p53 and senescence

Senescence inducers such as telomeric attrition and oncogenic or oxidative stress cause DNA damage. DNA damage results in increased deposition of γH2Ax and 53BP1 in chromatin that in turn activates a kinase cascade involving first ATM and ATR and then CHK1 and CHK2, eventually resulting in p53 activation (d’Adda di Fagagna, 2008; Fumagalli et al., 2012). p53 induces transcription of the cyclin-dependent kinase inhibitor p21^CIP1. In turn, p21^CIP1 blocks CDK4/6 activity, resulting in hypophosphorylated Rb and cell cycle exit (d’Adda di Fagagna, 2008). Although transient increases in p53 levels can enact a quiescent state and activate DNA repair processes, during senescence, there is a sustained induction of p53 (Salama et al., 2014; Kruiswijk et al., 2015). This is a result of damage occurring in repair-resistant regions of the genome known as DNA segments with chromatin alterations reinforcing senescence, such as telomeres (Rodier et al., 2011; Fumagalli et al., 2012), that allow for a permanent arrest of the cell cycle by persistent induction of p21^cip1. Given the key roles of p53, additional regulatory layers exist. For example, the induction of ARF, a product of the INK4/ARF locus, sequesters the ubiquitin ligase MDM2, contributing to increased levels of p53. Recently, the interaction between Forkhead box protein O4 (FOXO4) and p53 has been shown to play an important role in modulating p53 localization and transcriptional activity during senescence (Baar et al., 2017). Interestingly FOXO transcription factors regulate aging, with FOXO activity in Drosophila melanogaster leading to delayed aging in response to disrupted protein homeostasis and oxidative stress (Demontis and Perrimon, 2010).

Regulation of the SASP

The specific combination of secreted factors is thought to depend on the cell type and the senescent inducer. However, many of the key effectors of the SASP and its regulatory mechanism seemed to be shared. Nuclear factor κB (NF-κB) and CCAAT/enhancer–binding protein beta are the key transcriptional SASP regulators (Acosta et al., 2008; Kuilman et al., 2008). DNA damage (Rodier et al., 2009), p38α MAPK (Freund et al., 2011), mTOR (Herranz et al., 2015; Laberge et al., 2015), mixed lineage leukemia 1 (Capell et al., 2016), and GATA4 (Kang et al., 2015) are also able to regulate the SASP. Recently, sensing of cytoplasmic chromatin by the cGAS/STING pathway has been suggested as a trigger for SASP induction (Dou et al., 2017; Glück et al., 2017). There are additional layers of SASP regulation. For example, mTOR controls IL-1α translation to regulate the SASP (Laberge et al., 2015). In addition, mTOR indirectly regulates the activity of ZFP36L1, an RNA-binding protein that binds to AU-rich elements in the 5′-end of inflammatory transcripts, targeting them for degradation (Herranz et al., 2015). There is also a global remodeling of enhancers in senescent cells, and the recruitment of BRD4 to superenhancers adjacent to SASP genes is needed for their induction (Tasdemir et al., 2016).

The complex composition of the SASP means that different subsets of the SASP, such as the proinflammatory and TGF-β secretomes, can be regulated independently. The proinflammatory arm of the SASP is regulated by IL-1 signaling (Acosta et al., 2013). IL-1α partially recapitulates the inflammatory SASP in vitro, and inhibiting the NLRP3 inflammasome, which processes IL-1β, can blunt the SASP (Acosta et al., 2013). Conversely, the juxtacrine Notch signaling pathway promotes the secretion of a TGF-β–enriched secretome (Hoare et al., 2016).

Senescence regulatory networks and aging in vivo

Expression of the components of the INK4/ARF locus correlates with aging, and p16^INK4a can be used as a prognostic marker for some age-related diseases, such as IPF and glomerulosclerosis (Melk et al., 2004; Lomas et al., 2012). Furthermore, p16^INK4a accumulates during aging. For example, p16^INK4a expression increases with age in the islet of Langerhans (Helman et al., 2016), renal cortex (Melk et al., 2004), and fat tissue (Xu et al., 2015; Baker et al., 2016), among other areas (Krishnamurthy et al., 2004). Its knockout also mitigates functional decline and proliferative exhaustion upon HSC transplantation (Janzen et al., 2006). Although these studies suggest that p16^INK4a accumulation is detrimental during aging, increased gene dosage of INK4/ARF in super-p16 mice does not result in reduced life span. The possible detrimental effects cause by p16^INK4a overexpression may be outweighed by their clear tumor suppressive benefits, with a threefold reduction in tumor incidence (Matheu et al., 2009). Similarly, p53^−/− can ameliorate some of the effects of severe progeria mutants such as Ku80^−/−, mTR^−/−, and Zmpste24^−/− (Chin et al., 1999; Lim et al., 2000; Varela et al., 2005), but the resulting increase in tumorigenesis obscures potential increases in life span. Mice with extra copies of p53 or p19^ARF are more resistant to tumors and display delayed aging (García-Cao et al., 2002; Matheu et al., 2007).

