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Aging is the outcome of a balance between damage and repair. The rate of aging and the appearance of age-related pathology are modulated by stress response and repair pathways that gradually decline, including the proteostasis and DNA damage repair networks and mitochondrial respiratory metabolism. Highly conserved insulin/IGF-1, TOR, and sirtuin signaling pathways in turn, control these critical cellular responses. The coordinated action of these signaling pathways maintains cellular and organismal homeostasis in the face of external perturbations, such as changes in nutrient availability, temperature and oxygen level, as well as internal perturbations, such as protein misfolding and DNA damage. Studies in model organisms suggest that changes in signaling can augment these critical stress response systems, increasing lifespan and reducing age-related pathology. The systems biology of stress response signaling thus provides a new approach to the understanding and potential treatment of age-related diseases.
A phylogenetically conserved feature of aging is the induction of stress response pathways. Microarray studies of gene expression show that age-dependent induction of stress response genes occurs in all systems studied, including whole organism analysis of Caenorhabditis elegans (C. elegans) and Drosophila, and analysis of the brain in mouse, rat, chimpanzee and human (Bishop et al., 2010; Yankner et al., 2008). Cellular stress response pathways are controlled at the molecular level by a number of highly conserved signaling molecules and transcriptional regulators, including proteins involved in insulin/insulin-like growth factor (IGF) signaling, sirtuins, target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) pathways (Kenyon, 2010). The molecules involved possess features enabling them to both sense changes in inputs, such as energy status, DNA damage, protein damage, and hypoxia, and transmit information to molecules that allow for adaptive cellular responses. These adaptive responses often involve coordinated regulation of protein synthesis and turnover, autophagy, and mitochondrial function. This highly regulated circuitry thus maintains protein and DNA integrity in the face of stress and declining function during aging.
This review explores the connection between stress response pathways and aging. We begin by discussing the evidence that aging is a regulated process that is controlled by a few highly conserved signaling mechanisms, including the insulin/IGF-1, TOR, and AMPK pathways, as well as sirtuins. Evidence is discussed for coordinated regulation by these signaling pathways of stress responses that play a role in aging, including nutrient sensing (Sengupta, 2010), mitochondrial function, redox metabolism (Majmundar et al., 2010; Wellen et al., 2010), the DNA damage response (Ciccia, 2010), proteostasis (Buchberger, 2010; Richter, 2010) and autophagy (Kroemer, 2010). The potential for harnessing the phenomenon of hormesis, in which low level stressors enhance organismal resistance and reduce physiological decline, is described as an emerging concept. Recent findings suggest that stress response signaling can be manipulated to extend lifespan and retard the onset of age-related pathology, suggesting a new approach to the degenerative disorders of aging humans.
Lifespan is regulated by highly conserved nutrient sensing pathways providing evidence for a pivotal role of nutrient signaling in the control of aging and aging-related diseases. High caloric intake shortens lifespan and accelerates the onset of aging-associated disorders, including diabetes, metabolic syndrome, cancer and neurodegenerative disorders. By contrast, a dietary regimen of moderate calorie restriction with adequate nutrient intake delays aging in a wide variety of organisms from yeast to primates, and may delay or attenuate age-related diseases such as diabetes, cancer and Alzheimer’s disease (AD) (Comfort, 1963; Haigis and Sinclair, 2010; McCay et al., 1989; Weindruch and Walford, 1988). Caloric restriction also activates stress pathways that increase organismal resistance to subsequent stress or nutritional limitation, an effect known as hormesis. Energy sensing pathways are linked to the aging process and are regulated by insulin/IGF-1, sirtuins, TOR, and AMPK signaling (Kenyon, 2010). Moreover, recent studies demonstrate that these pathways coordinately regulate each other, as well as a variety of stress response pathways that impact organismal survival and lifespan. Understanding how organisms sense nutrient intake and stress and coordinate these signals is likely to increase our understanding of mechanisms that underlie aging and age-related diseases.
Mutations that reduce insulin/IGF-1 signaling extend lifespan in a variety of model organisms. In C. elegans, loss of function mutations in daf-2, a homolog of mammalian insulin/ IGF receptors, extends lifespan by more than 2-fold (Kenyon et al., 1993; Kimura et al., 1997). The regulation of aging by insulin-like factors involves downstream signaling through phosphatidylinositol 3-OH kinase (PI(3)K), AKT and FOXOs; in worms, the AGE-1 mutation in an insulin/IGF-regulated PI(3)K ortholog increases lifespan (Morris et al., 1996). Furthermore, lifespan extension requires signaling through the transcription factors DAF-16, HSF-1 (Heat Shock Factor-1), and SKN-1 that induce the expression of a broad network of genes involved in anti-oxidant defense, mitochondrial function, proteostasis and autophagy (Kenyon, 2010). Thus, signaling through insulin/IGF-1 modulates cellular and organismal stress responses by controlling key transcriptional programs.
