mutated in PD patients disrupts parkin function (150). Under cellular stress, parkin also increases linear ubiquitination of NF-κB essential modulator (NEMO) that up-regulates the mitochondrial GTPase OPA1 for the maintenance of mitochondrial integrity (151). Loss of either NEMO or OPA1 (but not the mitophagy pathway) entrains the loss of parkin-induced stress protection, suggesting that parkin ubiquitination of NEMO, activation of NF-κB and up-regulation of OPA1 are part of the physiological relevance of parkin regulating of cell aging pathway (151). Parkin initiates a neuronal ubiquitin ligase cascade involving the multi-subunit ubiquitin ligase SCF (Fbw7beta) on the MOM, with the SCF substrate adapter Fbw7beta being engaged for proteasomal degradation, so the Fbw7beta substrate mitochondrial pro-survival factor Mcl-1 is stabilized (152). Thus, loss of parkin function may lead to death of dopaminergic neurons through unregulated SCF (Fbw7beta)-mediated ubiquitination-dependent proteolysis of the pro-survival protein Mcl-1 (152).

Interestingly, the latest studies show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis, suggesting an unexpected functional link between mitophagy and infectious disease (153). While the ubiquitin ligases responsible for catalyzing ubiquitination of intracellular bacteria are poorly understood, parkin-deficient mice and flies are sensitive to various intracellular bacterial infections (153). Consistently, genetic polymorphisms in the PARK2 regulatory region in humans are associated with increased susceptibility to intracellular pathogens (153). These This article is protected by copyright. All rights reserved.

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findings imply a new mechanism of parkin mutation in bacterial pathogen-induced cell aging (56). In searching for the regulators of parkin translocation to damaged mitochondria, Hasson et al. used genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy and found that TOMM7 stabilizes PINK1 on the MOM, but SIAH3 localized to mitochondria inhibits PINK1 accumulation after mitochondrial insult reducing parkin translocation (148). HSPA1L (HSP70 family member) and BAG4 then play an opposing role in regulating parkin translocation (148). Burchell et al. reported that Fbxo7, an F-box domain-containing protein encoded by PARK15 and mutated in early-onset autosomal recessive PD, interacts with PINK1 and parkin (147). Under-expression of Fbxo7 expression in cells leads to defects of parkin translocation to mitochondria, ubiquitination of Mfn 1 and mitophagy, whereas ectopic over-expression of Fbxo7 rescues loss of parkin and PD-causing mutations in Fbxo7 in Drosophila (147). As shown by Chen et al, ablation of Mfn2 in mouse cardiac myocytes also prevents depolarization-induced parkin translocation to mitochondria and suppresses mitophagy (18). Parkin bound to Mfn2 in a manner dependent on PINK1 phosphorylation of Mfn2 on MOM (18). Thus, Mfn2 is found to function as a mitochondrial receptor for parkin and is required for quality control of mitochondria by PINK1 and parkin (18).

Ions are recently found to regulate PINK1 function. Deprivation of iron using iron chelators generates a heightened mitophagy response in primary human fibroblasts as well as those isolated from a Parkinson's patient with parkin mutations, without This article is protected by copyright. All rights reserved.

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requiring PINK1 stabilization or parkin activation (154). In Drosophila, a dominant suppressor of PINK1 was identified as aconitase (acon) that is localized to mitochondria and acts independent of parkin, downstream of PINK1, by harboring a labile iron-sulfur cluster that can scavenge superoxide to release hydrogen peroxide and iron. Mutation of PINK1 disrupts acon protection of the rpm-sufur cluster and increases superoxide leading to oxidative stress and mitochondrial injuries (155). These findings link iron toxicity to mitochondrial failure in PD-relevant models.

Therapeutic approaches to enhance the activity of PINK1 would improve the PINK1-parkin pathway for improving mitochondrial function. The latest research shows that an alternative neo-substrate using the ATP analog kinetin triphosphate (KTP) increases the activity of both PD-related mutant PINK1 (G309D) and wild type PINK1, and application of the KTP precursor kinetin to cells results in significantly higher levels of PINK1 activity and parkin recruitment to depolarized mitochondria in association with reduced mitochondrial motility in axons and apoptosis (156).

Diabetes

Diabetes is presented with absolute or relative insufficiency of insulin due to pancreatic islet insulin-producing β cell aging or insulin receptor signaling insensitivity (desensitization and resistance) in other aging tissues respectively. Differentiated cell aging may be the primary pathological phenotype of pancreatic β cells in the adults. This article is protected by copyright. All rights reserved.

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Several cell death modes that define apoptosis have not been observed for the β cells, and upon serum removal no apoptosis is observed for β cells, suggesting that apoptosis is not the primary mode of adult primary β cell fate (157). The precise causes of insulin resistance are varied. Currently the heterogeneous etiology of insulin resistance involves the roles of inflammation and lipid metabolism in the aging hepatocytes and adipocytes, glucose metabolisms in the aging skeletal muscle cells, and metabolic disorders associated with the gastrointestinal microbiota in the aging digestive tracts. Molecular mechanisms underpinning the differentiated aging cells take account of the intracellular signaling pathway, protein folding in the endoplasmic reticulum (ER), and exocytotic pathways (56).

The mechanisms of pancreatic β cell aging remain poorly understood despite many decades of research. Wolfram syndrome is an autosomal recessive disorder caused by mutations in WFS1 gene and characterized by insulin-dependent diabetes mellitus, optic atrophy and deafness (158). Using pluripotent stem (iPS) cells to create insulin-producing cells from individuals with Wolfram syndrome, Shang et al. showed that WFS1-deficient β cells exhibits increased ER stress and decreased insulin, upon exposure to experimental ER stress Wolfram β cells display impaired insulin processing and secretion in response to glucose and other secretagogues, and 4-phenyl butyric acid, a chemical protein folding and trafficking chaperone improves insulin synthesis and secretion, indicating that ER stress plays a key role in β cell failure in Wolfram syndrome and other forms of diabetes (158). As a key regulator of This article is protected by copyright. All rights reserved.

