Nuclear Genomic Instability and Aging.

The nuclear genome decays as organisms age. Numerous studies demonstrate that the burden of several classes of DNA lesions is greater in older mammals than in young mammals. More challenging is proving this is a cause rather than a consequence of aging. The DNA damage theory of aging, which argues that genomic instability plays a causal role in aging, has recently gained momentum. Support for this theory stems partly from progeroid syndromes in which inherited defects in DNA repair increase the burden of DNA damage leading to accelerated aging of one or more organs. Additionally, growing evidence shows that DNA damage accrual triggers cellular senescence and metabolic changes that promote a decline in tissue function and increased susceptibility to age-related diseases. Here, we examine multiple lines of evidence correlating nuclear DNA damage with aging. We then consider how, mechanistically, nuclear genotoxic stress could promote aging. We conclude that the evidence, in toto, supports a role for DNA damage as a nidus of aging.

Retraction Note: Selective killing of cancer cells by a small molecule targeting the stress response to ROS.

This Letter is being retracted owing to issues with Fig. 1d and Supplementary Fig. 31b, and the unavailability of original data for these figures that raise concerns regarding the integrity of the figures. Nature published two previous corrections related to this Letter1,2. These issues in aggregate undermine the confidence in the integrity of this study. Authors Michael Foley, Monica Schenone, Nicola J. Tolliday, Todd R. Golub, Steven A. Carr, Alykhan F. Shamji, Andrew M. Stern and Stuart L. Schreiber agree with the Retraction. Authors Lakshmi Raj, Takao Ide, Aditi U. Gurkar, Anna Mandinova and Sam W. Lee disagree with the Retraction. Author Xiaoyu Li did not respond.

Forestalling age-impaired angiogenesis and blood flow by targeting NOX: Interplay of NOX1, IL-6, and SASP in propagating cell senescence.

In an aging population, intense interest has shifted toward prolonging health span. Mounting evidence suggests that cellular reactive species are propagators of cell damage, inflammation, and cellular senescence. Thus, such species have emerged as putative provocateurs and targets for senolysis, and a clearer understanding of their molecular origin and regulation is of paramount importance. In an inquiry into signaling triggered by aging and proxy instigator, hyperglycemia, we show that NADPH Oxidase (NOX) drives cell DNA damage and alters nuclear envelope integrity, inflammation, tissue dysfunction, and cellular senescence in mice and humans with similar causality. Most notably, selective NOX1 inhibition rescues age-impaired blood flow and angiogenesis, vasodilation, and the endothelial cell wound response. Indeed, NOX1i delivery in vivo completely reversed age-impaired hind-limb blood flow and angiogenesis while disrupting a NOX1-IL-6 senescence-associated secretory phenotype (SASP) proinflammatory signaling loop. Relevant to its comorbidity with age, clinical samples from diabetic versus nondiabetic subjects reveal as operant this NOX1-mediated vascular senescence and inflammation in humans. On a mechanistic level, our findings support a previously unidentified role for IL-6 in this feedforward inflammatory loop and peroxisome proliferator-activated receptor gamma (PPARγ) down-regulation as inversely modulating p65-mediated NOX1 transcription. Targeting this previously unidentified NOX1-SASP signaling axis in aging is predicted to be an effective strategy for mitigating senescence in the vasculature and other organ systems.

Cellular Senescence: The Trojan Horse in Chronic Lung Diseases.

Senescence is a cell fate decision characterized by irreversible arrest of proliferation accompanied by a senescence-associated secretory phenotype. Traditionally, cellular senescence has been recognized as a beneficial physiological mechanism during development and wound healing and in tumor suppression. However, in recent years, evidence of negative consequences of cellular senescence has emerged, illuminating its role in several chronic pathologies. In this context, senescent cells persist or accumulate and have detrimental consequences. In this review, we discuss the possibility that in chronic obstructive pulmonary disease, persistent senescence impairs wound healing in the lung caused by secretion of proinflammatory senescence-associated secretory phenotype factors and exhaustion of progenitor cells. In contrast, in idiopathic pulmonary fibrosis, chronic senescence in alveolar epithelial cells exacerbates the accumulation of senescent fibroblasts together with production of extracellular matrix. We review how cellular senescence may contribute to lung disease pathology.

