Enhanced microglial pro‐inflammatory response to lipopolysaccharide correlates with brain infiltration and blood–brain barrier dysregulation in a mouse model of telomere shortening

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self‐renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as ‘priming’. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first‐generation G1 mTerc^?/?)‐ and late‐generation (third‐generation G3 and G4 mTerc^?/?) telomerase‐deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late‐generation mTerc^?/? microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc^?/? microglia are comparable with microglia derived from G1 mTerc^?/? mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc^?/? microglia mice show an enhanced pro‐inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age‐associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood–brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.     

Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan

Progressive attrition of telomeres triggers DNA damage response (DDR) and limits the regenerative capacity of adult stem cells during mammalian aging. Intriguingly, telomere integrity is not only determined by telomere length but also by the epigenetic status of telomeric/sub‐telomeric regions. However, the functional interplay between DDR induced by telomere shortening and epigenetic modifications in aging remains unclear. Here, we show that deletion of Gadd45a improves the maintenance and function of intestinal stem cells (ISCs) and prolongs lifespan of telomerase‐deficient mice (G3Terc^?/?). Mechanistically, Gadd45a facilitates the generation of a permissive chromatin state for DDR signaling by inducing base excision repair‐dependent demethylation of CpG islands specifically at sub‐telomeric regions of short telomeres. Deletion of Gadd45a promotes chromatin compaction in sub‐telomeric regions and attenuates DDR initiation at short telomeres of G3Terc^?/? ISCs. Treatment with a small molecule inhibitor of base excision repair reduces DDR and improves the maintenance and function of G3Terc^?/? ISCs. Taken together, our study proposes a therapeutic approach to enhance stem cell function and prolong lifespan by targeting epigenetic modifiers.     

In vivo role of checkpoint kinase 2 in signaling telomere dysfunction

Checkpoint kinase 2 (CHK2) is a downstream effector of the DNA damage response (DDR). Dysfunctional telomeres, either owing to critical shortening or disruption of the shelterin complex, activate a DDR, which eventually results in cell cycle arrest, senescence and/or apoptosis. Successive generations of telomerase‐deficient (Terc) mice show accelerated aging and shorter lifespan due to tissue atrophy and impaired organ regeneration associated to progressive telomere shortening. In contrast, mice deficient for the shelterin component TRF1 in stratified epithelia show a rapid and massive induction of DDR, leading to perinatal lethality and severe skin defects. In both mouse models, p53 deficiency can rescue survival. Here, we set to address the role of CHK2 in signaling telomere dysfunction in both mouse models. To this end, we generated mice doubly deficient for Chk2 and either Terc (Chk2^?/? Terc^?/?) or Trf1 (Trf1^Δ/Δ K5Cre Chk2^?/?). We show that Chk2 deletion improves Terc‐associated phenotypes, including lifespan and age‐associated pathologies. Similarly, Chk2 deficiency partially rescues perinatal mortality and attenuates degenerative pathologies of Trf1^Δ/Δ K5Cre mice. In both cases, we show that the effects are mediated by a significant attenuation of p53/p21 signaling pathway. Our results represent the first demonstration of a role for CHK2 in the in vivo signaling of dysfunctional telomeres.     

