Time-dependent accumulation of damage to cells and macromolecules is thought to drive aging (2
). DNA damage is one type of damage implicated in aging based on the fact that mutations affecting a diverse array of DNA repair mechanisms lead to accelerated aging of one or more tissues (66
). However, what is not known is the mechanism by which, for instance, damage to the nuclear genome drives aging. The mechanism could be via loss of functional cells once a threshold of damage is reached. Alternatively, activation of conserved stress response pathways may promote aging. Herein, to decipher how damage drives aging, we used a well-defined murine system: mice that spontaneously age rapidly as a consequence of failure to repair endogenous DNA damage (44
The NF-κB family consists of transcription factors activated in response to a diverse array of cellular stressors (68
). NF-κB activity increases with chronologic age in a variety of tissues of mammals (24
). Thus, NF-κB activation could drive aging in response to time-dependent accumulation of cell damage. However, prior studies do not demonstrate a causal relationship between NF-κB activation and aging; neither do they reveal what drives NF-κB activation with aging.
Using a knockin NF-κBEGFP
reporter system, we discovered a significant increase in the percentage of cells in which NF-κB was activated in old and DNA repair–deficient, progeroid mice relative to that in young WT mice. The progeroid mice had increased EGFP expression in kidneys, skeletal muscle, pancreata, and livers (Figure and Supplemental Figure 1). There was not significantly greater NF-κB activation in the spleen, suggesting that inflammatory cells are not the primary driver of NF-κB activation. These data strongly support the conclusion that spontaneous, endogenous DNA damage is sufficient to drive NF-κB activation in vivo. The resolution afforded by the reporter construct revealed that age-related activation of NF-κB is stochastic, meaning that there is activation in one cell while none is detected in adjacent cells, rather than pan-activation throughout a tissue. This is consistent with the stochastic theory of aging, which posits that cellular damage occurs randomly in a fraction of cells (2
We also demonstrate a key causal role for NF-κB in driving multiple age-related pathologies. Inhibition of the IKK/NF-κB pathway genetically, through deletion of one copy of p65, or pharmacologically, using an IKK inhibitory peptide, delayed the onset and severity of aging-related pathologies in the musculoskeletal, hepatobiliary, renal, and nervous systems (Figure D and Figure D). Aging-related symptoms caused by these pathologies were also delayed or attenuated (Tables and ). This provides strong experimental evidence that an increase of IKK/NF-κB activity plays a causal role in aging.
We further demonstrate that genetic reduction of NF-κB reduced the amount of mitochondrial-derived ROS (Figure A). This could be mediated through upregulation of antioxidants. Expression of catalase and targets of NRF2 was significantly increased in mice chronically treated with the IKK inhibitor 8K-NBD (Figure ) compared with that in mice treated with an inactive mutant peptide. In further support of this, oxidative damage to lipids (lipofuscin; Figure B) and DNA (Figure , C and D) was significantly reduced in Ercc1–/–p65+/– mice and Ercc1–/– mice chronically treated with the IKK inhibitor 8K-NBD. This corresponded with a reduction in multiple markers of cellular senescence, including reduced proliferation of primary cells, increased γH2AX foci, and senescence-associated β-galactosidase (SA β-gal) activity. These data support a mechanism by which accumulated cellular damage (in particular DNA damage) with aging leads to activation of NF-κB. This in turn drives increased ROS production and even more cellular damage. Inhibiting NF-κB activation in response to stress is sufficient to attenuate damage and extend healthspan of a murine model of accelerated aging.