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mitochondrial fission, dynamin-related protein 1 (DRP1) is found as an ER resident regulating ER morphology in stressed β cells (159). Interestingly, inhibition of DRP1 activity with a GTP hydrolysis-defective mutant (Ad-K38A) inhibits both fatty acid-induced ER expansion and mitochondrial fission (159). Stimulating the key energy-sensor AMP-activated protein kinase (AMPK) however increases the phosphorylation at the anti-fission site Serine 637 and largely prevents the alterations in ER and mitochondrial morphology, but expression of a DRP1 mutant resistant to phosphorylation at this position partially prevents the recovery of ER and mitochondrial morphology by AMPK (159). So, DRP1 may be a quality control GTPase for both ER and mitochondria, serving downstream of AMPK involved in differentiated cell aging.

Recent studies also show that in stimulating β cell function and insulin secretion, the fractalkine (FKN)/CX3CR1 ligand/receptor signaling pathway represents a regulatory mechanism that failed in diabetes. FKN is significantly decreased in the islets in aging and high-fat diet/obesity, implicating FKN/CX3CR1 signaling as playing an important role in controlling β cell dysfunction in type 2 diabetes (160). In mice, CX3CR1 deficiency exhibits a marked defect in glucose and Glucagon-likepeptide1 (GLP1)-stimulated insulin secretion, whereas administration of FKN improves glucose tolerance with an increase in insulin secretion in association with FKN-increased intracellular Ca(2+) (160).


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Skeletal muscle accounts for 70-90% of insulin-stimulated glucose disposal, one mechanism underlying muscle insulin resistance possibly in various metabolic disorders including obesity and type 2 diabetes has been unveiled recently. In mice, the muscle-specific E3 ubiquitin ligase mitsugumin 53 (MG53; also called TRIM72) mediates the degradation of the insulin receptor and insulin receptor substrate 1 (IRS1), and when up-regulated, causes metabolic syndrome featuring insulin resistance, obesity, hypertension and dyslipidaemia (161). In contrast, disrupting MG53 prevents diet-induced metabolic syndrome by preserving the insulin receptor, IRS1 and insulin signalling integrity (161). While the full spectrum of MG53 substrates require further investigation, ER stress in skeletal muscles increases tribbles 3, a putative protein kinase downstream of NF-κB and inhibitory to Akt. High-fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases tribbles 3 and ER stress in skeletal muscle (162). Consistently ER stressors thapsigargin and tunicamycin also increases tribbles 3, and increased tribbles 3 in C2C12 myotubes and in the mouse tibialis anterior muscles impairs insulin signalling and glucose uptake, effects reversed in cells overexpressing RNAi for tribbles 3 and in muscles from tribbles 3 knockout mice (162). Furthermore, tribbles 3 knockout mice are protected from high-fat diet-induced insulin resistance in skeletal muscle, demonstrating tribbles 3 in mediating ER stress-induced insulin resistance in skeletal muscle (162)

Obesity-induced insulin resistance is the major determinant of metabolic syndrome, which precedes the development of type 2 diabetes mellitus and is thus the driving force behind the emerging diabetes epidemic. Kinase suppressors of Ras 1 and 2 (KSR1 This article is protected by copyright. All rights reserved.

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and KSR2) function as molecular scaffolds to bind Raf-MEK-ERK complex and potently regulate multiple cell fates. Recently, targeted deletion of Ksr2 leads to obesity in mice, and multiple rare variants in KSR2 occurs to early-onset obesity in human, with disrupted signaling through the Raf-MEK-ERK pathway and impaired cellular fatty acid oxidation and glucose oxidation - effects that can be ameliorated by the widely prescribed anti-diabetic drug metformin (163). Most importantly, mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance (163). These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans by governing the Raf-MEK-ERK signaling (163). Although KSR2 mutation has no difference from the wild type in affecting AMPK (163), KSR2 interacts with AMP kinase probably contributing to insulin functionality and AMPK-mediated regulation of energy metabolism under particular conditions (163, 164).

Adipocyte aging biology is an area requiring intensive investigation. The adipocyte nuclear receptor PPARγ, peroxisome proliferator activated receptor gamma, has been identified to be the target of the thiazolidinedione class of insulin sensitizing drugs. Recently, fibroblast growth factor 1 (FGF1) is involved in regulating adipose homeostasis by linking to PPARγ (165). Mice deficient of FGF1 develop an aggressive diabetes-like phenotype with adipose expansion when challenged with a high-fat diet, whereas in response to a high-fat diet, FGF1 is highly induced by PPARγ through a conserved proximal PPAR response element in the FGF1 gene in adipose tissue (165). This article is protected by copyright. All rights reserved.

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Lack of FGF1 induces an inflammatory process with the vasculature network, aberrant adipocyte size distribution and ectopic expression of pancreatic lipases (165). Thus, the feast and famine cycles regulate adipose tissue remodeling by regulating the PPARγ and FGF1 axis. However, if the inflammatory phenotype in adipose tissues with insulin resistance is a feature of adipose aging involving SASP is unknown. Chronic, low-grade inflammation has been a major link common to both type 1 and type 2 diabetes (166). Prolonged nutrient excess triggers leukocyte accumulation in visceral adipose tissue and ultimately other tissues, leading to insulin resistance, type2 diabetes and fatty-liver. Recently, a population of Treg cells has been implicated in adipose inflammation and insulin sensitivity (166, 167). PPARγ expression by Treg cells is necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione drug pioglitazone (166).