Because insulin/ signaling functions as a nutrient sensor and controls transcription of stress response genes, this pathway provides a molecular connection between dietary intake and cellular stress response pathways. Indeed, experimental data support that idea that insulin/IGF-1 pathways mediate at least part of the beneficial effect of calorie restriction. For example, caloric restriction decreases the levels of insulin/IGF-1 in mammals and may be perceived by an organism as a mild type of stress, providing hormetic benefits (Comfort, 1963; Haigis and Sinclair, 2010; McCay et al., 1989; Weindruch and Walford, 1988). Interestingly, DAF-16 and SKN1, which are regulated by insulin/IGF-1 signaling, are required for lifespan extension in some models of calorie restriction in worms (Bishop and Guarente, 2007; Greer and Brunet, 2009). In flies, the lifespan extension by dietary conditions seems to be mediated, in part, by FOXO activity (Giannakou et al., 2008). The insulin/IGF-1 signaling pathway also appears to play a central role in the beneficial effects of caloric restriction in mammals. Growth hormone receptor knockout mice are long-lived, and their lifespan cannot be further extended by dietary restriction (Bonkowski et al., 2009), suggesting that reduced IGF-1 signaling and caloric restriction extend lifespan through similar mechanisms.
Recent genetic studies have found inherited SNPs in genes of the insulin/IGF-1 signaling pathway that correlate with longevity. Polymorphisms in the IGF-1 receptor gene have been identified in Ashkenazi Jewish centenarians (Suh et al., 2008). SNPS have also been identified in the insulin signaling genes AKT1, FOXO1 and FOXO3a in multiple centenarian cohorts (Wilcox et al., 2008;Flachsbart et al., 2009; Pawlikowska et al., 2009). Larger population based studies will be required to determine whether these genetic associations represent true human longevity traits.
Sirtuins are a highly conserved family of proteins that connect metabolic status to the regulation of aging and age-related phenotypes (Haigis and Sinclair, 2010). Sirtuins possess NAD-dependent protein deacetylase and/or ADP-ribosyltransferase activity. The requirement for NAD is one mechanism by which sirtuins sense and respond to metabolic status (Guarente, 2006; Schwer and Verdin, 2008). A number of studies in model organisms, including yeast, worms and flies, suggest that the sirtuin Sir2 can extend lifespan (Kaeberlein et al., 1999; Lin et al., 2000) (Rogina and Helfand, 2004; Tissenbaum and Guarente, 2001). There are seven mammalian sirtuins (SIRT1-7) that play various roles in the regulation of stress resistance, metabolism and cell survival. However, their roles in the regulation of mammalian lifespan are still unresolved. Nonetheless, many reports suggest that sirtuins regulate stress-response pathways that contribute to aging and age-related diseases.
The best studied mammalian sirtuin is SIRT1, the mammalian ortholog of yeast Sir2 (Bordone and Guarente, 2005; Frye, 2000). SIRT1 activity is regulated by cellular nutrient status and triggers stress response pathways and changes in energy metabolism. Moreover, SIRT1 expression and activity decrease with age in a number of tissues, and can also be reduced by a high fat diet (Ramsey et al., 2008; Sasaki et al., 2006). Conversely, SIRT1 activity is increased during times of nutrient deprivation, such as fasting and calorie restriction. When activated, SIRT1 deacetylates many different substrate proteins that are involved in aging, stress responses and metabolic regulation, including PGC-1α, Ku70, NF-κB, AceCS1, MEF2 and p53 (Haigis and Sinclair, 2010) Moreover, SIRT1 deacetylates and activates FOXO transcription factors, providing a level of transcriptional control of stress response genes.
SIRT1-deficient mice develop insulin resistance and metabolic deficits that may relate, in part, to impaired energy metabolism (Haigis and Sinclair, 2010). Mitochondria isolated from SIRT1-deficient mice show reduced respiratory function and increased generation of reactive oxygen species (ROS) (Boily et al., 2008). By contrast, mice overexpressing SIRT1 show improved metabolic parameters that resemble the metabolic changes associated with caloric restriction (Banks et al., 2008; Bordone et al., 2007). Moreover, activation of SIRT1 by resveratrol may contribute to lifespan extension in mice fed a high fat diet (Baur et al., 2006; Lagouge et al., 2006). However, overexpression of SIRT1 in transgenic mice fed a normal diet does not extend lifespan (Herranz et al., 2010), raising the possibility that SIRT1 function is most relevant to lifespan regulation under stress-related conditions in mammals. In addition to SIRT1, studies of the other sirtuins implicate this family of proteins in the regulation of multiples aspects of stress resistance and metabolism, including DNA repair, mitochondrial function, protein quality control, and cell survival (Haigis and Sinclair, 2010). Taken together, these studies suggest that sirtuins activate protective stress responses in a variety of model systems, but the role of sirtuins in the regulation of mammalian lifespan remains to be determined.
AMP-activated protein kinase (AMPK) is activated under conditions of elevated intracellular AMP or reduced ATP, enabling this kinase to serve as a rheostat for cellular energy status. Stressors such as glucose deprivation, ischemia, hypoxia and exercise that deplete cellular ATP lead to the activation of AMPK (Kahn et al., 2005). AMPK activation results in transcriptional and post-translational signaling responses that increase catabolic metabolic pathways in response to the stress of a low energy state (Nilsson et al., 2006; Osler and Zierath, 2008). For example, AMPK activity promotes fatty acid oxidation through phosphorylation and inhibition of ACC, an enzyme that synthesizes malonyl CoA from acetyl CoA. The regulation of ACC activity is a pivotal node in the switch between anabolic and catabolic processes. The malonyl CoA generated by ACC provides a precursor for fatty acid synthesis, while also inhibiting mitochondrial fatty acid oxidation via allosteric inhibition of carnitine palmitoyltransferase-1 (CPT1), the rate-limiting enzyme in mitochondrial fatty acid uptake. (Abu-Elheiga et al., 2001; Saggerson, 2008). Thus, during periods of nutrient stress resulting in low ATP, the switch from fatty acid synthesis to oxidation is mediated in a large part by the increased activity of AMPK.
