Progeroid Mice Research Articles

Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.

Vascular Smooth Muscle Cell-Specific Progerin Expression Provokes Contractile Impairment in a Mouse Model of Hutchinson-Gilford Progeria Syndrome that Is Ameliorated by Nitrite Treatment.

Cardiovascular disease (CVD) is the main cause of death worldwide, and aging is its leading risk factor. Aging is much accelerated in Hutchinson–Gilford progeria syndrome (HGPS), an ultra-rare genetic disorder provoked by the ubiquitous expression of a mutant protein called progerin. HGPS patients die in their teens, primarily due to cardiovascular complications. The primary causes of age-associated CVD are endothelial dysfunction and dysregulated vascular tone; however, their contribution to progerin-induced CVD remains poorly characterized. In the present study, we found that progeroid LmnaG609G/G609G mice with ubiquitous progerin expression show both endothelial dysfunction and severe contractile impairment. To assess the relative contribution of specific vascular cell types to these anomalies, we examined LmnaLCS/LCSTie2Cretg/+ and LmnaLCS/LCSSm22αCretg/+ mice, which express progerin specifically in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. Whereas vessel contraction was impaired in mice with VSMC-specific progerin expression, we observed no endothelial dysfunction in mice with progerin expression restricted to VSMCs or ECs. Vascular tone regulation in progeroid mice was ameliorated by dietary sodium nitrite supplementation. Our results identify VSMCs as the main cell type causing contractile impairment in a mouse model of HGPS that is ameliorated by nitrite treatment.

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice.

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.

Endothelial progeria induces adipose tissue senescence and impairs insulin sensitivity through senescence associated secretory phenotype

Vascular senescence is thought to play a crucial role in an ageing-associated decline of organ functions; however, whether vascular senescence is causally implicated in age-related disease remains unclear. Here we show that endothelial cell (EC) senescence induces metabolic disorders through the senescence-associated secretory phenotype. Senescence-messaging secretomes from senescent ECs induced a senescence-like state and reduced insulin receptor substrate-1 in adipocytes, which thereby impaired insulin signaling. We generated EC-specific progeroid mice that overexpressed the dominant negative form of telomeric repeat-binding factor 2 under the control of the Tie2 promoter. EC-specific progeria impaired systemic metabolic health in mice in association with adipose tissue dysfunction even while consuming normal chow. Notably, shared circulation with EC-specific progeroid mice by parabiosis sufficiently transmitted the metabolic disorders into wild-type recipient mice. Our data provides direct evidence that EC senescence impairs systemic metabolic health, and thus establishes EC senescence as a bona fide risk for age-related metabolic disease.

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.

Long-Term Clearance of Senescent Cells Prevents the Hematopoietic Stem Cell Aging in Naturally Aged Mice