A role of gut microbiota has been identified recently in type 2 diabetes. Analysis on gut microbial content in patients with type 2 diabetes, Qin et al. have developed a protocol for a metagenome-wide association study (MGWAS) and undertook a two-stage MGWAS based on deep shotgun sequencing of the gut microbial DNA from 345 Chinese individuals (168). Patients with type 2 diabetes were characterized by a moderate degree of gut microbial dysbiosis, a decrease in the abundance of some universal butyrate-producing bacteria and an increase in various opportunistic pathogens, as well as an enrichment of other microbial functions conferring sulphate reduction and oxidative stress resistance (168). By altering microbial folate and This article is protected by copyright. All rights reserved.

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methionine metabolism, Cabreiro et al. reported also recently, the widely prescribed biguanide drug metformin to treat type 2 diabetes and metabolic syndrome increases lifespan (169). Metformin also increases lifespan in C. elegans co-cultured with Escherichia coli depending on E. coli strain metformin sensitivity and glucose concentration (169). In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic strategies of diabetes. (169)

In addition to gut microbiota and metformin, exercise has long been demonstrated to have beneficial effects on diabetes. Recent studies revealed lysosomes as checkpoints for organelle and protein quality control underlying the exercise-induced effects. Mice with acute exercise show induced autophagy in skeletal and cardiac muscle of fed mice, but mice deficient in stimulus (exercise- or starvation)-induced autophagy, termed BCL2 AAA mice containing knock-in mutations in BCL2 phosphorylation sites, show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise(and starvation)-induced autophagy in mice (170).


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Cell renaissance?

Recent studies that a number of life species do not appear to undergo aging with age-progressive reduction in mortality suggest that there are mechanisms of cell renaissance in the life kingdoms (33). Comparing fertility and mortality patterns of 46 different species including 11 mammals, 12 other vertebrates, 10 invertebrates, 12 vascular plants and one green alga, Jones et al. found that there is no significant association between the length of life and the degree of aging (33). There is great variation among these species, including increasing, constant, decreasing, humped and bowed trajectories for both long- and short-lived species. However, most of the 46 species can be roughly classified into strong deterioration with age, negligible deterioration, negative senescence and improvement with age (33). Lifespans range from 1,400 years for the hydra to just 25 days for nematode worms (33). Of the 24 species showing the most abrupt increase in mortality with age, 11 had relatively long lifespans and 13 had relatively short lifespans, whereas among 24 species with less senescence, 13 had relatively long life spans and 11 had relatively short life spans. For approximately constant mortality and fertility, vertebrates include collared flycatchers and red-legged frogs, invertebrates include hermit crabs (Pagurus longicarpus) and red abalone, and vascular plants include great rhododendron (Rhododendron maximum) and armed saltbush (Atriplex acanthocarpa) (33).

The mechanisms of constant mortality and cell renaissance await investigations. They might be just the reverse of the mechanisms of aging, including post-birth continuously gained the ability of cell regeneration and tissue repair, and age-progressively This article is protected by copyright. All rights reserved.

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maintained or reduced loss of genetic materials (such as telomere shortening, gene mutations, RNA and protein degradation). It has been discussed that asexual reproduction, modularity, lack of germ-line sequestration from the soma, the importance of protected niches, regenerative capacity and paucity of diverse cell types may facilitate the escape from senescence in some clades (33). The central to the mechanisms against aging in mammalian tissues could thus be the up-regulated capacity of intracellular repair, recycling and reconstruction, and the increased capacity of cell renewal and reproduction. Most mammalian tissues harbor stem cells, but the extent to which they contribute to normal homeostasis and repair varies widely (171). There is an overall decline in tissue regenerative potential with age, and it is not known if the stem cell aging is due to the intrinsic mechanism of ageing or to the aged tissue environment (171).

Evolution might not inevitably lead to senescence, or a decline in intrinsic physiological function leading to an increase in age-specific mortality rate and a decrease in age-specific reproductive rate (56). That some mechanisms were enabled, and others are maintained through evolution to confer mortality and fertility in an inverse proportionality to lifespan on certain forms of life may be also possible. Many experts in the biology of ageing believe that interventions to slow ageing are a matter of when rather than if (172). With the currently delayed death and constant rate of deterioration with age across individuals and over time, humans are reaching old age in


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better health and further progress is likely to be made in advancing the frontier of healthy survival to even greater ages (173).

Acknowledgements

The author wishes to thank members of his research team for supports and helpful discussions. This work was supported by grants from National Basic Research Program of China (2012CB911204), the National Natural Science Foundation of China (81170313, 81272889), and the National Health and Medical Research Council of Australia.

References 1.

Arends M, van Dussen L, Biegstraaten M, Hollak CE. Malignancies and monoclonal gammopathy

in Gaucher disease; a systematic review of the literature. Br J Haematol; 161:832-42. 2.

Cox CJ, Sharma M, Leckman JF, et al. Brain human monoclonal autoantibody from sydenham

chorea targets dopaminergic neurons in transgenic mice and signals dopamine d2 receptor: implications in human disease. J Immunol; 191:5524-41. 3.

Kakizoe T, Tanooka H, Tanaka K, Sugimura T. Single-cell origin of bladder cancer induced by

N-butyl-N-(4-hydroxybutyl) nitrosamine in mice with cellular mosaicism. Gann 1983; 74:462-5. 4.

Rinaldi F, Mairs RJ, Wheldon TE, Katz F, Chessells JM, Gibson BE. Clonality analysis suggests that

early-onset acute lymphoblastic leukaemia is of single-cell origin and implies no major role for germ cell mutations in parents. Br J Cancer 1999; 80:909-13. 5.