Declining AMPK activity during aging may contribute to insulin resistance and metabolic syndrome. AMPK activity declines in aging skeletal muscle, and is associated with insulin resistance that can be reversed by treatment with AICAR, an AMP analog that activates AMPK (Qiang et al., 2007). AMPK may regulate insulin sensitivity by stimulating GLUT4 translocation, increasing glucose uptake and metabolism. These observations suggest that AMPK is a potential therapeutic target for age-related metabolic disorders (McCarty, 2004).
The target of rapamycin (TOR) pathway is a conserved nutrient sensor that is linked to lifespan regulation. TOR integrates environmental cues, such as growth factors and nutrients to control eukaryotic growth, metabolism and cell division (Cunningham et al., 2007; Wullschleger et al., 2006). TOR activity is suppressed by conditions of nutrient limitation, consistent with the notion that decreasing TOR signaling mimics aspects of caloric restriction. Moreover, mTOR signaling is reduced in the Ames Dwarf mouse, a model of extended longevity (Sharp and Bartke, 2005). Indeed, TOR inhibition extends lifespan in a variety of model organisms, and can even extend lifespan when inhibited during a limited period of adult life in mice (Harrison et al., 2009).
The mechanisms by which TOR exerts its effects on aging may involve the modulation of protein synthesis and autophagy (Medvedik et al., 2007; Steffen et al., 2008) (Chen et al., 2007; Hansen et al., 2007; Kapahi et al., 2004; Pan et al., 2007; Steffen et al., 2008). In mammals, mTOR functions in two distinct signaling complexes - mTORC1 and mTORC2. The mTORC1 complex is composed of mTOR, RAPTOR, PRAS40 and mLST8 and regulates cell growth, protein synthesis, ribosome biogenesis and autophagy through the activation of key downstream targets that include ribosomal S6 kinase and 4E-BP1 (Guertin and Sabatini, 2007). The mTORC2 complex is composed of mTOR, RICTOR, mSIN1, PROTOR and mLST8. Downregulation of mTORC1 activity by rapamycin reduces glycolysis and facilitates a switch in fat metabolism by increasing fatty acid oxidation in skeletal muscle cells (Brown et al., 2007; Sipula et al., 2006). S6K1−/− mice have reduced fat stores and are protected from weight gain on a high fat diet (Um et al., 2004), while mice lacking mTORC1 inhibitory targets 4E-BP1 and 4E-BP2 display diminished lipolysis, increased fatty acid synthesis and enhanced sensitivity to diet-induced obesity (Le Bacquer et al., 2007). TOR signaling therefore stands at the crossroads of metabolism, stress responses and aging, with potentially important implications for the pathogenesis and treatment of age-related metabolic disorders.
Research spanning decades has investigated the idea that reactive oxygen species (ROS) generation leads to the macromolecular damage that underlies the aging process, known as the “Free Radical Theory of Aging” (Harmon, 1956). While many studies find a correlation between oxidative damage and lifespan, there are enough exceptions and contradictions to rule out simple causality (Lapointe and Hekimi, 2010). For example, in worms, a mutation of a subunit in complex II of the electron transport chain, succinate dehydrogenase cytochrome b, results in reduced respiration and decreased lifespan, especially under high oxygen conditions (Ishii et al., 1998), demonstrating a correlation between ROS and lifespan. In flies, decreasing superoxide dismutase (SOD) 2 levels in skeletal muscle decreases locomotion and lifespan (Martin et al., 2009), while increasing SOD2 promotes lifespan extension (Sun and Tower, 1999). By contrast, decreasing SOD isoforms in worms increases both ROS and lifespan (Van Raamsdonk and Hekimi, 2009). Manipulating MnSOD in mice also leads to mixed results; mice hetereozygous for MnSOD deletion demonstrate elevated ROS, but display normal lifespan, and MnSOD overexpression decreases lipid peroxidation and increases resistance against paraquot-induced oxidative stress, but does not extend lifespan (Jang et al., 2009). However, some mammalian studies support a link between ROS generation and lifespan. This was dramatically demonstrated by overexpression of catalase targeted specifically to mitochondria, which reduced oxidative stress and extended lifespan (Schriner et al., 2005). Catalase expression in the nucleus or peroxisomal compartments did not affect lifespan, emphasizing the central role of mitochondria in oxidative metabolism and lifespan regulation (Schriner et al., 2005).