Senescent cells (SnCs) accumulate with age. Genetic or pharmacological clearance of SnCs delays several age-associated disorders and extends the healthspan of both progeroid and wild-type (WT) mice (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). In addition, our previous study showed that transient depletion of SnCs could rejuvenated aged hematopoietic stem cells (HSCs) and muscle stem cells in normally aged mice (Chang J et al. Nat Med, 22:78, 2016). These findings suggest that SnCs play a causative role in aging and certain aging-related pathologies, and that selective clearance of SnCs with a senolytic agent that can selectively kill SnCs can be beneficial in part by rejuvenating aged tissue stem cells. Thus, senolytic drugs are potential novel anti-aging agents. However, SnCs can re-accumulate within a few weeks after depletion in mice, suggesting that long-term and intermittent clearance of SnCs is required to extend healthspan, as shown in previous studies (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). It is not known how long-term and intermittent clearance of SnCs affects tissue stem cells such as HSCs because HSCs are mostly quiescent in order to self-renew and prevent exhaustion. We used p16-3MR transgenic mice to evaluate the effect on HSCs of long-term and intermittent clearance of SnCs by ganciclovir (GCV), which selectively kills SnCs in these mice. We treated WT and p16-3MR mice with GCV (25 mg/kg/day x 5 days/per cycle with a 2-week interval between the cycles by ip injection) or saline (vehicle control) from 12 to 24 months of age. GCV effectively prevented SnC accumulation in p16-3MR mice but had no effect in WT mice. In general, aged p16-3MR mice treated with GCV looked healthier than old vehicle-treated p16-3MR mice and old WT mice treated with vehicle or GCV as expected (Baker DJ et al. Nature, 530: 184, 2016). Importantly, GCV abrogated aging-induced increases in p16 expression, p38MAPK activation, and accumulation of DNA double-strand breaks in HSCs in p16-3MR mice. This abrogation was associated with a significant improvement in HSC clonogenicity, suggesting that long-term and intermittent clearance of SnCs prevents HSC aging in naturally aged mice. This suggestion is confirmed by competitive serial and limited dilution HSC transplantation assays, which revealed that HSCs from GCV-treated old p16-3MR mice were functionally indistinguishable from those of young mice. To elucidate the mechanism by which SnC clearance prevents HSC aging, we evaluated the clonogenicity of Lin−Sca1+c-Kit+ (LSK) cells from young or vehicle- or GCV-treated old p16-3MR mice in co-cultures with bone marrow stromal cells (BMSCs) from these mice. We found that clearance of SnCs not only prevented the decline in HSC clonogenic function with aging but also preserved the hematopoietic supporting function of BMSCs. This preservation was associated with reduced levels of Cdkn2a and Cdkn1a mRNAs and senescence-associated secretory phenotype (SASP) factors (such as Il1β, Tnfα and Ccl5), which can adversely affect HSC self-renewal, and decrease of adipogenic and osteoclastic differentiation. In addition, the levels of the HSC self-renewal promoting factor Spp1 mRNA was higher in BMSCs from GCV-treated old p16-3MR mice than in the cells from vehicle-treated old p16-3MR mice. These findings suggest that long-term and intermittent clearance of SnCs can also inhibit BMSC senescence, which contributes to the prevention of HSC aging. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Chang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Kim:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Almeida:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Campisi:Unity Biotechnology: Equity Ownership, Other: co-founder of Unity Biotechnology which develops small-molecule senolytic drugs. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents.

P4500Cellular senescence of endothelial cells impairs angiogenesis by altering energy metabolism through p53-tigar axis

Abstract Background Ischemic disease is prevalent in elderly population due to impaired angiogenesis. Endothelial cell (EC) generates energy largely via glycolysis, which is further activated when angiogenesis actively occurs. PFK-1 is one of the most important regulatory enzymes for glycolysis, which is activated by PFKFB3. On the other hand, TIGAR inhibits PFK-1 under the control of p53. Crucial roles of PFKFB3 in EC functions under physiological and pathological conditions have been reported; however, a role of TIGAR in EC angiogenic functions remains to be elucidated. Furthermore, it remains unknown whether and how cellular senescence affect the energy metabolism in EC. Purpose The purpose of this study is to investigate molecular mechanisms underlying EC dysfunction associated with ageing, especially by focusing on endothelial energy metabolism. Method and result Senescent EC showed reduced glucose consumption assessed by [U-13C]-glucose tracer assay in association with increased expression of p53 and TIGAR. Angiogenic capacity assessed by tube-formation assay was reduced in senescent EC. Of note, either silencing of TIGAR by siRNA or lentivirus-mediated overexpression of PFKFB3 improved angiogenic capacity in senescent EC. These results collectively suggest that senescence impairs glycolysis in EC by activating p53-TIGAR axis, which leads to senescence-associated endothelial dysfunction. To analyze an impact of EC senescence in angiogenesis in vivo, we generated EC-specific progeroid mice in which dominant negative form of telomere repeat-binding factor 2 (TRF2) was overexpressed in EC under the control of the TIE2 promoter. After confirming EC-specific senescence in these endothelial progeroid mice, we generated hind-limb ischemia model. Recovery of blood flow assessed by laser doppler velocimeter was significantly impaired in endothelial progeroid mice, indicating that EC senescence is directly and causally implicated in age-related angiogenic dysfunction. Of note, genetic inactivation of TIGAR completely rescued the impaired ischemia-induced neovessel formation in EC-specific progeroid mice. Conclusion Using unique endothelial progeroid mice, we revealed that EC senescence is a bona fide risk for ischemic disease, largely by reducing glycolysis in EC through p53-TIGAR axis. Our data suggest that endothelial energy metabolism is an attracting therapeutic target for the prevention and/or treatment of ischemic diseases, especially in elderly population.