Pistoia V, Roncella S, Di Celle PF, et al. Emergence of a B-cell lymphoblastic lymphoma in a

patient with B-cell chronic lymphocytic leukemia: evidence for the single-cell origin of the two tumors. Blood 1991; 78:797-804.


Accepted Article

6.

Cleary ML, Galili N, Trela M, Levy R, Sklar J. Single cell origin of bigenotypic and biphenotypic B

cell proliferations in human follicular lymphomas. J Exp Med 1988; 167:582-97. 7.

Tanooka H, Tanaka K. Evidence for single-cell origin of 3-methylcholanthrene-induced

fibrosarcomas in mice with cellular mosaicism. Cancer Res 1982; 42:1856-8. 8.

Tchkonia T, Zhu Y, van Deursen J, Campisi J, Kirkland JL. Cellular senescence and the senescent

secretory phenotype: therapeutic opportunities. J Clin Invest; 123:966-72. 9.

Acosta JC, Banito A, Wuestefeld T, et al. A complex secretory program orchestrated by the

inflammasome controls paracrine senescence. Nat Cell Biol; 15:978-90. 10. Hoare M, Narita M. Transmitting senescence to the cell neighbourhood. Nat Cell Biol; 15:887-9. 11. Zhao Y, Chen F, Chen S, Liu X, Cui M, Dong Q. The Parkinson's disease-associated gene PINK1 protects neurons from ischemic damage by decreasing mitochondrial translocation of the fission promoter Drp1. J Neurochem. 12. Yoshimoto S, Loo TM, Atarashi K, et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature; 499:97-101. 13. Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol; 75:685-705. 14. Coppe JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol; 5:99-118. 15. Mitteldorf J, Pepper JW. How can evolutionary theory accommodate recent empirical results on organismal senescence? Theory Biosci 2007; 126:3-8. 16. de Jesus BB, Blasco MA. Assessing cell and organ senescence biomarkers. Circ Res; 111:97-109. 17. Fine RE. Vesicles without clathrin: intermediates in bulk flow exocytosis. Cell 1989; 58:609-10. 18. Chen Y, Dorn GW, 2nd. PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria. Science; 340:471-5. 19. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res 1961; 25:585-621. 20. Liu JP, Nicholls C, Chen SM, Li H, Tao ZZ. Strategies of treating cancer by cytokine regulation of chromosome end remodelling. Clin Exp Pharmacol Physiol; 37:88-92. 21. Bayne S, Jones ME, Li H, Pinto AR, Simpson ER, Liu JP. Estrogen deficiency leads to telomerase inhibition, telomere shortening and reduced cell proliferation in the adrenal gland of mice. Cell research 2008; 18:1141-50.


Accepted Article

22. Li H, Liu JP. Signaling on telomerase: a master switch in cell aging and immortalization. Biogerontology 2002; 3:107-16. 23. Liu JP. Studies of the molecular mechanisms in the regulation of telomerase activity. FASEB J 1999; 13:2091-104. 24. Kaplon J, Zheng L, Meissl K, et al. A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence. Nature; 498:109-12. 25. Di Micco R, Fumagalli M, Cicalese A, et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 2006; 444:638-42. 26. Bartkova J, Rezaei N, Liontos M, et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006; 444:633-7. 27. Jiang P, Du W, Mancuso A, Wellen KE, Yang X. Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence. Nature; 493:689-93. 28. Ye C, Zhang X, Wan J, et al. Radiation-induced cellular senescence results from a slippage of long-term G2 arrested cells into G1 phase. Cell Cycle 2013; 12:1424-32. 29. Banito A, Lowe SW. A new development in senescence. Cell; 155:977-8. 30. Munoz-Espin D, Canamero M, Maraver A, et al. Programmed Cell Senescence during Mammalian Embryonic Development. Cell; 155:1104-18. 31. Senescence and cell death. Nature 2001; 409:1123-4. 32. Baker DJ, Wijshake T, Tchkonia T, et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature; 479:232-6. 33. Jones OR, Scheuerlein A, Salguero-Gomez R, et al. Diversity of ageing across the tree of life. Nature. 34. Flatt T. A new definition of aging? Front Genet; 3:148. 35. Signer RA, Morrison SJ. Mechanisms that regulate stem cell aging and life span. Cell Stem Cell; 12:152-65. 36. Rodgers JT, Rando TA. Sprouting a new take on stem cell aging. EMBO J; 31:4103-5. 37. Jones LS, Berndtson WE. A quantitative study of Sertoli cell and germ cell populations as related to sexual development and aging in the stallion. Biol Reprod 1986; 35:138-48. 38. Cohen G. The pathobiology of Parkinson's disease: biochemical aspects of dopamine neuron senescence. J Neural Transm Suppl 1983; 19:89-103.