Mice with a homozygous mutation in the exonuclease domain of mitochondria DNA (mtDNA) polymerase gamma (POLG) have been used as a model of mitochondrial dysfunction and aging. These mice possess a mtDNA mutator phenotype, accumulating large numbers of deletions and point mutations in mtDNA (Kujoth et al., 2005; Trifunovic et al., 2004). Surprisingly, these mice do not display signs of elevated ROS generation, but instead exhibit increased apoptosis, a number of age-related phenotypes, and a shortened lifespan (Kujoth et al., 2005; Trifunovic et al., 2004). Subsequent studies revealed that mtDNA deletions that accumulate in the brain and heart and provide the driving force behind the progeroid-like phenotype of the mutator mice (Vermulst et al., 2007; Vermulst et al., 2008). Interestingly, catalase overexpression can attenuate the age-dependent cardiomyopathy observed in POLG mutant mice (Dai et al., 2010), suggesting that oxidative stress may in fact contribute to some of the age-related phenotypes in this mouse model. Taken together, these studies demonstrate the critical but complex connection between mitochondrial function and lifespan.
Studies in model organisms show that mtDNA mutations can both reduce or extend lifespan, depending on severity, context and developmental stage. Surprisingly, complete absence of mtDNA in yeast, the so-called petite mutation, is associated with increased lifespan (Powell et al., 2000). Similarly in worms, RNA interference (RNAi) studies showed that decreasing the expression of mitochondrial genes increased lifespan (Dillin et al., 2002; Lee et al., 2003). Furthermore, timing studies showed that respiration must be decreased during development for the life-extending benefits (Dillin et al., 2002). Rea and colleagues demonstrated the importance of the level of mitochondrial gene expression in this effect (Rea et al., 2007). Moderate inhibition extended lifespan significantly, whereas high levels of RNAi inhibition reduced lifespan. The exact mechanisms that mediate lifespan extension in these models are still not known, but may involve a mitochondrial-driven stress response that is similar to hormesis. This idea is supported by glucose restriction in worms, an intervention that increases lifespan but is associated with increased mitochondrial respiration (Schulz et al., 2007). Remarkably, in this model, increased oxidative stress is required for lifespan benefits, as antioxidant treatment of glucose-restricted worms blocks lifespan extension (Schulz et al., 2007). Along these lines, feeding worms low doses of the ROS-generating compound juglone induces small heat shock protein HSP-16.2 and increases lifespan in a DAF-16-dependent manner. Administration of higher juglone concentrations results in decreased lifespan (Hartwig et al., 2009). Furthermore, in worms, SOD2 deletion and ubiquinone-defective clk-1mutants both show increased lifespan despite elevated ROS generation and oxidative stress (Lapointe and Hekimi, 2008; Van Raamsdonk and Hekimi, 2009). Taken together, these studies demonstrate that mild forms of mitochondrial dysfunction may activate stress response pathways that promote a protective environment, conducive to long life. A greater understanding of the mechanisms involved in mitochondrial stress response pathways might provide new therapeutic opportunities for aging and age-related pathology.
Mitochondrial function is controlled by a number of signaling pathways and transcriptional regulators that sense energetic stress and contribute to lifespan regulation, including peroxisome proliferation-activated receptor coactivator 1 α (PGC-1α), sirtuins, mTOR, and AMPK. When energy is low, these pathways allow the cell to adjust fuel utilization and mitochondrial number. Many aspects of mitochondrial dysfunction associated with aging can be blocked by caloric restriction. (Hunt et al., 2006; Sohal and Weindruch, 1996). For example, caloric restriction partially prevents the age-related decline in mitochondrial gene expression in mouse heart, brain and skeletal muscle (Lee et al., 2002; Lee et al., 1999; Lee et al., 2000). These metabolic effects are mediated by a complex interplay between signaling pathways that converge on the transcriptional co-activator PGC-1 α. PGC-1 α is a central regulator of mitochondrial biogenesis and function that is induced by a variety of metabolic stressors, including low energy availability and oxidative stress (Kelly and Scarpulla, 2004). Studies in mammalian cells and tissues have shown that caloric restriction induces mitochondrial biogenesis through the up-regulation of PGC-α (Lopez-Lluch et al., 2006) and endothelial nitric oxide synthase (eNOS) (Nisoli et al., 2005). Moreover, the severe muscle wasting phenotype of mice lacking a subunit of electron transport complex IV in skeletal muscle can be partially rescued by PGC-1 α overexpression, suggesting that boosting the total number of mitochondria may compensate for a mutation in the electron transport chain (Wenz et al., 2008). Likewise, age-associated sarcopenia and metabolic dysfunction can be rescued by PGC-1α overexpression (Wenz et al., 2009). SIRT1 also activates PGC-1α by deacetylation and promotes mitochondrial biogenesis (Mattagajasingh et al., 2007; Rodgers et al., 2005). Treatment with resveratrol results in increases the lifespan of mice fed a high fat diet (Baur et al., 2006; Feige et al., 2008), and this may be mediated, in part, via activation of PGC-1α by SIRT1. PGC-1α can also be activated by phosphorylation by AMPK, increasing the expression of target genes involved in mitochondrial biogenesis and fatty acid oxidation (Jager et al., 2007; Long et al., 2005). Hence, PGC-1α is a central node of regulation for several signaling pathways that regulate both mitochondrial function and lifespan.