Visualizing senescence in vivo

One of the biggest hindrances to investigating senescence in vivo has been the lack of robust, consistent markers. Most studies of senescence in aged tissues have relied on usage of senescence-associated β-galactosidase (SA-β-Gal) staining or the lack of proliferative markers such as Ki67. However, these may yield mixed results. For example, macrophages display elevated levels of SA-β-Gal activity. The use of additional senescence markers, such as lipofuscin, which accumulates in the cytoplasm of senescent cells, could be applied to bridge this gap (Sharpless and Sherr, 2015).

Another useful tool that has emerged is the use of bioluminescent senescence reporters. With the advent of p16^INK4a-LUC mice expressing a luciferase reporter under the control of a p16^INK4a promoter, there is now confirmation that multiple tissues show an exponential age-related increase in p16^INK4a expression that correlates with higher levels of proinflammatory factors or SASP components (Yamakoshi et al., 2009; Burd et al., 2013).

Evaluating the consequences of targeting senescence during aging

Establishing causality of a gene in diseases such as cancer is usually a matter of generating appropriate knockout or overexpression mouse models. To evaluate the role of senescence in aging that approach gets complicated by the tumor suppressive roles of the INK4/ARF locus and p53 (see Pathways regulating the senescence growth arrest section). Seminal studies by Baker et al., first in progeroid BubR1 mice (Baker et al., 2011) and later in naturally aged mice (Baker et al., 2016), demonstrated that by expressing an inducible suicide gene under the control of the p16^INK4a promoter it is possible to ablate senescent cells and improve health span. The elimination of senescent cells improved several age-associated conditions, delayed tumor formation, and ameliorated the side effects of chemotherapy (Baker et al., 2016; Demaria et al., 2016; Baar et al., 2017). The role of p16^INK4a-positive senescent cells in age-related pathologies has been further confirmed by additional studies using other p16^INK4a-based senescence ablation systems such as p16-3MR and INK-NTR mice (Demaria et al., 2014; Childs et al., 2016). These studies have finally confirmed that senescence causes, or at least contributes to, aging.

Opposing roles for senescence in cancer

Age is a strong prognostic marker of reduced survival across many cancers (de Magalhães, 2013). Senescence is a strong tumor suppressor mechanism that limits cancer initiation through both cell-intrinsic (Collado and Serrano, 2010) and cell-extrinsic mechanisms (Kang et al., 2011). However, there is strong evidence suggesting that through the SASP, aged tissues provide a supportive niche for cancer (Coppé et al., 2010). Senescent cells can contribute to tumor progression by enhancing the proliferative potential of cancer cells (Krtolica et al., 2001) or contributing to epithelial to mesenchymal transition (Coppé et al., 2008). Therefore, the increased numbers of senescent cells present in aged tissues could contribute to the increased incidence of cancer with age. Supporting this, a delayed onset in tumor formation is observed when senescent cells are eliminated (Baker et al., 2016). Senolytic therapy also reduces the incidence of metastasis, the leading cause of cancer-related deaths (Demaria et al., 2016).

Renal dysfunction

Aged individuals often display a reduced glomerular filtration rate and cortical volume that can result in glomerulosclerosis and nephron atrophy, both of which are associated with increased expression of p16^INK4a and p53 (Melk et al., 2003, 2004). Senescence has detrimental effects in most renal diseases analyzed (Sturmlechner et al., 2017). Ablation of senescent cells protects against glomerulosclerosis and improves kidney function in aged mice (Baker et al., 2016).

Type 2 diabetes

One of the largest risk factors for the development of type 2 diabetes is age. Several genome-wide association studies of type 2 diabetes have highlighted variants at the INK4/ARF locus, suggesting a possible link between senescence and diabetes (Zeggini et al., 2007; Jeck et al., 2012). In addition, senescence markers and IL-1β are elevated in β cells from diabetic mice (Sone and Kagawa, 2005; Dinarello et al., 2010). Surprisingly, although p16^INK4a expression drove a decline in β cell regenerative capacity and predisposed mice to mild diabetes (Krishnamurthy et al., 2006; Chen et al., 2009), senescent β islets increased insulin secretion, making it unclear how senescence contributes to maintain glucose homeostasis (Helman et al., 2016).