Accepted Article

39. Yang X, Doser TA, Fang CX, et al. Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress. FASEB J 2006; 20:1024-6. 40. Dong W, Cheng S, Huang F, et al. Mitochondrial dysfunction in long-term neuronal cultures mimics changes with aging. Med Sci Monit; 17:BR91-6. 41. Geng YQ, Guan JT, Xu XH, Fu YC. Senescence-associated beta-galactosidase activity expression in aging hippocampal neurons. Biochem Biophys Res Commun; 396:866-9. 42. Uehara T, Nakamura T, Yao D, et al. S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature 2006; 441:513-7. 43. Johnson MS, Mitchell R, Thomson FJ. The priming effect of luteinizing hormone-releasing hormone (LHRH) but not LHRH-induced gonadotropin release, can be prevented by certain protein kinase C inhibitors. Mol.Cell.Endocrinol. 1992; 85:183-93. 44. Ross JM, Stewart JB, Hagstrom E, et al. Germline mitochondrial DNA mutations aggravate ageing and can impair brain development. Nature; 501:412-5. 45. Trifunovic A, Wredenberg A, Falkenberg M, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 2004; 429:417-23. 46. Demontis F, Perrimon N. FOXO/4E-BP signaling in Drosophila muscles regulates organism-wide proteostasis during aging. Cell; 143:813-25. 47. Schaetzlein S, Kodandaramireddy NR, Ju Z, et al. Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice. Cell 2007; 130:863-77. 48. Lundblad V, Blackburn EH. An alternative pathway for yeast telomere maintenance rescues est1senescence. Cell 1993; 73:347-60. 49. Lundblad V, Szostak JW. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 1989; 57:633-43. 50. d'Adda di Fagagna F, Reaper PM, Clay-Farrace L, et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003; 426:194-8. 51. Wong KK, Maser RS, Bachoo RM, et al. Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing. Nature 2003; 421:643-8. 52. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990; 346:866-8.


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53. Yu GL, Bradley JD, Attardi LD, Blackburn EH. In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 1990; 344:126-32. 54. Karlseder J, Smogorzewska A, de Lange T. Senescence induced by altered telomere state, not telomere loss. Science 2002; 295:2446-9. 55. Nicholls C, Pinto AR, Li H, et al. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) induces cancer cell senescence by interacting with telomerase RNA component. Proc Natl Acad Sci U S A; 109:13308-13. 56. Liu JP. Aging and related diseases: research progress. Progress in Biochemistry and Biophysics 2014; 41:215-30. 57. Kang TW, Yevsa T, Woller N, et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature; 479:547-51. 58. Storer M, Mas A, Robert-Moreno A, et al. Senescence Is a Developmental Mechanism that Contributes to Embryonic Growth and Patterning. Cell; 155:1119-30. 59. Acosta JC, Banito A, Wuestefeld T, et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol 2013; 15:978-90. 60. Li H, Xu D, Li J, Berndt MC, Liu JP. Transforming Growth Factor beta Suppresses Human Telomerase Reverse Transcriptase (hTERT) by Smad3 Interactions with c-Myc and the hTERT Gene. J Biol Chem 2006; 281:25588-600. 61. Li H, Xu D, Toh BH, Liu JP. TGF-beta and cancer: is Smad3 a repressor of hTERT gene? Cell research 2006; 16:169-73. 62. Cassar L, Li H, Pinto AR, Nicholls C, Bayne S, Liu JP. Bone morphogenetic protein-7 inhibits telomerase activity, telomere maintenance, and cervical tumor growth. Cancer research 2008; 68:9157-66. 63. Katik I, Mackenzie-Kludas C, Nicholls C, et al. Activin inhibits telomerase activity in cancer. Biochem Biophys Res Commun 2009; 389:668-72. 64. Liu JP, Nicholls C, Chen SM, Li H, Tao ZZ. Strategies of Treating Cancer by Cytokine Regulation of Chromosome End Remodelling. Clin Exp Pharmacol Physiol 2009. 65. Liu JP, Chen SM, Cong YS, et al. Regulation of telomerase activity by apparently opposing elements. Ageing Res Rev 2010; 9:245-56. 66. Wang Y, Sharpless N, Chang S. p16(INK4a) protects against dysfunctional telomere-induced ATR-dependent DNA damage responses. J Clin Invest; 123:4489-501.


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67. Yasaei H, Gilham E, Pickles JC, Roberts TP, O'Donovan M, Newbold RF. Carcinogen-specific mutational and epigenetic alterations in INK4A, INK4B and p53 tumour-suppressor genes drive induced senescence bypass in normal diploid mammalian cells. Oncogene; 32:171-9. 68. Chuprin A, Gal H, Biron-Shental T, et al. Cell fusion induced by ERVWE1 or measles virus causes cellular senescence. Genes Dev 2013; 27:2356-66. 69. Blackburn EH. Structure and function of telomeres. [Review]. Nature 1991; 350:569-73. 70. de Lange T. Telomeres. Nature 1998; 392:753-4. 71. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990; 345:458-60. 72. Karlseder J, Broccoli D, Dai Y, Hardy S, de Lange T. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 1999; 283:1321-5. 73. Hemann MT, Strong MA, Hao LY, Greider CW. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 2001; 107:67-77. 74. Munoz P, Blanco R, Flores JM, Blasco MA. XPF nuclease-dependent telomere loss and increased DNA damage in mice overexpressing TRF2 result in premature aging and cancer. Nature genetics 2005; 37:1063-71. 75. Denchi EL, de Lange T. Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature 2007; 448:1068-71. 76. Hewitt G, Jurk D, Marques FD, et al. Telomeres are favoured targets of a persistent DNA damage response in ageing and stress-induced senescence. Nat Commun 2012; 3:708. 77. Cesare AJ, Hayashi MT, Crabbe L, Karlseder J. The telomere deprotection response is functionally distinct from the genomic DNA damage response. Molecular cell 2013; 51:141-55. 78. Wang Y, Sharpless N, Chang S. p16(INK4a) protects against dysfunctional telomere-induced ATR-dependent DNA damage responses. The Journal of clinical investigation 2013; 123:4489-501. 79. Epel ES, Blackburn EH, Lin J, et al. Accelerated telomere shortening in response to life stress. Proceedings of the National Academy of Sciences of the United States of America 2004; 101:17312-5. 80. Ornish D, Lin J, Chan JM, et al. Effect of comprehensive lifestyle changes on telomerase activity and telomere length in men with biopsy-proven low-risk prostate cancer: 5-year follow-up of a descriptive pilot study. The lancet oncology 2013; 14:1112-20.