Interestingly, our data indicate that treating Ercc1–/Δ
mice with 8K-NBD, beginning at 5 weeks of age, has a greater beneficial effect than genetic depletion of p65 from conception. The delay in aging symptoms, the attenuation of osteoporosis, and the maintenance of weight (Supplemental Figure 4) were greater in mice in which NF-κB activity was inhibited pharmacologically compared with those with genetic inhibition. This could be because p65/NF-κB has a positive role during development and/or early in life. Also, p65
was heterozygous in our mice, and therefore it is possible that the remaining copy of p65
is sufficient to initiate a stress response. Alternatively, 8K-NBD may be more effective at inhibiting NF-κB in response to stress, possibly because inhibition of IKK would act upstream of p65 and could lead to the cytoplasmic sequestration of more than just the p65 subunit of NF-κB. Targeting IKK also may affect other pathways in addition to NF-κB. For example, IKK phosphorylates BCL-10, β-catenin, cyclin D1, FOXO3A, p53, ERα, mTOR, and HIF-1α in addition to NF-κB (69
The magnitude of the effect on healthspan elicited by NF-κB inhibition has only been observed in outbred mice treated with rapamycin (73
), which targets the mTOR pathway, or by genetic deletion of S6K1, a downstream target of mTOR (74
). Interestingly, mTOR has been shown to activate NF-κB via interaction with IKK (75
). In contrast to the results with rapamycin and our results with NBD, treatment with resveratrol and other SIRT agonists appears only able to extend healthspan and life span in mice on high-fat diets (76
Interestingly, chronic inhibition of NF-κB with the 8K-NBD peptide caused a dramatic change in gene expression compared with that in Ercc1–/Δ
littermates treated with a less active control peptide. Expression of 5% of all genes was significantly altered. Based on ontology analysis of these genes, the biological process most significantly affected by NF-κB inhibition was the immune response, which was not surprisingly downregulated. In addition, many processes previously demonstrated to be altered in progeroid or old WT mice, including suppression of the growth hormone/IGF-1 axis, inhibition of cell cycle progression, activation of proapoptotic mechanisms, and DNA damage/stress response (45
), were at least partially corrected by NF-κB inhibition. This provides experimental evidence that NF-κB is indeed a master regulator of aging-related transcriptional reprogramming.
Gene expression analysis also confirmed the efficacy of inhibition of IKK/NF-κB by 8K-NBD, demonstrating decreased expression of numerous genes with known NF-κB promoter sequences, including Gadd45b
, and Prkcd
). The NF-κB–regulated genes that were most downregulated by 8K-NBD were Apod
, both of which have been shown to have roles in cellular senescence and age-related disease (78
). Similarly, a number of cytokines and other proinflammatory genes expressed by senescent cells (80
) are downregulated in Ercc1–/Δ
mice chronically treated with 8K-NBD, notably Il16
, and Il6st
. 8K-NBD treatment also reduced liver expression of p16 at both the mRNA and protein level. Moreover, chronic 8K-NBD treatment significantly upregulated catalase, genes regulated by NRF2, and genes involved in mitochondrial respiration, all important in regulating ROS levels. These observations are consistent with the recent demonstration of a key role for p16-expressing senescent cells in driving aging, suggesting that 8K-NBD treatment can reduce senescence (81
). 8K-NBD treatment also suppressed expression of chemokines known to regulate the trafficking of immune cells during inflammation. Overall, the expression data demonstrate that inhibition of IKK/NF-κB leads to suppression of numerous processes that are known to modulate healthspan, including inflammation and cellular senescence.
In conclusion, these studies demonstrate that spontaneous, endogenous DNA damage can activate NF-κB. Activation of NF-κB is stochastic, occurring only in a subset of cells. Chronic inhibition of IKK/NF-κB activation is sufficient to delay the onset of aging symptoms and chronic aging-related diseases that arise spontaneously in DNA repair–deficient Ercc1–/Δ mice that model a human progeroid syndrome. Moreover, inhibiting NF-κB activation reduces ROS production and oxidative damage to lipids and DNA. This demonstrates a direct causal role for NF-κB in driving aging-related changes in response to cellular damage by promoting continued damage. Inhibition of NF-κB offers what we believe to be a novel strategy for simultaneously delaying and/or attenuating multiple chronic degenerative diseases in patients with progeroid syndromes and potentially in old age.