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 β–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.
Senescent cells contribute to aging and its associated morbidities. Senescent cells accumulate in vertebrates with aging. Here, we survey where (in what tissues) senescence occurs with aging in mice, by measuring p16Ink4a and p21Cip1 mRNA. A similar survey in Ercc1−/Δ mice illustrates where (in what tissues) senescence occurs in vivo as a consequence of endogenous DNA damage.
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.
With the loss of Ercc1, endogenous DNA damage is not repaired, leading to activation of p53. In response to chronic genotoxic stress, p53 activation overcomes antioxidant defenses, inducing cardiac myocyte apoptosis, leading to dilated cardiomyopathy and death. Either genetic or pharmacologic inhibition of p53 attenuates cardiac myocyte death and ventricular remodeling.
ABSTRACT
Previously, we demonstrated that intratumoral delivery of adenoviral vector encoding single‐chain (sc) IL‐23 (Ad.scIL‐23) was able to induce systemic antitumor immunity. Here, we examined ...the role of IL‐23 in diabetes in nonobese diabetic mice. Intravenous delivery of Ad.scIL‐23 did not accelerate the onset of hyperglycemia but instead resulted in the development of psoriatic arthritis. Ad.scIL‐23–treated mice developed erythema, scales, and thickening of the skin, as well as intervertebral disc degeneration and extensive synovial hypertrophy and loss of articular cartilage in the knees. Immunological analysis revealed activation of conventional T helper type 17 cells and IL‐17–producing γδ T cells along with a significant depletion and suppression of T cells in the pancreatic lymph nodes. Furthermore, treatment with anti–IL‐17 antibody reduced joint and skin psoriatic arthritis pathologies. Thus, these Ad.scIL‐23–treated mice represent a physiologically relevant model of psoriatic arthritis for understanding disease progression and for testing therapeutic approaches.—Flores, R. R., Carbo, L., Kim, E., Van Meter, M., De Padilla, C. M. L., Zhao, J., Colangelo, D., Yousefzadeh, M. J., Angelini, L. A., Zhang, L., Pola, E., Vo, N., Evans, C. H., Gambotto, A., Niedernhofer, L. J., Robbins, P. D. Adenoviral gene transfer of a single‐chain IL‐23 induces psoriatic arthritis–like symptoms in NOD mice. FASEB J. 33, 9505–9515 (2019). www.fasebj.org
An insulin-like growth factor-1 receptor (IGF1R) variant in exon 6 (Arg-407-His) in Ashkenazi Jewish centenarians was previously found to be associated with reduced IGF1R activity. To further study ...this longevity associated IGF1R variant, we generated a novel mouse line carrying the R407H variant in exon 6 of the
gene by employing CRISPR/Cas9 genome editing technology. Here, we show that the
gene can be edited in mouse embryos by zygotic electroporation of Cas9 protein and a single-guide RNAs together with a single stranded oligonucleotide donor containing the desired key nucleotide changes at the
locus. Sequence analysis of F0 and F1 mice following targeted editing demonstrated the robustness of this approach in mice using CRISPR/Cas9 directed homologous recombination (HDR). Western blot analysis indicates that mice heterozygous for the variant have a significant decrease in IGF1R phosphorylation in various tissues, including skeletal muscle, compared to wildtype. In addition, depletion of IGF1R signaling specifically in skeletal muscle of progeroid
mice resulted in extended health span and median lifespan providing the rationale for long term lifespan studies in
hR407H variant mice. This mouse line will be a valuable genetic tool to help determine the impact of IGF1R signaling on aging and longevity. The CRISPR editing approach represents a prototype for generating additional longevity associated gene variant mouse lines to study relevance to human exceptional longevity.
Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly
. To define the contribution of immune system ageing to organism ageing, here we ...selectively deleted Ercc1, which encodes a crucial DNA repair protein
, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence
in the immune system only. We show that Vav-iCre
;Ercc1
mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice
. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre
;Ercc1
or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre
;Ercc1
mice with rapamycin reduced markers of senescence in immune cells and improved immune function
. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.
