Cheryl L. Clauson

NF-κB inhibition delays DNA damage–induced senescence and aging in mice

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.

Advances in Understanding the Complex Mechanisms of DNA Interstrand Cross-Link Repair

DNA interstrand cross-links (ICLs) are lesions caused by a variety of endogenous metabolites, environmental exposures, and cancer chemotherapeutic agents that have two reactive groups. The common feature of these diverse lesions is that two nucleotides on opposite strands are covalently joined. ICLs prevent the separation of two DNA strands and therefore essential cellular processes including DNA replication and transcription. ICLs are mainly detected in S phase when a replication fork stalls at an ICL. Damage signaling and repair of ICLs are promoted by the Fanconi anemia pathway and numerous posttranslational modifications of DNA repair and chromatin structural proteins. ICLs are also detected and repaired in nonreplicating cells, although the mechanism is less clear. A unique feature of ICL repair is that both strands of DNA must be incised to completely remove the lesion. This is accomplished in sequential steps to prevent creating multiple double-strand breaks. Unhooking of an ICL from one strand is followed by translesion synthesis to fill the gap and create an intact duplex DNA, harboring a remnant of the ICL. Removal of the lesion from the second strand is likely accomplished by nucleotide excision repair. Inadequate repair of ICLs is particularly detrimental to rapidly dividing cells, explaining the bone marrow failure characteristic of Fanconi anemia and why cross-linking agents are efficacious in cancer therapy. Herein, recent advances in our understanding of ICLs and the biological responses they trigger are discussed.

The oxidative DNA lesions 8,5'-cyclopurines accumulate with aging in a tissue-specific manner.

Accumulation of DNA damage is implicated in aging. This is supported by the fact that inherited defects in DNA repair can cause accelerated aging of tissues. However, clear‐cut evidence for DNA damage accumulation in old age is lacking. Numerous studies report measurement of DNA damage in nuclear and mitochondrial DNA from tissues of young and old organisms, with variable outcomes. Variability results from genetic differences between specimens or the instability of some DNA lesions. To control these variables and test the hypothesis that elderly organisms have more oxidative DNA damage than young organisms, we measured 8,5′‐cyclopurine‐2′‐deoxynucleosides (cPu), which are relatively stable, in tissues of young and old wild‐type and congenic progeroid mice. We found that cPu accumulate spontaneously in the nuclear DNA of wild‐type mice with age and to a greater extent in DNA repair–deficient progeroid mice, with a similar tissue‐specific pattern (liver > kidney > brain). These data, generated under conditions where genetic and environmental variables are controlled, provide strong evidence that DNA repair mechanisms are inadequate to clear endogenous lesions over the lifespan of mammals. The similar, although exaggerated, results obtained from progeroid, DNA repair–deficient mice and old normal mice support the conclusion that DNA damage accumulates with, and likely contributes to, aging.

Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration

Oxidative damage is a well‐established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial‐derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria‐targeted reactive oxygen species (ROS) scavenger XJB‐5‐131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δ mice, previously established to be a useful in vivo model to study age‐related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial‐derived ROS contributes to age‐associated IDD in Ercc1−/Δ mice. Collectively, these data provide strong experimental evidence that mitochondrial‐derived ROS play a causal role in driving changes linked to aging‐related IDD and a potentially important role for radical scavengers in preventing IDD.