P2255Senescence associated secretory phenotype exacerbates overload pressure-cardiac hypertrophy

Abstract Background Advanced age is a significant risk factor for cardiovascular diseases such as hypertension and cardiac hypertrophy. The vascular system forms an essential component of cardiac tissue, to provide routes for circulation and transportation of nutrients and oxygen throughout the cardiac muscle. In addition to its function in vascular biology such as vasodilation and neovessel formation, endothelial cell (EC) also provides many secreted angiocrine factors that are crucially involved in maintaining tissue homeostasis. Ageing induces cellular senescence in various cells including EC. Senescent cells produce senescence-messaging secretomes that have deleterious effects on the tissue microenvironment, referred to as the senescence-associated secretory phenotype (SASP). Because of the crucial roles of EC in tissue homeostasis, EC senescence is presumed to play significant roles in age-related cardiac dysfunction, however, whether and the mechanism by which EC senescence affects age-related cardiac dysfunction remains to be elucidated. Purpose We aimed to investigate the role of senescent ECs in cardiac hypertrophy and heart function. Methods To investigate a contribution of senescent EC in age-related cardiac tissue dysfunction in vivo, we generated EC-specific progeroid mice that overexpress the dominant negative form of telomeric repeat-binding factor 2 (TRF2), which play a central role in the protection of chromosome ends, under the control of the vascular endothelial cadherin promoter (VEcad-TRF2DN-Tg). To induce pathological cardiac remodeling, Transverse Aortic Constriction (TAC) was performed in mice at the age of 10–12 weeks old. Cardiac function was assessed using fractional shortening percentage and ejection fraction measured with echocardiography every week until sacrifice day. Mice were sacrificed 4 weeks after TAC, heart tissue was collected for histological analysis, cardiac morphometry analysis, gene expression and protein expression analysis. In vitro, H9C2 rat cardiomyoblast cells were incubated with conditioned medium derived from control or senescent EC in the presence or absence of angiotensin II to induce cardiac hypertrophy. Results The serial echocardiographic analysis after TAC revealed the exacerbated LV dysfunction in VEcad-TRF2DN-Tg compared to that in wild-type mice. Morphometric and histological analysis 4 weeks after TAC showed increased heart weight and aggravated cardiac fibrosis in VEcad-TRF2DN-Tg mice. In vitro studies demonstrated that conditioned medium derived from senescent ECs enhanced cardiomyocyte hypertrophy in H9C2 cells. Of note, we found that treatment with Y2762, a Rho Kinase inhibitor, canceled the exacerbated cardiac hypertrophy caused by endothelial SASP. Conclusion These findings demonstrate for the first time that senescent ECs play causative roles in age-related cardiac disorders through the SASP, potentially by activating Rho-ROCK pathway in cardiomyocytes.

3306Endothelial cell senescence accelerates atherosclerosis by enhancing monocyte recruitment via hyper-reactivity to inflammatory stimuli