Senescence is a tumor suppressor mechanism activated in stressed cells to prevent replication of damaged DNA. Senescent cells have been demonstrated to play a causal role in driving aging and ...age-related diseases using genetic and pharmacologic approaches. We previously demonstrated that the combination of dasatinib and the flavonoid quercetin is a potent senolytic improving numerous age-related conditions including frailty, osteoporosis and cardiovascular disease. The goal of this study was to identify flavonoids with more potent senolytic activity.
A panel of flavonoid polyphenols was screened for senolytic activity using senescent murine and human fibroblasts, driven by oxidative and genotoxic stress, respectively. The top senotherapeutic flavonoid was tested in mice modeling a progeroid syndrome carrying a p16INK4a-luciferase reporter and aged wild-type mice to determine the effects of fisetin on senescence markers, age-related histopathology, disease markers, health span and lifespan. Human adipose tissue explants were used to determine if results translated.
Of the 10 flavonoids tested, fisetin was the most potent senolytic. Acute or intermittent treatment of progeroid and old mice with fisetin reduced senescence markers in multiple tissues, consistent with a hit-and-run senolytic mechanism. Fisetin reduced senescence in a subset of cells in murine and human adipose tissue, demonstrating cell-type specificity. Administration of fisetin to wild-type mice late in life restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan.
The natural product fisetin has senotherapeutic activity in mice and in human tissues. Late life intervention was sufficient to yield a potent health benefit. These characteristics suggest the feasibility to translation to human clinical studies.
NIH grants P01 AG043376 (PDR, LJN), U19 AG056278 (PDR, LJN, WLL), R24 AG047115 (WLL), R37 AG013925 (JLK), R21 AG047984 (JLK), P30 DK050456 (Adipocyte Subcore, JLK), a Glenn Foundation/American Federation for Aging Research (AFAR) BIG Award (JLK), Glenn/AFAR (LJN, CEB), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK), Robert J. and Theresa W. Ryan (JLK), and a Minnesota Partnership Grant (AMAY-UMN#99)-P004610401–1 (JLK, EAA).
Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone ...marrow‐derived mesenchymal stem cells (BM‐MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM‐MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM‐MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM‐MSC CM extended life span of Ercc1−/− mice similarly to injection of young BM‐MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC‐derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.
Extracellular vesicles from young bone marrow‐derived mesenchymal stem cells (MSC) reduce markers of senescence in vitro. EVs derived from MSCs generated from human embryonic stem cells reduced expression of senescence markers in culture and in vivo in accelerated and naturally aged mice and improved measures of healthspan. This work demonstrates the senotherapeutic potential of extracellular vesicles in suppressing senescence‐driven age related disease.
Constitutive NF‐κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF‐κB family members are enriched in centenarians. We recently identified a novel ...small molecule (SR12343) that inhibits IKK/NF‐κB activation by disrupting the association between IKKβ and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence‐associated beta‐galactosidase (SA‐β‐gal) activity in oxidative stress‐induced senescent mouse embryonic fibroblasts as well as in etoposide‐induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1−/∆ and Zmpste24−/− mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2‐year‐old WT mice. Taken together, these results demonstrate that IKK/NF‐κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age‐related diseases.
Constitutive IKK/NF‐κB activation is associated with cellular senescence and aging. Here we demonstrate that pharmacological inhibition of IKK/NF‐κB activation by the small molecule SR12343 reduced senescence and SAPS factors in cell culture, improved healthspan in mice with accelerated aging, and reduced markers of senescence and pathology in multiple tissues of both accelerated and naturally aged mice. Therefore, IKK/NF‐κB pathway is a promising target for aging interventions to treat age‐related diseases.
Senescent cells accumulate in multiple tissues as virtually all vertebrate organisms age. Senescence is a highly conserved response to many forms of cellular stress intended to block the propagation ...of damaged cells. Senescent cells have been demonstrated to play a causal role in aging via their senescence-associated secretory phenotype and by impeding tissue regeneration. Depletion of senescent cells either through genetic or pharmacologic methods has been demonstrated to extend murine lifespan and delay the onset of age-related diseases. Measuring the burden and location of senescent cells in vivo remains challenging, as there is no marker unique to senescent cells. Here, we describe multiple methods to detect the presence and extent of cellular senescence in preclinical models, with a special emphasis on murine models of accelerated aging that exhibit a more rapid onset of cellular senescence.
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
-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
MEFs.
mice heterozygous for
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
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.
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