DNA damage drives accelerated bone aging via an NF‐κB–dependent mechanism

Advanced age is one of the most important risk factors for osteoporosis. Accumulation of oxidative DNA damage has been proposed to contribute to age‐related deregulation of osteoblastic and osteoclastic cells. Excision repair cross complementary group 1–xeroderma pigmentosum group F (ERCC1‐XPF) is an evolutionarily conserved structure‐specific endonuclease that is required for multiple DNA repair pathways. Inherited mutations affecting expression of ERCC1‐XPF cause a severe progeroid syndrome in humans, including early onset of osteopenia and osteoporosis, or anomalies in skeletal development. Herein, we used progeroid ERCC1‐XPF–deficient mice, including Ercc1‐null (Ercc1−/−) and hypomorphic (Ercc1−/Δ) mice, to investigate the mechanism by which DNA damage leads to accelerated bone aging. Compared to their wild‐type littermates, both Ercc1−/− and Ercc1−/Δ mice display severe, progressive osteoporosis caused by reduced bone formation and enhanced osteoclastogenesis. ERCC1 deficiency leads to atrophy of osteoblastic progenitors in the bone marrow stromal cell (BMSC) population. There is increased cellular senescence of BMSCs and osteoblastic cells, as characterized by reduced proliferation, accumulation of DNA damage, and a senescence‐associated secretory phenotype (SASP). This leads to enhanced secretion of inflammatory cytokines known to drive osteoclastogenesis, such as interleukin‐6 (IL‐6), tumor necrosis factor α (TNFα), and receptor activator of NF‐κB ligand (RANKL), and thereby induces an inflammatory bone microenvironment favoring osteoclastogenesis. Furthermore, we found that the transcription factor NF‐κB is activated in osteoblastic and osteoclastic cells of the Ercc1 mutant mice. Importantly, we demonstrated that haploinsufficiency of the p65 NF‐κB subunit partially rescued the osteoporosis phenotype of Ercc1−/Δ mice. Finally, pharmacological inhibition of the NF‐κB signaling via an I‐κB kinase (IKK) inhibitor reversed cellular senescence and SASP in Ercc1−/Δ BMSCs. These results demonstrate that DNA damage drives osteoporosis through an NF‐κB–dependent mechanism. Therefore, the NF‐κB pathway represents a novel therapeutic target to treat aging‐related bone disease.

ISSLS prize winner: inhibition of NF-κB activity ameliorates age-associated disc degeneration in a mouse model of accelerated aging.

Study Design. NF-&kgr;B activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. Objective. To study the mediatory role of NF-&kgr;B-signaling pathway in age-dependent intervertebral disc degeneration. Summary of Background Data. Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-&kgr;B is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-&kgr;B activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-&kgr;B inhibition on IDD, using a DNA repair-deficient mouse model of accelerated aging (Ercc1−/&Dgr; mice) previously been reported to exhibit age-related IDD. Methods. Systemic inhibition of NF-&kgr;B activation was achieved either genetically by deletion of 1 allele of the NF-&kgr;B subunit p65 (Ercc1−/&Dgr;p65+− mice) or pharmacologically by chronic intraperitoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-&kgr;B, I&kgr;B Inducible Kinase (IKK), in Ercc1−/&Dgr; mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice, and untreated controls were assessed. Results. Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-&kgr;B activity were observed in discs of progeroid Ercc1−/&Dgr; mice and naturally aged wild-type mice compared with young wild-type mice. Systemic inhibition of NF-&kgr;B by the 8K-NBD peptide in Ercc1−/&Dgr; mice increased disc proteoglycan synthesis and ameriolated loss of disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1−/&Dgr;p65+/− mice. Conclusion. These findings demonstrate that the IKK/NF-&kgr;B signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.

Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs.

Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1(-/Δ) mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5× in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments was significantly increased. Additionally, new PG synthesis was reduced 2-3× in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1(-/Δ) mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

Expansion of myeloid-derived suppressor cells with aging in the bone marrow of mice through a NF-κB-dependent mechanism

With aging, there is progressive loss of tissue homeostasis and functional reserve, leading to an impaired response to stress and an increased risk of morbidity and mortality. A key mediator of the cellular response to damage and stress is the transcription factor NF‐κB. We demonstrated previously that NF‐κB transcriptional activity is upregulated in tissues from both natural aged mice and in a mouse model of a human progeroid syndrome caused by defective repair of DNA damage (ERCC1‐deficient mice). We also demonstrated that genetic reduction in the level of the NF‐κB subunit p65(RelA) in the Ercc1−/∆ progeroid mouse model of accelerated aging delayed the onset of age‐related pathology including muscle wasting, osteoporosis, and intervertebral disk degeneration. Here, we report that the largest fraction of NF‐κB ‐expressing cells in the bone marrow (BM) of aged (>2 year old) mice (C57BL/6‐NF‐κBEGFP reporter mice) are Gr‐1+CD11b+myeloid‐derived suppressor cells (MDSCs). There was a significant increase in the overall percentage of MDSC present in the BM of aged animals compared with young, a trend also observed in the spleen. However, the function of these cells appears not to be compromised in aged mice. A similar increase of MDSC was observed in BM of progeroid Ercc1−/∆ and BubR1H/H mice. The increase in MDSC in Ercc1−/∆ mice was abrogated by heterozygosity in the p65/RelA subunit of NF‐κB. These results suggest that NF‐κB activation with aging, at least in part, drives an increase in the percentage of MDSCs, a cell type able to suppress immune cell responses.