Selective killing of cancer cells by a small molecule targeting the stress response to ROS.

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.

A molecular index for biological age identified from the metabolome and senescence-associated secretome in humans.

Unlike chronological age, biological age is a strong indicator of health of an individual. However, the molecular fingerprint associated with biological age is ill-defined. To define a high-resolution signature of biological age, we analyzed metabolome, circulating senescence-associated secretome (SASP)/inflammation markers and the interaction between them, from a cohort of healthy and rapid agers. The balance between two fatty acid oxidation mechanisms, ß-oxidation and ω-oxidation, associated with the extent of functional aging. Furthermore, a panel of 25 metabolites, Healthy Aging Metabolic (HAM) index, predicted healthy agers regardless of gender and race. HAM index was also validated in an independent cohort. Causal inference with machine learning implied three metabolites, ß-cryptoxanthin, prolylhydroxyproline, and eicosenoylcarnitine as putative drivers of biological aging. Multiple SASP markers were also elevated in rapid agers. Together, our findings reveal that a network of metabolic pathways underlie biological aging, and the HAM index could serve as a predictor of phenotypic aging in humans.

DNA damage-induced stalling of transcription drives aging through gene expression imbalance.

Age-related changes in gene expression have long been examined to understand the biology of aging. The hallmarks of aging are biological processes known to be associated with aging, but whether there is a unifying driver of these attributes, is not well understood. With the advent of technology over the last few years, it is quite clear that aging leads to global decline in transcription. In this Perspective, we highlight a new study in Nature Genetics that aimed to determine why global transcription rate reduces with age and how this phenomenon is the driver that interconnects multiple hallmarks of aging. This study recognizes that age-related accumulation of DNA damage, particularly transcription-blocking lesions, stalls RNA polymerase. This phenomenon affects longer genes leading to a gradual loss of transcription and skewing the transcriptome. In order to design a successful aging intervention, future work will be needed to test how some promising therapies in pre-clinical trials target affect transcriptional rate.

A Chemoptogenetic Tool for Spatiotemporal Induction of Oxidative DNA Lesions In Vivo.

Oxidative nuclear DNA damage increases in all tissues with age in multiple animal models, as well as in humans. However, the increase in DNA oxidation varies from tissue to tissue, suggesting that certain cells/tissues may be more vulnerable to DNA damage than others. The lack of a tool that can control dosage and spatiotemporal induction of oxidative DNA damage, which accumulates with age, has severely limited our ability to understand how DNA damage drives aging and age-related diseases. To overcome this, here we developed a chemoptogenetic tool that produces 8-oxoguanine (8-oxoG) at DNA in a whole organism, Caenorhabditis elegans. This tool uses di-iodinated malachite green (MG-2I) photosensitizer dye that generates singlet oxygen, 1O2, upon fluorogen activating peptide (FAP) binding and excitation with far-red light. Using our chemoptogenetic tool, we are able to control generation of singlet oxygen ubiquitously or in a tissue-specific manner, including in neurons and muscle cells. To induce oxidative DNA damage, we targeted our chemoptogenetic tool to histone, his-72, that is expressed in all cell types. Our results show that a single exposure to dye and light is able to induce DNA damage, promote embryonic lethality, lead to developmental delay, and significantly reduce lifespan. Our chemoptogenetic tool will now allow us to assess the cell autonomous versus non-cell autonomous role of DNA damage in aging, at an organismal level.

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy.

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1-/D mice). Ckmm-Cre+/- ;Ercc1-/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre+/- ;Ercc1-/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre+/- ;Ercc1-/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre+/- ;Ercc1-/fl and Ercc1-/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.

Spatial mapping of cellular senescence: emerging challenges and opportunities.