Several studies have linked the TOR signaling pathway to altered mitochondrial function, nutrient sensing and lifespan regulation. In yeast, TOR inhibition regulates mitochondrial respiration and increases chronological lifespan (Bonawitz et al., 2007). Mice deficient for the TOR target S6K1 show increased expression of genes that mediate mitochondrial respiratory function and fatty acid oxidation in white adipose tissue and skeletal muscle (Um et al., 2004). Likewise, inhibition of mTOR signaling with rapamycin treatment decreases transcription of genes that augment mitochondrial function, such as PGC-1α, PGC-1β, NRF-1 and ERRα, resulting in reduced oxygen consumption and mitochondrial number. Under normal conditions, mTORC1 complexes with PGC-1α and the transcription factor YY1, leading to the transcription of nuclear-encoded mitochondrial genes. Upon treatment with rapamycin, the complex is dissociated downregulating the transcription of mitochondrial genes (Cunningham et al., 2007). These studies suggest that mTOR may link the control of lifespan to the regulation of cell growth and mitochondrial metabolism.
A distinct category of genetic disorders involving impaired sensing or repair of DNA damage has provided evidence for a central role of genome maintenance in the aging process. These disorders are known as segmental progerias because they are associated with a subset of the phenotypic changes that occur during normal aging, often including neurodegeneration and cancer. A prototypical example is Werner syndrome, which typically starts at puberty with accelerated onset of many different features of normal aging including cataracts, skin atrophy, hair loss, osteoporosis, atherosclerosis, type II diabetes and a variety of different neoplasms (Martin, 2005). Werner syndrome is caused by autosomal recessive mutations in a RecQ helicase that is involved in transcription, DNA replication and DNA repair (Rossi et al., 2010). In culture, cells from Werner syndrome patients exhibit genomic instability and accelerated senescence, features shared by other progeroid disorders caused by loss of function mutations in DNA repair genes, including Xeroderma pigmentosa, Cockayne syndrome, and trichothiodystrophy. Conversely, increased longevity may be associated with more efficient DNA repair. The lifespan regulating protein SIRT1 can promote DNA repair by deacetylation of repair proteins (Fan and Luo, 2010) and the double strand break sensor NBS1 (Yuan et al., 2007), and possibly by epigenetic modification of chromatin (Oberdoerffer et al., 2008).
The study of human progeroid syndromes has provided evidence for a link between genotoxic stress responses and signaling pathways that regulate the aging process. This was illustrated by a recent mouse model of a human progeroid syndrome caused by mutation of the XPF-ERCC1 endonuclease, which is involved in nucleotide excision repair. The transcriptional profile of the liver in XPF-ERCC1-deficient mice showed reduced expression of genes in the insulin/IGF-1 signaling pathway, and a shift to anabolic metabolism and increased anti-oxidant defense (Niedernhofer et al., 2006). Downregulation of insulin/IGF-1 signaling is also a feature of other mouse models with genomic instability, such as the SIRT6-knockout and a transgenic mouse with overexpression of a truncated p53 isoform (Maier et al., 2004; Mostoslavsky et al., 2006). An expression profile suggesting reduced insulin/IGF-1 signaling was also observed in cultured fibroblasts from Werner syndrome patients (Kyng and Bohr, 2005). The downregulation of this central signaling pathway may be a mechanism for shifting cellular resources from growth to repair and protection in the setting of genomic instability. Interestingly, caloric restriction or genetic mutations that extend lifespan have a similar effect on insulin/IGF-1 signaling in a variety of model organisms. Hence, genotoxic stress and aging may stimulate similar stress response pathways.
The broad spectrum of genotoxic stress responses and their potential relationship to aging is exemplified by the disorder ataxia telangiectasia (AT), which is caused by autosomal recessive mutations in the PI3 kinase-related kinase ataxia telangiectasia mutated (ATM). AT is characterized by immunodeficiency, skin lesions, pigmentary changes, neurodegeneration and a variety of neoplasms (Martin, 2005). The clinical manifestations of AT suggest that an impaired DNA damage response has different consequences in mitotic versus post-mitotic cells. Loss of efficient ATM-mediated signaling in mitotic cells leads to genomic instability, giving rise to immunodeficiency and a variety of tumors (Lombard et al., 2005). In post-mitotic neurons, however, the phenotype is one of degeneration, with a predilection for the large metabolically active Purkinje cell neurons of the cerebellum.
A recent proteomic analysis of the substrates of the ATM and ATR kinases suggests that DNA damage can activate a broad range of signaling pathways, some of which also regulate aging (Matsuoka et al., 2007). ATM is activated through autophosphorylation following interaction with the MRN complex (mre11-rad50-nbs1) at double strand breaks, whereas ATR is activated through interaction with ATR-interacting protein (ATRIP) at sites of stalled replication forks or single stranded DNA associated with double strand breaks. A proteome-wide analysis identified over 700 protein substrates of ATM and ATR that are phosphorylated in response to ionizing radiation (Matsuoka et al., 2007). In addition to replicating previously known DNA damage response proteins, many new substrates were identified including components of signaling pathways that regulate aging. For example, several proteins in the insulin/IGF-1 signaling pathway were identified, including IRS2, the kinase AKT3, and the transcription factor FOXO1. Multiple components of the protein translation regulatory pathway regulated by TOR signaling were also identified as ATM/ATR substrates, including TSC1, 4E-BP1 and p70S6K (ribosomal protein S6 kinase). Activation of these signaling pathways would be predicted to augment short-term cell survival by preventing apoptosis and increasing macromolecular biosynthesis. Surprisingly, an opposite effect, reduced insulin/IGF signaling, has been described in progeroid syndromes with chronic genomic instability. Hence, the DNA damage response network may have evolved to facilitate repair and survival in the short-term following acute DNA damage. However, repetitive or sustained activation of the DNA damage response during aging may compromise normal tissue homeostasis, leading to apoptosis or cellular senescence.