Effect of oxidative stress on telomere maintenance in aortic smooth muscle cells

Reactive oxygen species (ROS) and telomere dysfunction are both associated with aging and the development of age-related diseases. Although there is evidence for a direct relationship between ROS and telomere dysfunction as well as an independent association of oxidative stress and telomere attrition with age-related disorders, there has not been sufficient exploration of how the interaction between oxidative stress and telomere function may contribute to the pathophysiology of cardiovascular diseases (CVD). To better understand the complex relationships between oxidative stress, telomerase biology and pathophysiology, we examined the telomere biology of aortic smooth muscle cells (ASMCs) isolated from mutant mouse models of oxidative stress. We discovered that telomere lengths were significantly shorter in ASMCs isolated from superoxide dismutase 2 heterozygous (Sod2^+/?) mice, which exhibit increased arterial stiffness with aging, and the observed telomere attrition occurred over time. Furthermore, the telomere erosion occurred even though telomerase activity increased. In contrast, telomeres remained stable in wild-type and superoxide dismutase 1 heterozygous (Sod1^+/?) mice, which do not exhibit CVD phenotypes. The data indicate that mitochondrial oxidative stress, in particular elevated superoxide levels and decreased hydrogen peroxide levels, induces telomere erosion in the ASMCs of the Sod2^+/? mice. This reduction in telomere length occurs despite an increase in telomerase activity and correlates with the onset of disease phenotype. Our results suggest that the oxidative stress caused by imbalance in mitochondrial ROS, from deficient SOD2 activity as a model for mitochondrial dysfunction results in telomere dysfunction, which may contribute to pathogenesis of CVD.     

CHK2‐independent induction of telomere dysfunction checkpoints in stem and progenitor cells

Telomere shortening limits the proliferation of primary human fibroblasts by the induction of senescence, which is mediated by ataxia telangiectasia mutated‐dependent activation of p53. Here, we show that CHK2 deletion impairs the induction of senescence in mouse and human fibroblasts. By contrast, CHK2 deletion did not improve the stem‐cell function, organ maintenance and lifespan of telomere dysfunctional mice and did not prevent the induction of p53/p21, apoptosis and cell‐cycle arrest in telomere dysfunctional progenitor cells. Together, these results indicate that CHK2 mediates the induction of senescence in fibroblasts, but is dispensable for the induction of telomere dysfunction checkpoints at the stem and progenitor cell level in vivo.     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

Knockout of receptor for advanced glycation end‐products attenuates age‐related renal lesions

Pro‐aging effects of endogenous advanced glycation end‐products (AGEs) have been reported, and there is increasing interest in the pro‐inflammatory and ‐fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill‐defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML‐enriched diet on renal aging. Two‐month‐old male, wild‐type (WT) and RAGE^?/? C57Bl/6 mice were fed a control or a CML‐enriched diet (200 μg CML/g_food) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE^?/? mice, with a predominantly tubular localization. The CML‐rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE^?/? mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A‐II (ApoA‐II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA‐II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE^?/? mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE^?/? mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE^?/? mice, suggesting RAGE is an important receptor in so‐called inflamm‐aging.     

A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance

RAP1 is one of the components of shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere length maintenance and protection has remained elusive. RAP1 also binds subtelomeric repeats and along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres, and its deficiency causes a premature aging in humans and mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact on mouse survival, mice lacking both RAP1 and telomerase show a progressively decreased survival with increasing mouse generations compared to telomerase single mutants. Telomere shortening is more pronounced in Rap1^?/? Terc^?/? doubly deficient mice than in the single‐mutant Terc^?/? counterparts, leading to an earlier onset of telomere‐induced DNA damage and degenerative pathologies. Telomerase deficiency abolishes obesity and liver steatohepatitis provoked by RAP1 deficiency. Using genomewide ChIP sequencing, we find that progressive telomere shortening owing to telomerase deficiency leads to re‐localization of RAP1 from telomeres and subtelomeric regions to extratelomeric sites in a genomewide manner. These findings suggest that although in the presence of sufficient telomere reserve RAP1 is not a key factor for telomere maintenance and protection, it plays a crucial role in the context of telomerase deficiency, thus in agreement with its evolutionary conservation as a telomere component from yeast to humans.     