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81. Cohen S, Janicki-Deverts D, Turner RB, et al. Association between telomere length and experimentally induced upper respiratory viral infection in healthy adults. JAMA : the journal of the American Medical Association 2013; 309:699-705. 82. Valdes AM, Andrew T, Gardner JP, et al. Obesity, cigarette smoking, and telomere length in women. Lancet 2005; 366:662-4. 83. Vasan RS, Demissie S, Kimura M, et al. Association of leukocyte telomere length with circulating biomarkers of the renin-angiotensin-aldosterone system: the Framingham Heart Study. Circulation 2008; 117:1138-44. 84. Mirabello L, Huang WY, Wong JY, et al. The association between leukocyte telomere length and cigarette smoking, dietary and physical variables, and risk of prostate cancer. Aging cell 2009; 8:405-13. 85. Du M, Prescott J, Kraft P, et al. Physical activity, sedentary behavior, and leukocyte telomere length in women. American journal of epidemiology 2012; 175:414-22. 86. Frenck RW, Jr., Blackburn EH, Shannon KM. The rate of telomere sequence loss in human leukocytes varies with age. Proceedings of the National Academy of Sciences of the United States of America 1998; 95:5607-10. 87. Aubert G, Baerlocher GM, Vulto I, Poon SS, Lansdorp PM. Collapse of telomere homeostasis in hematopoietic cells caused by heterozygous mutations in telomerase genes. PLoS genetics 2012; 8:e1002696. 88. Benetos A, Kark JD, Susser E, et al. Tracking and fixed ranking of leukocyte telomere length across the adult life course. Aging cell 2013; 12:615-21. 89. Daniali L, Benetos A, Susser E, et al. Telomeres shorten at equivalent rates in somatic tissues of adults. Nat Commun 2013; 4:1597. 90. Cusanelli E, Romero CA, Chartrand P. Telomeric noncoding RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres. Molecular cell 2013; 51:780-91. 91. Martadinata H, Phan AT. Structure of human telomeric RNA (TERRA): stacking of two G-quadruplex blocks in K(+) solution. Biochemistry 2013; 52:2176-83. 92. Pfeiffer V, Crittin J, Grolimund L, Lingner J. The THO complex component Thp2 counteracts telomeric R-loops and telomere shortening. The EMBO journal 2013; 32:2861-71. 93. Pfeiffer V, Lingner J. TERRA promotes telomere shortening through exonuclease 1-mediated resection of chromosome ends. PLoS genetics 2012; 8:e1002747.


Accepted Article

94. Balk B, Maicher A, Dees M, et al. Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence. Nature structural & molecular biology 2013; 20:1199-205. 95. Flynn RL, Centore RC, O'Sullivan RJ, et al. TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA. Nature 2011; 471:532-6. 96. Redon S, Zemp I, Lingner J. A three-state model for the regulation of telomerase by TERRA and hnRNPA1. Nucleic acids research 2013; 41:9117-28. 97. Deng Z, Wang Z, Xiang C, et al. Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma. Journal of cell science 2012; 125:4383-94. 98. Armanios M. Syndromes of Telomere Shortening. Annu Rev Genomics Hum Genet 2009. 99. Calado RT, Young NS. Telomere diseases. N Engl J Med 2009; 361:2353-65. 100. Codd V, Nelson CP, Albrecht E, et al. Identification of seven loci affecting mean telomere length and their association with disease. Nature genetics 2013; 45:422-7, 7e1-2. 101. Mourkioti F, Kustan J, Kraft P, et al. Role of telomere dysfunction in cardiac failure in Duchenne muscular dystrophy. Nature cell biology 2013; 15:895-904. 102. Sauerwald A, Sandin S, Cristofari G, Scheres SH, Lingner J, Rhodes D. Structure of active dimeric human telomerase. Nature structural & molecular biology 2013; 20:454-60. 103. Jiang J, Miracco EJ, Hong K, et al. The architecture of Tetrahymena telomerase holoenzyme. Nature 2013; 496:187-92. 104. Pinto AR, Li H, Nicholls C, Liu JP. Telomere protein complexes and interactions with telomerase in telomere maintenance. Front Biosci 2011; 16:187-207. 105. Wang F, Podell ER, Zaug AJ, et al. The POT1-TPP1 telomere complex is a telomerase processivity factor. Nature 2007; 445:506-10. 106. Zaug AJ, Podell ER, Nandakumar J, Cech TR. Functional interaction between telomere protein TPP1 and telomerase. Genes & development 2010; 24:613-22. 107. Alder JK, Cogan JD, Brown AF, et al. Ancestral mutation in telomerase causes defects in repeat addition processivity and manifests as familial pulmonary fibrosis. PLoS genetics 2011; 7:e1001352. 108. Gramatges MM, Qi X, Sasa GS, Chen JJ, Bertuch AA. A homozygous telomerase T-motif variant resulting in markedly reduced repeat addition processivity in siblings with Hoyeraal Hreidarsson syndrome. Blood 2013; 121:3586-93.