The notion that chronic activation of the DNA damage response may contribute to the aging process is supported by the phenotypes of different p53 gain of function mouse models. It was originally reported that a mutant p53 allele with enhanced tumor suppressor activity accelerated aging in a variety of different tissues in a mouse model (Tyner et al., 2002). However, accelerated aging was not observed in a different mouse model that overexpressed wild-type p53 under the control of the endogenous promoter (Garcia-Cao et al., 2002). A salient difference between the two models was the constitutive expression of the gain of function p53 allele versus the regulated expression of the allele under the control of the endogenous promoter, which was overexpressed under stress-related conditions, but not in the absence of stress. The constitutive overexpressing mutant mouse showed accelerated aging, whereas the stress-related expressor had a normal lifespan with augmented tumor suppressor activity. Hence, persistent stimulation of the DNA damage response resulting in chronic p53 activation, even at a low level, may be deleterious, potentially leading to apoptosis or cellular senescence (Lombard et al., 2005). Moreover, recent studies suggest that chronic DNA damage and ATM signaling in senescent cells leads to secretion of pro-inflammatory cytokines, possibly through activation of the ATM target NF-κB (Rodier et al., 2009). A persistent DNA damage response may therefore contribute to systemic inflammation, a known contributory factor for many age-related degenerative disorders.
A consequence of chronic DNA damage in the aging brain may be transcriptional repression and altered neuronal function (Lu et al., 2004). Transcriptional repression of actively expressed synaptic genes is pronounced in the aging human brain, especially for genes involved in cognitive and affective functions (Loerch et al., 2008). These genes may be selectively vulnerable to DNA damage owing to their activated euchromatic state in neurons, resulting in greater access of DNA to reactive oxygen species and other damaging agents. It is unclear, however, why DNA damage becomes persistent in aging neurons but is efficiently repaired in younger neurons. Another unresolved issue is the role of DNA damage in the pathogenesis of age-related neurodegenerative disorders, particularly Alzheimer’s and Parkinson’s disease, which are accompanied by substantial oxidative stress (Moreira et al., 2008).
A set of transcription factors, molecular chaperones and cofactors function together to promote correct protein folding and protect the cell by sequestering misfolded proteins in a process collectively known as proteostasis. The efficiency of this quality control system declines with age together with changes in protein structure due to oxidative modification, missense mutations and misincorporation of amino acids during translation. Compartments that are highly sensitive to redox state, such as the endoplasmic reticulum and mitochondria, are particularly vulnerable and have their own distinct protein folding quality control systems. The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is activated by misfolding of newly synthesized proteins, and can be induced by exogenous toxins or by metabolic disorders such as type II diabetes (Ron and Walter, 2007). A similar but less well-defined pathway is induced by protein misfolding in mitochondria (Broadley and Hartl, 2008). In the cytoplasm and nucleus, a number of molecular chaperones, exemplified by hsp40, 70 and 90, monitor, sequester and promote the refolding of misfolded proteins. The cross-talk between protein quality control pathways across different cellular compartments is not well understood, although excessive protein misfolding in one compartment can globally affect proteostasis and may contribute to aging and the pathogenesis of neurodegenerative disorders (Bennett et al., 2005; Bennett et al., 2007; Kaganovich et al., 2008; Morimoto, 2008).
Genetic studies in C. elegans suggest that aging and proteostasis are closely linked and coordinately regulated. Aging worms show impaired activation of heat shock and unfolded protein responses and the accumulation of aggregated proteins (Ben-Zvi et al., 2009; David et al., 2010). Proteostasis in aging worms can be restored by manipulations that also extend lifespan, such as downregulation of insulin/IGF-1 signaling by RNAi or activation of the transcription factors DAF-16 and HSF-1 (David et al., 2010; Hsu et al., 2003; Morley et al., 2002). HSF-1 is a member of a family of transcription factors that act in worms and mammals to transcriptionally activate the proteostasis network. Lifespan extension from reduced insulin/IGF-1 signaling is suppressed by RNAi for HSF-1 or chaperones that are transcriptional targets of HSF-1, suggesting that proteostasis is a major component of lifespan regulation in the worm (Morley and Morimoto, 2004). In mammalian cells, the related transcription factor HSF is subject to feedback inhibition through interaction with shock proteins and by direct acetylation. Deacetylation of HSF by the stress resistance protein SIRT1 potentiates the transactivation of heat shock genes (Westerheide et al., 2009). Proteostasis is also a component or hormesis, in which mild stress results in a long-term increase in stress resistance. In worms, hormetic heat shock in young adult animals extends lifespan through the induction of heat shock proteins (Olsen et al., 2006).