Abstract Background Ageing is a significant risk factor for atherosclerotic diseases. Vascular senescence has been considered to play an important role in the progression of atherosclerosis; however, it remains unclear whether endothelial cell (EC) senescence is causally involved in the pathogenesis of atherosclerosis. Purpose The purpose of this study is to elucidate a causative role of senescent EC in the progression of atherosclerosis. Methods Telomeric repeat-binding factor 2 (TRF2) plays a central role in telomere maintenance and protection against end-to-end fusion of chromosome. We previously reported that overexpression of TRF2-dominant negative mutant (TRF2DN) induced premature senescence in EC. We recently generated EC-specific progeroid mice by overexpressing TRF2DN specifically in EC (TRF2DN-Tg), and then generated ApoE-KO/TRF2DN-Tg mice to analyze a role of EC senescence in atherosclerosis. These mice were fed with a high cholesterol-diet, and atherosclerosis was assessed by en face analysis of whole aorta and histological analysis of aortic sinus. In vitro studies to analyze the underlying mechanisms were performed using replicative senescent HUVECs. Results En face analysis of aorta revealed that atherosclerotic lesions were significantly increased in ApoE-KO/TRF2DN-Tg mice comparing with that in ApoE-KO mice at as early as 2 weeks after high-cholesterol diet. Histological analysis of aortic sinus also exhibited accelerated atherosclerosis in ApoE-KO/TRF2DN-Tg mice in association with increased macrophage infiltration. Mechanistically, we found that the induction of adhesion molecules such as VCAM-1, ICAM-1, and E-selectin in response to low-grade inflammatory stimuli was substantially augmented in senescent ECs comparing with that in young ECs. As a result, monocyte adhesion was significantly enhanced in senescent ECs. Of note, eNOS inhibition did not affect the hyper-reactivity of senescent EC to inflammation, while antagonizing NF-kB abolished it. Nuclear translocation of NF-kB in response to inflammation was not different between young and senescent ECs, suggesting that NF-kB transcriptional activity might be enhanced in senescent ECs. Conclusion We revealed a causative role of EC senescence in the progression of atherosclerosis in vivo using unique EC-specific progeroid mice. Our findings revealed that EC senescence is a bona fide risk for atherosclerosis, and thus senescent ECs are attracting pharmacotherapeutic targets for the prevention and/or treatment of atherosclerotic disease in elderly population.

5219A novel senolytic drug, seno-7284 ameliorates age-related cardiometabolic diseases

Abstract Background Senescence at cellular level develops with various genotoxic stresses and it plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so called “senolysis” has potential to become a next generation therapy for age-related disorders including cardiovascular diseases, pulmonary emphysema, Alzheimer's diseases, etc. However, currently there is no senolytic agent available in clinical settings. Purpose Present study was aimed to identify a novel senolytic agent effective for cardiometabolic diseases in compounds already available in clinical settings. Here we demonstrate a compound called “seno-7284” exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis and progeroid. Methods We generated 1) diet-induced obase and diabetic model by imposing a high fat diet for two months, 2) atherosclerosis mice model by imposing western diet to ApoE homozygous knockout mice (ApoE-KO mice) for three months, and 3) Zmpste24 homozygous knockout mice (Zmpste24-KO mice) as a progeroid mice model. We administrated seno-7284 by mixing it into the diet (0.03% w/w). In one, two or four weeks after the administration of seno-7284 to each mice model, we collected tissue samples for further analyses. Results Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of dietary obese mice as senescence-associated beta-galactosidase (SA-beta-gal) staining exhibits (Figure a). This effect was associated with the suppression in systemic glucose intolerance (Figure b), and adipose tissue inflammation in four weeks after the administration of seno-7284. Administrating seno-7284 for two weeks also reduced accumulation of senescent cells in atherosclerotic lesion in aorta of ApoE-KO mice (Figure c), and inhibited the progression of atherosclerosis (Figure d). Surprisingly, this drug significantly improved the lifespan of Zmpste24-KO mice by administering it from 12 weeks old. Further analysis including RNA-seq or metabolomic analysis suggested that seno-7284 stimulates endogenous senolytic function of NK cells and CD8+ T cells. Conclusion Our results indicate that seno-7284 mediates its biological effects by inducing senolysis in some murine aging models. Seno-7284 would become a promising therapeutic agent for age-related cardiometabolic diseases.

Voluntary exercise normalizes the proteomic landscape in muscle and brain and improves the phenotype of progeroid mice.