Oxidative stress‐induced renal telomere shortening as a mechanism of cyclosporine‐induced nephrotoxicity

Due to the association of oxidative stress and telomere shortening, it was aimed in the present study to investigate the possibility whether cyclosporine‐A exerts its nephrotoxic side effects via induction of oxidative stress‐induced renal telomere shortening and senescent phenotype in renal tissues of rats. Renal oxidative stress markers, 8‐hydroxydeoxyguanosine, malondialdehyde, and protein carbonyl groups were measured by standard methods. Telomere length and telomerase activity were also evaluated in kidney tissue samples. Results showed that cyclosporine‐A treatment significantly (P < 0.05) enhanced renal malondialdehyde, 8‐hydroxydeoxyguanosine, and protein carbonyl groups levels, decreased renal telomere length, and deteriorated renal function compared with the controls. Renal telomerase activity was not affected by cyclosporine‐A. Renal telomere length could be considered as an important parameter of both oxidative stress and kidney function. Telomere shortening and accelerated kidney aging may be caused by cyclosporine‐induced oxidative stress, indicating the potential mechanism of cyclosporine‐induced nephrotoxicity.     

KCa3.1 upregulation preserves endothelium‐dependent vasorelaxation during aging and oxidative stress

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium‐dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial K_Ca3.1, which contributes to EDR, is upregulated by H₂O₂. We investigated whether K_Ca3.1 upregulation compensates for diminished EDR to NO during aging‐related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1‐phosphate were increased in aged wild‐type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild‐type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age‐matched wild‐type mice. Increased H₂O₂ levels induced Fyn and extracellular signal‐regulated kinases (ERKs) phosphorylation and K_Ca3.1 upregulation. Catalase/GPX1 double knockout (catalase^?/?/GPX1^?/?) upregulated K_Ca3.1 in MAECs. NO production was decreased in aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? MAECs. However, K_Ca3.1 activation‐induced, N^G‐nitro‐l ‐arginine‐, and indomethacin‐resistant EDR was increased without a change in acetylcholine‐induced EDR in aortic rings from aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1‐phosphate induced similar changes in levels of the antioxidant enzymes and upregulated K_Ca3.1. Our findings suggest that, during aging‐related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H₂O₂ and thereby upregulate K_Ca3.1 expression and function via a H₂O₂/Fyn‐mediated pathway. Altogether, enhanced K_Ca3.1 activity may compensate for decreased NO signaling during vascular aging.     

Oxidative stress contributes to arsenic-induced telomere attrition,chromosome instability,and apoptosis

The environmental contaminant arsenic causes cancer, developmental retardation, and other degenerative diseases and, thus, is a serious health concern worldwide. Paradoxically, arsenic also may serve as an anti-tumor therapy, although the mechanisms of its antineoplastic effects remain unclear. Arsenic exerts its toxicity in part by generating reactive oxygen species. We show that arsenic-induced oxidative stress promotes telomere attrition, chromosome end-to-end fusions, and apoptotic cell death. An antioxidant, N-acetylcysteine, effectively prevents arsenic-induced oxidative stress, telomere erosion, chromosome instability, and apoptosis, suggesting that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic-induced chromosome instability and genotoxicity. Embryos with shortened telomeres from late generation telomerase-deficient mice exhibit increased sensitivity to arsenic-induced oxidative damage, suggesting that telomere attrition mediates arsenic-induced apoptosis. Unexpectedly, arsenite did not cause chromosome end-to-end fusions in telomerase RNA knockout mouse embryos despite progressively damaged telomeres and disrupting embryo viability. Together, these findings may explain why arsenic can initiate oxidative stress and telomere erosion, leading to apoptosis and anti-tumor therapy on the one hand and chromosome instability and carcinogenesis on the other.     

Rag1 immunodeficiency‐induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1^?/? zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity‐ and apoptosis‐related genes was increased in the rag1^?/? fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1^?/? zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1^?/? zebrafish compared to their wild‐type (wt) siblings, rag1^?/? showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence‐related genes and senescence‐associated β‐galactosidase, respectively, diminished telomere length, and abnormal self‐renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1^?/? fish, as was the deficiency of the antioxidant metabolite l ‐acetylcarnitine (ALCAR) in rag1^?/? fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT‐263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging.     