Loss of function in the protein quality control network contributes to the pathogenesis of a group of age-dependent disorders known as conformational diseases that are exemplified by Alzheimer’s and Huntington’s disease. A characteristic feature is age-dependent accumulation of protein aggregates in the brain that may contribute to neurodegeneration and neurological decline. Huntington’s disease (HD) is the prototype of a group of human genetic diseases caused by polyglutamine-containing proteins that aggregate, leading to neurodegeneration. The length of the polyglutamine tract is inversely related to the age of onset of disease, suggesting a critical interplay between aging, proteostasis, and neurodegenerative pathology. Moreover, signaling pathways that regulate aging also control polyglutamine-related pathology in C. elegans models; loss of function mutants in the insulin/IGF-1 regulated transcription factors DAF-16 and HSF-1 reduce lifespan and accelerate polyglutamine aggregation and toxicity (Hsu et al., 2003). Conversely, the AGE-1 mutation that extends lifespan in worms reduces polyglutamine aggregation and toxicity (Morley et al., 2002). Conformational diseases also illustrate the fragility of the proteostasis network during aging. Misfolding and aggregation of a single metastable protein, such as mutant huntingtin, can impair the folding of other proteins (David et al., 2010; Gidalevitz et al., 2006) and globally inhibit the ubiquitin proteosome system (Bennett et al., 2007).
Recent studies in worms and mice suggest that stress response pathways which regulate proteostasis during aging may also contribute to the pathogenesis of Alzheimer’s disease (AD) (De Strooper, 2010). One of the first examples was a worm model in which the 42 amino acid form of the amyloid β-peptide (Aβ42), which forms deposits in the brain in AD, was expressed in muscle cells. This resulted in age-dependent aggregation and toxicity that could be prevented by downregulating the insulin/IGF-1 signaling pathway. The protective effect was mediated by activation of the transcription factors DAF-16 and HSF-1. Surprisingly, DAF-16 and HSF-1 had opposite effects on Aβ aggregation. HSF-1 activation resulted in the inhibition of Aβ aggregation, whereas DAF-16 promoted Aβ aggregation, but presumably through a more benign pathway that generated non-toxic fibrillar aggregates (Cohen et al., 2006). A significant modulating effect of insulin/IGF-1 signaling was also observed in APP transgenic mouse models, where downregulation of the pathway was also protective, reducing neuronal loss and inflammation, and improving cognitive function. This was paradoxically associated with increased Aβ aggregation in dense core amyloid plaques, similar to the protective effect of DAF-16 activation in the worm model. It was suggested that this unique proteostatic mechanism may protect against Aβ toxicity by inducing the formation of larger, more inert aggregates, resulting in less accumulation of smaller more toxic oligomeric forms (Cohen et al., 2006). Hence, age-related neurodegeneration may be modulated by insulin/IGF-1 signaling through the control of proteostasis and other stress response pathways.
Autophagy declines in nearly all cells and tissue types as organisms age, likely contributing to the accumulation of dysfunctional organelles and damaged proteins (Cuervo et al., 2010). Studies using model organisms have firmly established a direct role for autophagy in the regulation of lifespan extension. A recent genome-wide screen in yeast identified autophagy genes as a requirement for normal chronological lifespan (Fabrizio et al., 2010). In worms, increased autophagy alone is not sufficient to promote lifespan extension, but is required for the increased lifespan extension due to calorie restriction or decreased insulin signaling (Hansen et al., 2008). In flies, the expression of autophagy genes declines in neurons, and increasing autophagy in these cells promotes lifespan extension, while reducing autophagy in neurons decreases lifespan, increases oxidative stress and results in neurodegeneration (Juhasz et al., 2007; Simonsen et al., 2008). Feeding rapamycin to adult flies increases stress resistance and extends their lifespan via alterations in both autophagy and translation (Bjedov et al., 2010).
Mouse studies demonstrate the central importance of autophagy in mammalian aging and age-related neurodegeneration. Mice deficient for autophagy-related genes (ATGs) exhibit reduced autophagy, high levels of protein inclusion bodies, and neurodegenerative pathology (Hara et al., 2006; Komatsu et al., 2006). These phenotypes are also common features of human neurodegenerative diseases associated with protein aggregation, such as Alzheimer’s disease and Huntington’s disease (Bishop et al., 2010; Cuervo et al., 2010), suggesting a close relationship between the regulation of autophagy, aging and neurodegeneration. Moreover, blocking mTOR in a mouse model of AD decreases Aβ levels and improves cognitive function, and these effects may be due, in part, to increased autophagy (Spilman et al., 2010). Thus, the lifespan extension observed in mammals treated with mTOR inhibitor rapamycin (Harrison et al., 2009) may also be mediated, in part, by the up-regulation of autophagy. Taken together, these studies suggest a role for autophagy in the coordinated regulation of aging and age-related stress responses, as well as the pathogenesis of neurodegenerative disorders.