The accumulation of mitochondrial DNA (mtDNA) mutations is a suspected driver of aging and age‐related diseases, but forestalling these changes has been a major challenge. One of the best‐studied models is the prematurely aging mtDNA mutator mouse, which carries a homozygous knock‐in of a proofreading deficient version of the catalytic subunit of mtDNA polymerase‐γ (PolgA). We investigated how voluntary exercise affects the progression of aging phenotypes in this mouse, focusing on mitochondrial and protein homeostasis in both brain and peripheral tissues. Voluntary exercise significantly ameliorated several aspects of the premature aging phenotype, including decreased locomotor activity, alopecia, and kyphosis, but did not have major effects on the decreased lifespan of mtDNA mutator mice. Exercise also decreased the mtDNA mutation load. In‐depth tissue proteomics revealed that exercise normalized the levels of about half the proteins, with the majority involved in mitochondrial function and nuclear–mitochondrial crosstalk. There was also a specific increase in the nuclear‐encoded proteins needed for the tricarboxylic acid cycle and complex II, but not in mitochondrial‐encoded oxidative phosphorylation proteins, as well as normalization of enzymes involved in coenzyme Q biosynthesis. Furthermore, we found tissue‐specific alterations, with brain coping better as compared to muscle and with motor cortex being better protected than striatum, in response to mitochondrial dysfunction. We conclude that voluntary exercise counteracts aging in mtDNA mutator mice by counteracting protein dysregulation in muscle and brain, decreasing the mtDNA mutation burden in muscle, and delaying overt aging phenotypes.

Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.

The gut microbiome is emerging as a key regulator of several metabolic, immune and neuroendocrine pathways1,2. Gut microbiome deregulation has been implicated in major conditions such as obesity, type 2 diabetes, cardiovascular disease, non-alcoholic fatty acid liver disease and cancer3-6, but its precise role in aging remains to be elucidated. Here, we find that two different mouse models of progeria are characterized by intestinal dysbiosis with alterations that include an increase in the abundance of Proteobacteria and Cyanobacteria, and a decrease in the abundance of Verrucomicrobia. Consistent with these findings, we found that human progeria patients also display intestinal dysbiosis and that long-lived humans (that is, centenarians) exhibit a substantial increase in Verrucomicrobia and a reduction in Proteobacteria. Fecal microbiota transplantation from wild-type mice enhanced healthspan and lifespan in both progeroid mouse models, and transplantation with the verrucomicrobia Akkermansia muciniphila was sufficient to exert beneficial effects. Moreover, metabolomic analysis of ileal content points to the restoration of secondary bile acids as a possible mechanism for the beneficial effects of reestablishing a healthy microbiome. Our results demonstrate that correction of the accelerated aging-associated intestinal dysbiosis is beneficial, suggesting the existence of a link between aging and the gut microbiota that provides a rationale for microbiome-based interventions against age-related diseases.

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

SummaryHematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.

Pak2 kinase promotes cellular senescence and organismal aging

Cellular senescence defines an irreversible cell growth arrest state linked to loss of tissue function and aging in mammals. This transition from proliferation to senescence is typically characterized by increased expression of the cell-cycle inhibitor p16INK4a and formation of senescence-associated heterochromatin foci (SAHF). SAHF formation depends on HIRA-mediated nucleosome assembly of histone H3.3, which is regulated by the serine/threonine protein kinase Pak2. However, it is unknown if Pak2 contributes to cellular senescence. Here, we show that depletion of Pak2 delayed oncogene-induced senescence in IMR90 human fibroblasts and oxidative stress-induced senescence of mouse embryonic fibroblasts (MEFs), whereas overexpression of Pak2 accelerated senescence of IMR90 cells. Importantly, depletion of Pak2 in BubR1 progeroid mice attenuated the onset of aging-associated phenotypes and extended life span. Pak2 is required for expression of genes involved in cellular senescence and regulated the deposition of newly synthesized H3.3 onto chromatin in senescent cells. Together, our results demonstrate that Pak2 is an important regulator of cellular senescence and organismal aging, in part through the regulation of gene expression and H3.3 nucleosome assembly.

Rapamycin Rescues Age-Related Changes in Muscle-Derived Stem/Progenitor Cells from Progeroid Mice