The association between stressors and telomeres in non‐human vertebrates: a meta‐analysis

Animal response to stressors such as harsh environmental conditions and demanding biological processes requires energy generated through increased mitochondrial activity. This results in the production of reactive oxygen species (ROS). In vitro and some in vivo studies suggest that oxidative damage of DNA caused by ROS is responsible for telomere shortening. Since telomere length is correlated with survival in many vertebrates, telomere loss is hypothesised to trigger cellular ageing and/ or to reflect the harshness of the environment an individual has experienced. To improve our understanding of stress‐induced telomere dynamics in non‐human vertebrates, we analysed 109 relevant studies in a meta‐analytical framework. Overall, the exposure to possible stressors was associated with shorter telomeres or higher telomere shortening rate (average effect size = ?0.16 ± 0.03). This relationship was consistent for all phylogenetic classes and for all a priori‐selected stressor categories. It was stronger in the case of pathogen infection, competition, reproductive effort and high activity level, which emphasises their importance in explaining intraspecific telomere length variability and, potentially, lifespan variability. Interestingly, the association between stressor exposure and telomeres in one hand, and oxidative stress in the other hand, covaried, suggesting the implication of oxidative stress in telomere dynamics.     

Insulin‐like growth factor 1 deficiency exacerbates hypertension‐induced cerebral microhemorrhages in mice,mimicking the aging phenotype

Clinical and experimental studies show that aging exacerbates hypertension‐induced cerebral microhemorrhages (CMHs), which progressively impair neuronal function. There is growing evidence that aging promotes insulin‐like growth factor 1 (IGF‐1) deficiency, which compromises multiple aspects of cerebromicrovascular and brain health. To determine the role of IGF‐1 deficiency in the pathogenesis of CMHs, we induced hypertension in mice with liver‐specific knockdown of IGF‐1 (Igf1^f/f + TBG‐Cre‐AAV8) and control mice by angiotensin II plus l ‐NAME treatment. In IGF‐1‐deficient mice, the same level of hypertension led to significantly earlier onset and increased incidence and neurological consequences of CMHs, as compared to control mice, as shown by neurological examination, gait analysis, and histological assessment of CMHs in serial brain sections. Previous studies showed that in aging, increased oxidative stress‐mediated matrix metalloprotease (MMP) activation importantly contributes to the pathogenesis of CMHs. Thus, it is significant that hypertension‐induced cerebrovascular oxidative stress and MMP activation were increased in IGF‐1‐deficient mice. We found that IGF‐1 deficiency impaired hypertension‐induced adaptive media hypertrophy and extracellular matrix remodeling, which together with the increased MMP activation likely also contributes to increased fragility of intracerebral arterioles. Collectively, IGF‐1 deficiency promotes the pathogenesis of CMHs, mimicking the aging phenotype, which likely contribute to its deleterious effect on cognitive function. Therapeutic strategies that upregulate IGF‐1 signaling in the cerebral vessels and/or reduce microvascular oxidative stress, and MMP activation may be useful for the prevention of CMHs, protecting cognitive function in high‐risk elderly patients.     

Amelioration of age‐related brain function decline by Bruton's tyrosine kinase inhibition

One of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age‐dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53‐induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24^?/? progeroid mice, which also showed a reduction in general age‐related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety‐like behaviour and better long‐term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24^?/? mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age‐related degeneration of organs such as the brain.     

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1^?/? mice. The lack of deleterious phenotypes in Surf1^?/? mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1^?/? vs. Surf1^+/+ mice despite substantial decreases in COX activity (22%–87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1^+/+ and Surf1^?/? mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1^+/+ and Surf1^?/? mice. Gene expression was differentially regulated in a tissue‐specific manner. Many proteins and metabolites are downregulated in Surf1^?/? adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1^?/? mice. Finally, mitochondrial unfolded protein response (UPR^mt)‐associated proteins were not uniformly altered by age or genotype, suggesting the UPR^mt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1^?/? mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.