Activation of autophagy is fundamentally linked to the nutrient status of the cell and impacts metabolism and stress response pathways. Autophagy increases during times of nutrient deprivation, such as in fasting or calorie restriction. This is likely to provide cells with an additional source of energy during times of nutrient scarcity; generating free amino acids can be utilized for protein synthesis. Autophagy is also important for mobilizing lipids in the liver during nutrient deprivation (Singh et al., 2009a), which can be metabolized as an alternate energy source, or used for new membrane biosynthesis. Strikingly, overexpression of LAMP-2A in mouse liver in the late stages of adult life prevents an age-related decline in autophagy, reducing the associated accumulation of damaged proteins and leading to improved liver function (Zhang and Cuervo, 2008).
The mammalian sirtuin SIRT1 stimulates rapamycin- and starvation-stimulated autophagy in cultured cells via deacetylation and activation of autophagy proteins Atg5, Atg7 and Atg8 (Lee et al., 2008), suggesting that at least some of the effects of SIRT1 on metabolism and stress responses could be due to regulation of this clearance pathway. SIRT1 was also required for the CR-mediated increase in mitochondrial autophagy in mouse kidney observed during hypoxia (Kume et al., 2010), demonstrating multiple levels by which protein turnover can be regulated by this sirtuin. Hence, autophagy may be a major component of age-dependent stress response pathways that are activated by central regulators such as SIRT1 and mTOR.
The necessity for post-mitotic neurons to survive an entire lifespan imposes a major challenge for protein quality control, in particular autophagic clearance of damaged or aggregated proteins. Age-related diseases of the nervous system are characterized by the accumulation of aggregated proteins in affected neurons and glial cells that may contribute to dysfunction and neurodegeneration. Autophagy can become impaired in the aging brain at a number of levels. The initial formation of the autophagosome is mediated by the Class III PI3 kinase complex composed of beclin-1, Vps15 and Vps34. Beclin-1 levels decline in the normal aging brain (Shibata et al., 2006) and fall further in early AD, potentially reducing autophagy and predisposing to Aβ aggregation (Pickford et al., 2008). Autophagosome formation may also be impaired at the level of cargo recognition. This has been observed in models of Huntington’s disease, resulting in impaired clearance of aggregated proteins despite normal autophagosome formation (Martinez-Vicente et al., 2010). There is also evidence of impaired autophagosome clearance in neurodegenerative disorders, resulting in the accumulation of autophagosomes that are not efficiently processed further (Wong and Cuervo, 2010). This may relate, in part, to disruption of microtubular or actin cytoskeletal elements that are required for autophagosome transport and fusion. Autophagosome accumulation may contribute directly to neurodegeneration by serving as a source of cytotoxic proteins, such as the 42 amino acid form of Aβ, or by disrupting cellular trafficking (Yu et al., 2005). Presenilin-1 mutations that cause familial AD have recently been shown to compromise both delivery to the lysosome and acidification of lysosomes, compromising the clearance of autophagic substrates at several levels (Lee et al., 2010). Deficits in autophagy can also occur in specific subcellular compartments. Two genes associated with familial Parkinson’s disease, PINK1 and parkin, may function together to promote autophagy of failing mitochondria, a process known as mitophagy (Geisler et al., 2010; Narendra et al., 2008). Disease-causing mutations in PINK1 and parkin impair this process, potentially leading to the persistence of dysfunctional mitochondria that generate high levels of reactive oxygen species and increase oxidative stress. These observations provide evidence for a broad range of mechanisms leading to impaired autophagy in age-related neurodegenerative disorders.
The complex role of autophagy in aging and age-related diseases is apparent in the varying outcomes and context-dependence of manipulating the autophagic pathway. Although activating autophagy is often beneficial, inhibiting autophagosome formation may also be beneficial by reducing neurodegeneration when autophagosome clearance is impaired, as in frontotemporal dementia (Lee and Gao, 2009). Another example is inhibition of the autophagy proteins ATG-5 or ATG-7 in white adipose tissue in the developing mouse. This decreases adipocyte differentiation, resulting in a lean mouse with increased insulin sensitivity. It also confers some of the positive metabolic features of brown adipocytes, such as increased fatty acid oxidation (Singh et al., 2009b). Hence, pharmacologic manipulation of the autophagic pathway has great therapeutic potential, but will likely need to be targeted to specific cell types in defined contexts.
The insulin/IGF, TOR and sirtuin networks integrate cellular and organismal homeostasis through coordinate regulation of nutrient sensing and stress response pathways. These signaling pathways respond to changes in nutrient status and various stressors by altering mitochondrial and metabolic function, and mobilizing the genome maintenance and proteostasis networks. The integrated action of these stress response and maintenance systems has been optimized in the early years of life to maximize fitness. Many studies suggest, however, that a decline in the effectiveness and integration of stress responses contributes to aging and age-related diseases. One of the more remarkable insights from aging research during the past two decades is that age-related decline is not invariably fixed, but can be modified by augmenting stress resistance through the conserved signaling pathways, leading to lifespan extension. A greater understanding of the systems biology of stress response signaling and its breakdown during aging may lead to new therapeutic approaches to the intractable degenerative disorders of aging.
We apologize for many studies that were not discussed because of limited space. This work is supported by grants from the National Institutes of Health/National Institute on Aging (AG26651, AG27916 and AG036106) to B.A.Y. and (AG032375) to M.C.H, a New Scholar Award from the Ellison Medical Foundation to M.C.H., and funding from the Glenn Foundation for Medical Research to B.A.Y. and M.C.H.
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