Accepted Article

109. Bryan TM, Goodrich KJ, Cech TR. A mutant of Tetrahymena telomerase reversetranscriptase with increased processivity. J Biol Chem 2000. 110. Xie M, Podlevsky JD, Qi X, Bley CJ, Chen JJ. A novel motif in telomerase reverse transcriptase regulates telomere repeat addition rate and processivity. Nucleic acids research 2010; 38:1982-96. 111. D'Souza Y, Lauzon C, Chu TW, Autexier C. Regulation of telomere length and homeostasis by telomerase enzyme processivity. Journal of cell science 2013; 126:676-87. 112. Bojesen SE, Pooley KA, Johnatty SE, et al. Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer. Nature genetics 2013; 45:371-84, 84e1-2. 113. Zheng H, Seit-Nebi A, Han X, et al. A posttranslational modification cascade involving p38, Tip60, and PRAK mediates oncogene-induced senescence. Mol Cell 2013; 50:699-710. 114. Rokudai S, Laptenko O, Arnal SM, Taya Y, Kitabayashi I, Prives C. MOZ increases p53 acetylation and premature senescence through its complex formation with PML. Proceedings of the National Academy of Sciences of the United States of America 2013; 110:3895-900. 115. Zhu B, Ferry CH, Blazanin N, et al. PPARbeta/delta promotes HRAS-induced senescence and tumor suppression by potentiating p-ERK and repressing p-AKT signaling. Oncogene 2013. 116. Wang Z, Liu Y, Takahashi M, et al. N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence. Proc Natl Acad Sci U S A 2013; 110:312-7. 117. Xie Q, Chen J, Feng H, et al. YAP/TEAD-mediated transcription controls cellular senescence. Cancer Res 2013; 73:3615-24. 118. Tu Z, Zhuang X, Yao YG, Zhang R. BRG1 is required for formation of senescence-associated heterochromatin foci induced by oncogenic RAS or BRCA1 loss. Mol Cell Biol 2013; 33:1819-29. 119. Martin N, Raguz S, Dharmalingam G, Gil J. Co-regulation of senescence-associated genes by oncogenic homeobox proteins and polycomb repressive complexes. Cell Cycle 2013; 12:2194-9. 120. Ribeiro JD, Morey L, Mas A, et al. ZRF1 controls oncogene-induced senescence through the INK4-ARF locus. Oncogene 2013; 32:2161-8. 121. Li Q, Zhang Y, Fu J, et al. FOXA1 mediates p16(INK4a) activation during cellular senescence. The EMBO journal 2013; 32:858-73. 122. Yamaguchi H, Calado RT, Ly H, et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med 2005; 352:1413-24.


Accepted Article

123. Yamaguchi H, Baerlocher GM, Lansdorp PM, et al. Mutations of the human telomerase RNA gene (TERC) in aplastic anemia and myelodysplastic syndrome. Blood 2003; 102:916-8. 124. Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 1999; 402:551-5. 125. Vulliamy T, Marrone A, Goldman F, et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 2001; 413:432-5. 126. Heiss NS, Knight SW, Vulliamy TJ, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nature genetics 1998; 19:32-8. 127. Tsakiri KD, Cronkhite JT, Kuan PJ, et al. Adult-onset pulmonary fibrosis caused by mutations in telomerase. Proceedings of the National Academy of Sciences of the United States of America 2007; 104:7552-7. 128. Armanios MY, Chen JJ, Cogan JD, et al. Telomerase mutations in families with idiopathic pulmonary fibrosis. N Engl J Med 2007; 356:1317-26. 129. Armanios M, Chen JL, Chang YP, et al. Haploinsufficiency of telomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita. Proc Natl Acad Sci U S A 2005; 102:15960-4. 130. Cao Y, Li H, Mu FT, Ebisui O, Funder JW, Liu JP. Telomerase activation causes vascular smooth muscle cell proliferation in genetic hypertension. FASEB J 2002; 16:96-8. 131. Noureddine H, Gary-Bobo G, Alifano M, et al. Pulmonary artery smooth muscle cell senescence is a pathogenic mechanism for pulmonary hypertension in chronic lung disease. Circ Res; 109:543-53. 132. Benetos A, Gardner JP, Zureik M, et al. Short telomeres are associated with increased carotid atherosclerosis in hypertensive subjects. Hypertension 2004; 43:182-5. 133. Huda N, Tanaka H, Herbert BS, Reed T, Gilley D. Shared environmental factors associated with telomere length maintenance in elderly male twins. Aging Cell 2007; 6:709-13. 134. Yang Z, Huang X, Jiang H, et al. Short telomeres and prognosis of hypertension in a chinese population. Hypertension 2009; 53:639-45. 135. Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ, Reddel RR. Protein composition of catalytically active human telomerase from immortal cells. Science 2007; 315:1850-3. 136. Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 1999; 402:551-5.


Accepted Article

137. Venteicher AS, Abreu EB, Meng Z, et al. A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science 2009; 323:644-8. 138. Ballew BJ, Yeager M, Jacobs K, et al. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita. Human genetics 2013; 132:473-80. 139. Walne AJ, Vulliamy T, Beswick R, Kirwan M, Dokal I. TINF2 mutations result in very short telomeres: analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes. Blood 2008; 112:3594-600. 140. Touzot F, Callebaut I, Soulier J, et al. Function of Apollo (SNM1B) at telomere highlighted by a splice variant identified in a patient with Hoyeraal-Hreidarsson syndrome. Proceedings of the National Academy of Sciences of the United States of America 2010; 107:10097-102. 141. Marrone A, Walne A, Tamary H, et al. Telomerase reverse-transcriptase homozygous mutations in autosomal recessive dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. Blood 2007; 110:4198-205. 142. Hoyeraal HM, Lamvik J, Moe PJ. Congenital hypoplastic thrombocytopenia and cerebral malformations in two brothers. Acta paediatrica Scandinavica 1970; 59:185-91. 143. Brault ME, Lauzon C, Autexier C. Dyskeratosis congenita mutations in dyskerin SUMOylation consensus sites lead to impaired telomerase RNA accumulation and telomere defects. Human molecular genetics 2013; 22:3498-507. 144. Deng Z, Glousker G, Molczan A, et al. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome. Proceedings of the National Academy of Sciences of the United States of America 2013; 110:E3408-16. 145. Walne AJ, Vulliamy T, Kirwan M, Plagnol V, Dokal I. Constitutional mutations in RTEL1 cause severe dyskeratosis congenita. American journal of human genetics 2013; 92:448-53. 146. Aviv A, Aviv H. Reflections on telomeres, growth, aging, and essential hypertension. Hypertension 1997; 29:1067-72. 147. Burchell VS, Nelson DE, Sanchez-Martinez A, et al. The Parkinson's disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy. Nat Neurosci; 16:1257-65. 148. Hasson SA, Kane LA, Yamano K, et al. High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy. Nature. 149. Okatsu K, Uno M, Koyano F, et al. A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment. J Biol Chem.