IPF

Fibrosis is a pathological condition whereby tissue accumulates ECM proteins such as collagen, resulting in tissue scarification, usually in response to damage. Senescence appears to have both beneficial and detrimental roles during fibrosis and wound healing. Secretion of MMPs, which occurs as part of the SASP, could help in the resolution of fibrotic plaques (Craig et al., 2015). Conversely, fibroblasts and tissues isolated from IPF patients display increased levels of SA-β-Gal staining and p21^CIP1, suggesting a link with senescence (Yanai et al., 2015; Schafer et al., 2017). The detrimental nature of senescence in IPF was recently demonstrated using senolytics. Elimination of senescent fibroblasts in a mouse model of lung fibrosis reduced expression of profibrotic SASP components and improved pulmonary function (Schafer et al., 2017).

Nonalcoholic fatty liver disease

Cirrhosis is the pathological outcome from liver fibrosis and nonalcoholic fatty liver disease, which in turn is a result of hepatic steatosis, the abnormal accumulation of lipids in hepatocytes (Pellicoro et al., 2014; Hardy et al., 2016). Senescence is associated with liver fibrosis (Kim et al., 2013) and cirrhosis (Wiemann et al., 2002). The risk of developing nonalcoholic fatty liver disease increases with age (Hardy et al., 2016) and is predicted by the presence of senescent hepatocytes (Pellicoro et al., 2014). The elimination of senescent cells using INK-ATTC mice reduces liver fat accumulation (Ogrodnik et al., 2017). The role of senescence in the liver is complex, however, because knocking out p53 or p16^INK4a increases liver fibrosis (Krizhanovsky et al., 2008). Moreover, senescent hepatic stellate cells down-regulate collagen and up-regulate MMPs and cytokines that could remodel fibrotic plaques and recruit macrophages (Krizhanovsky et al., 2008).

Cardiovascular disease

The risk of developing atherosclerosis and cardiomyopathy and their respective conditions, coronary heart disease and heart failure, increases with age. In the case of atherosclerosis, the role of senescence has been confirmed using senolytic models (Childs et al., 2016). Ablation of senescent cells improved the stability of plaques and reduced both the incidence and progression of plaque formation. Senescent cells were initially identified in atherosclerosis in vascular smooth muscle cells at the site of the plaque (Uryga and Bennett, 2016). Subsequent studies showed that macrophages were the primary senescent cell present with higher levels of SA-β-Gal staining and SASP production, suggesting their key contribution to coronary heart disease (Childs et al., 2016). Cardiomyocyte atrophy is one of the underlying causes of myocardial infarction in the elderly (Niccoli and Partridge, 2012). It is unclear how ablation of senescent cells protects against cardiomyocyte hypertrophy in aged mice and provides resistance to cardiac stress (Baker et al., 2016).

Osteoarthritis

Lifelong wear and tear on ligaments is a significant risk factor for the development of arthritis. Failure of chondrocytes to produce cartilage results in degradation of joints and immobilization. Expression of p16^INK4a in these cells correlates with severity and progression of the disease (Price et al., 2002). Moreover, when mice were subjected to an acute trauma to model osteoarthritis, senescent cells accumulated in the site of the injury (Kuyinu et al., 2016). Clearance of these senescent cells using senolytics resulted in the increased functionality of the remaining chondrocytes with rejuvenation of cartilage soon after (Jeon et al., 2017).

Decline in immune function with age

One of the primary risk factors for complications in end-of life care is infection. The inability of the body to raise a response to immune offenses is caused by a functional decline in HSCs. The accumulation of senescent HSCs with age contributes to immune decline and senescence bypass allows for stem cell rejuvenation. Interestingly, the removal of these cells restored the functionality of HSCs and increased myeloid, B, and T cell numbers in transplant experiments (Chang et al., 2016).

Sarcopenia

Muscle stem cells (MuSCs) undergo a decline in their ability to differentiate and facilitate repair of muscle tissue, which is hypothesized to be the underlying cause of age-dependent muscle wasting or sarcopenia. MuSCs are quiescent unless stimulated to repair muscle (Gopinath and Rando, 2008). However, with age, they become senescent, up-regulating p16^INK4a (Sousa-Victor et al., 2014). The elimination of senescent MuSCs increases the ability of the remaining MuSCs to form muscle cell colonies (Chang et al., 2016). Additionally, inhibition of p38 or p16^INK4a bypasses MuSC senescence and strengthens muscle in geriatric mice (Cosgrove et al., 2014; Sousa-Victor et al., 2014).