Cellular senescence is a well-established driver of aging and age-related diseases. There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity. Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights. The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space. The Cellular Senescence Network (SenNet), a National Institutes of Health (NIH) Common Fund initiative, aims to map senescent cells across the lifespan of humans and mice. Here, we provide a comprehensive review of the existing and emerging methodologies for spatial imaging and their application toward mapping senescent cells. Moreover, we discuss the limitations and challenges inherent to each technology. We argue that the development of spatially resolved methods is essential toward the goal of attaining an atlas of senescent cells.

Lipids as Regulators of Cellular Senescence.

Lipids are key macromolecules that perform a multitude of biological functions ranging from maintaining structural integrity of membranes, energy storage, to signaling molecules. Unsurprisingly, variations in lipid composition and its levels can influence the functional and physiological state of the cell and its milieu. Cellular senescence is a permanent state of cell cycle arrest and is a hallmark of the aging process, as well as several age-related pathologies. Senescent cells are often characterized by alterations in morphology, metabolism, chromatin remodeling and exhibit a complex pro-inflammatory secretome (SASP). Recent studies have shown that the regulation of specific lipid species play a critical role in senescence. Indeed, some lipid species even contribute to the low-grade inflammation associated with SASP. Many protein regulators of senescence have been well characterized and are associated with lipid metabolism. However, the link between critical regulators of cellular senescence and senescence-associated lipid changes is yet to be elucidated. Here we systematically review the current knowledge on lipid metabolism and dynamics of cellular lipid content during senescence. We focus on the roles of major players of senescence in regulating lipid metabolism. Finally, we explore the future prospects of lipid research in senescence and its potential to be targeted as senotherapeutics.

Integrated -omics approach reveals persistent DNA damage rewires lipid metabolism and histone hyperacetylation via MYS-1/Tip60.

Although DNA damage is intricately linked to metabolism, the metabolic alterations that occur in response to DNA damage are not well understood. We use a DNA repair-deficient model of ERCC1-XPF in Caenorhabditis elegans to gain insights on how genotoxic stress drives aging. Using multi-omic approach, we discover that nuclear DNA damage promotes mitochondrial ß-oxidation and drives a global loss of fat depots. This metabolic shift to ß-oxidation generates acetyl-coenzyme A to promote histone hyperacetylation and an associated change in expression of immune-effector and cytochrome genes. We identify the histone acetyltransferase MYS-1, as a critical regulator of this metabolic-epigenetic axis. We show that in response to DNA damage, polyunsaturated fatty acids, especially arachidonic acid (AA) and AA-related lipid mediators, are elevated and this is dependent on mys-1. Together, these findings reveal that DNA damage alters the metabolic-epigenetic axis to drive an immune-like response that can promote age-associated decline.

Rad18-mediated translesion synthesis of bulky DNA adducts is coupled to activation of the Fanconi anemia DNA repair pathway.

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

Analysis of representative mutants for key DNA repair pathways on healthspan in Caenorhabditis elegans.

Although the link between DNA damage and aging is well accepted, the role of different DNA repair proteins on functional/physiological aging is not well-defined. Here, using Caenorhabditis elegans, we systematically examined the effect of three DNA repair genes involved in key genome stability pathways. We assayed multiple health proxies including molecular, functional and resilience measures to define healthspan. Loss of XPF-1/ERCC-1, a protein involved in nucleotide excision repair (NER), homologous recombination (HR) and interstrand crosslink (ICL) repair, showed the highest impairment of functional and stress resilience measures along with a shortened lifespan. brc-1 mutants, with a well-defined role in HR and ICL are short-lived and highly sensitive to acute stressors, specifically oxidative stress. In contrast, ICL mutant, fcd-2 did not impact lifespan or most healthspan measures. Our efforts also uncover that DNA repair mutants show high sensitivity to oxidative stress with age, suggesting that this measure could act as a primary proxy for healthspan. Together, these data suggest that impairment of multiple DNA repair genes can drive functional/physiological aging. Further studies to examine specific DNA repair genes in a tissue specific manner will help dissect the importance and mechanistic role of these repair systems in biological aging.

Frataxin deficiency promotes endothelial senescence in pulmonary hypertension.