Accepted Article

150. Sarraf SA, Raman M, Guarani-Pereira V, et al. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature; 496:372-6. 151. Muller-Rischart AK, Pilsl A, Beaudette P, et al. The E3 ligase parkin maintains mitochondrial integrity by increasing linear ubiquitination of NEMO. Mol Cell; 49:908-21. 152. Ekholm-Reed S, Goldberg MS, Schlossmacher MG, Reed SI. Parkin-dependent degradation of the F-box protein Fbw7beta promotes neuronal survival in response to oxidative stress by stabilizing Mcl-1. Mol Cell Biol; 33:3627-43. 153. Manzanillo PS, Ayres JS, Watson RO, et al. The ubiquitin ligase parkin mediates resistance to intracellular pathogens. Nature; 501:512-6. 154. Allen GF, Toth R, James J, Ganley IG. Loss of iron triggers PINK1/Parkin-independent mitophagy. EMBO Rep; 14:1127-35. 155. Esposito G, Vos M, Vilain S, et al. Aconitase causes iron toxicity in Drosophila pink1 mutants. PLoS Genet; 9:e1003478. 156. Hertz NT, Berthet A, Sos ML, et al. A neo-substrate that amplifies catalytic activity of parkinson's-disease-related kinase PINK1. Cell; 154:737-47. 157. Yang YH, Johnson JD. Multi-parameter single-cell kinetic analysis reveals multiple modes of cell death in primary pancreatic beta-cells. J Cell Sci; 126:4286-95. 158. Shang L, Hua H, Foo K, et al. Beta cell dysfunction due to increased ER stress in a stem cell model of Wolfram syndrome. Diabetes. 159. Wikstrom JD, Israeli T, Bachar-Wikstrom E, et al. AMPK regulates ER morphology and function in stressed pancreatic beta-cells via phosphorylation of DRP1. Mol Endocrinol; 27:1706-23. 160. Lee YS, Morinaga H, Kim JJ, et al. The fractalkine/CX3CR1 system regulates beta cell function and insulin secretion. Cell; 153:413-25. 161. Song R, Peng W, Zhang Y, et al. Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders. Nature; 494:375-9. 162. Okada-Iwabu M, Yamauchi T, Iwabu M, et al. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature; 503:493-9. 163. Pearce LR, Atanassova N, Banton MC, et al. KSR2 Mutations Are Associated with Obesity, Insulin Resistance, and Impaired Cellular Fuel Oxidation. Cell. 164. Costanzo-Garvey DL, Pfluger PT, Dougherty MK, et al. KSR2 is an essential regulator of AMP kinase, energy expenditure, and insulin sensitivity. Cell Metab 2009; 10:366-78.


Accepted Article

165. Jonker JW, Suh JM, Atkins AR, et al. A PPARgamma-FGF1 axis is required for adaptive adipose remodelling and metabolic homeostasis. Nature; 485:391-4. 166. Cipolletta D, Feuerer M, Li A, et al. PPAR-gamma is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature; 486:549-53. 167. Eller K, Kirsch A, Wolf AM, et al. Potential role of regulatory T cells in reversing obesity-linked insulin resistance and diabetic nephropathy. Diabetes; 60:2954-62. 168. Qin J, Li Y, Cai Z, et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature; 490:55-60. 169. Cabreiro F, Au C, Leung KY, et al. Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism. Cell; 153:228-39. 170. He C, Bassik MC, Moresi V, et al. Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis. Nature; 481:511-5. 171. Rando TA. Stem cells, ageing and the quest for immortality. Nature 2006; 441:1080-6. 172. Johnson SC, Rabinovitch PS, Kaeberlein M. mTOR is a key modulator of ageing and age-related disease. Nature; 493:338-45. 173. Vaupel JW. Biodemography of human ageing. Nature; 464:536-42.

Figure legends: Fig. 1: Schematic presentation of the molecular pathways underlying different forms of cellular senescence. Stress- or oncogene-induced premature senescence (OIS), replicative senescence, and developmental and programmed senescence (DPS) are irreversible cell cycle arrests each induced by distinct mechanisms with the cyclin-dependent kinases (CDKs) being inhibited by specific CDK inhibitors. Oncogenic stimulations by mutated Ras small GTPase or Raf protein kinase, DNA damage responses (DDR), or physiological actions of growth factors and cytokines induce activations of a number of intermediate signaling kinases and transcription This article is protected by copyright. All rights reserved.

Accepted Article

regulators to mediate the gene expressions of CDK inhibitors such as p16INK4a and p21CIP1.

Fig. 2: Cartoon of recent understanding of p16INK4a gene expression regulation. The FOXA1 and p16INK4a loci are inhibited by the polycomb complexes recruited by the oncogenes HLX1 and HOXA9. The p16INK4a locus is however stimulated by the epigenetic factor ZRF1 and transcription factors BRG1 and FOXA1. ZRF1 and BRG1 are up-regulated by Ras.


Accepted Article This article is protected by copyright. All rights reserved.

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