The dynamic regulation of endothelial pathophenotypes in pulmonary hypertension (PH) remains undefined. Cellular senescence is linked to PH with intracardiac shunts; however, its regulation across PH subtypes is unknown. Since endothelial deficiency of iron-sulfur (Fe-S) clusters is pathogenic in PH, we hypothesized that a Fe-S biogenesis protein, frataxin (FXN), controls endothelial senescence. An endothelial subpopulation in rodent and patient lungs across PH subtypes exhibited reduced FXN and elevated senescence. In vitro, hypoxic and inflammatory FXN deficiency abrogated activity of endothelial Fe-S-containing polymerases, promoting replication stress, DNA damage response, and senescence. This was also observed in stem cell-derived endothelial cells from Friedreich's ataxia (FRDA), a genetic disease of FXN deficiency, ataxia, and cardiomyopathy, often with PH. In vivo, FXN deficiency-dependent senescence drove vessel inflammation, remodeling, and PH, whereas pharmacologic removal of senescent cells in Fxn-deficient rodents ameliorated PH. These data offer a model of endothelial biology in PH, where FXN deficiency generates a senescent endothelial subpopulation, promoting vascular inflammatory and proliferative signals in other cells to drive disease. These findings also establish an endothelial etiology for PH in FRDA and left heart disease and support therapeutic development of senolytic drugs, reversing effects of Fe-S deficiency across PH subtypes.

An alternatively spliced, non-signaling insulin receptor modulates insulin sensitivity via insulin peptide sequestration in C. elegans.

In the nematode C. elegans, insulin signaling regulates development and aging in response to the secretion of numerous insulin peptides. Here, we describe a novel, non-signaling isoform of the nematode insulin receptor (IR), DAF-2B, that modulates insulin signaling by sequestration of insulin peptides. DAF-2B arises via alternative splicing and retains the extracellular ligand binding domain but lacks the intracellular signaling domain. A daf-2b splicing reporter revealed active regulation of this transcript through development, particularly in the dauer larva, a diapause stage associated with longevity. CRISPR knock-in of mScarlet into the daf-2b genomic locus confirmed that DAF-2B is expressed in vivo and is likely secreted. Genetic studies indicate that DAF-2B influences dauer entry, dauer recovery and adult lifespan by altering insulin sensitivity according to the prevailing insulin milieu. Thus, in C. elegans alternative splicing at the daf-2 locus generates a truncated IR that fine-tunes insulin signaling in response to the environment.

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence-associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1-XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15-fold in peripheral lymphocytes from 4- to 5-month-old Ercc1-/∆ and 2.5-year-old wild-type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4- to 5-month-old Ercc1-/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence-associated ß-galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1-/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1-/∆ and aged WT mice support the conclusion that the DNA repair-deficient mice accurately model the age-related accumulation of senescent cells, albeit six-times faster.

ATM is a key driver of NF-κB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging.

NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.

Methods to Quantify the NF-κB Pathway During Senescence.

Nuclear factor κB (NF-κB) is a family of transcription factors important for regulating innate and adaptive immunity, cellular proliferation, apoptosis and senescence. The NF-κB family is comprised of five subunits, RelA/p65, RelB, C-Rel, p50 (p105/NF-κB1), and p52 (p100/NF-κB2). NF-κB activity goes up with age in multiple tissues. The two subunits RelA/p65 and p50 have been implicated in senescence and aging with genetic deletion of p65 and p50 reducing or increasing senescence respectively. Pharmacologic inhibition of NF-κB also extends health span and reduces senescence in mouse models of accelerated aging. In addition, NF-κB regulates expression of many of senescence associated secretory phenotype (SASP) factors released by certain types of senescent cells that drives loss of tissue homeostasis and secondary senescence. To measure NF-κB activity with aging in vivo, multiple methods can and need to be utilized including cellular localization of p65, EMSA analysis of NF-κB DNA binding, RNA in situ hybridization, and analysis of expression of NF-κB target genes. To colocalize NF-κB activation and senescence, p65 localization or transcriptional activity can be measured by immunostaining or RNA in situ hybridization for NF-κB regulated genes along with methods such as immunostaining for γH2AX or RNA in situ for senescence markers like p16INK4a and p21. These and related methods will be described in this chapter.