Aging-related loss of adult stem cell function contributes to impaired tissue regeneration. Mice deficient in zinc metalloproteinase STE24 (Zmpste24−/−) exhibit premature age-related musculoskeletal pathologies similar to those observed in children with Hutchinson-Gilford progeria syndrome (HGPS). We have reported that muscle-derived stem/progenitor cells (MDSPCs) isolated from Zmpste24−/− mice are defective in their proliferation and differentiation capabilities in culture and during tissue regeneration. The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth, and inhibition of the mTORC1 pathway extends the lifespan of several animal species. We therefore hypothesized that inhibition of mTORC1 signaling would rescue the differentiation defects observed in progeroid MDSPCs. MDSPCs were isolated from Zmpste24−/− mice, and the effects of mTORC1 on MDSPC differentiation and function were examined. We found that mTORC1 signaling was increased in senescent Zmpste24−/− MDSPCs, along with impaired chondrogenic, osteogenic, and myogenic differentiation capacity versus wild-type MDSPCs. Interestingly, we observed that mTORC1 inhibition with rapamycin improved myogenic and chondrogenic differentiation and reduced levels of apoptosis and senescence in Zmpste24−/− MDSPCs. Our results demonstrate that age-related adult stem/progenitor cell dysfunction contributes to impaired regenerative capacities and that mTORC1 inhibition may represent a potential therapeutic strategy for improving differentiation capacities of senescent stem and muscle progenitor cells.

Compression of morbidity in a progeroid mouse model through the attenuation of myostatin/activin signalling.

BackgroundOne of the principles underpinning our understanding of ageing is that DNA damage induces a stress response that shifts cellular resources from growth towards maintenance. A contrasting and seemingly irreconcilable view is that prompting growth of, for example, skeletal muscle confers systemic benefit.MethodsTo investigate the robustness of these axioms, we induced muscle growth in a murine progeroid model through the use of activin receptor IIB ligand trap that dampens myostatin/activin signalling. Progeric mice were then investigated for neurological and muscle function as well as cellular profiling of the muscle, kidney, liver, and bone.ResultsWe show that muscle of Ercc1 Δ/− progeroid mice undergoes severe wasting (decreases in hind limb muscle mass of 40–60% compared with normal mass), which is largely protected by attenuating myostatin/activin signalling using soluble activin receptor type IIB (sActRIIB) (increase of 30–62% compared with untreated progeric). sActRIIB‐treated progeroid mice maintained muscle activity (distance travel per hour: 5.6 m in untreated mice vs. 13.7 m in treated) and increased specific force (19.3 mN/mg in untreated vs. 24.0 mN/mg in treated). sActRIIb treatment of progeroid mice also improved satellite cell function especially their ability to proliferate on their native substrate (2.5 cells per fibre in untreated progeroids vs. 5.4 in sActRIIB‐treated progeroids after 72 h in culture). Besides direct protective effects on muscle, we show systemic improvements to other organs including the structure and function of the kidneys; there was a major decrease in the protein content in urine (albumin/creatinine of 4.9 sActRIIB treated vs. 15.7 in untreated), which is likely to be a result in the normalization of podocyte foot processes, which constitute the filtration apparatus (glomerular basement membrane thickness reduced from 224 to 177 nm following sActRIIB treatment). Treatment of the progeric mice with the activin ligand trap protected against the development of liver abnormalities including polyploidy (18.3% untreated vs. 8.1% treated) and osteoporosis (trabecular bone volume; 0.30 mm3 in treated progeroid mice vs. 0.14 mm3 in untreated mice, cortical bone volume; 0.30 mm3 in treated progeroid mice vs. 0.22 mm3 in untreated mice). The onset of neurological abnormalities was delayed (by ~5 weeks) and their severity reduced, overall sustaining health without affecting lifespan.ConclusionsThis study questions the notion that tissue growth and maintaining tissue function during ageing are incompatible mechanisms. It highlights the need for future investigations to assess the potential of therapies based on myostatin/activin blockade to compress morbidity and promote healthy ageing.

Vascular smooth muscle cell-specific progerin expression in a mouse model of Hutchinson-Gilford progeria syndrome promotes arterial stiffness: Therapeutic effect of dietary nitrite.

Vascular stiffness is a major cause of cardiovascular disease during normal aging and in Hutchinson–Gilford progeria syndrome (HGPS), a rare genetic disorder caused by ubiquitous progerin expression. This mutant form of lamin A causes premature aging associated with cardiovascular alterations that lead to death at an average age of 14.6 years. We investigated the mechanisms underlying vessel stiffness in LmnaG609G/G609G mice with ubiquitous progerin expression, and tested the effect of treatment with nitrites. We also bred LmnaLCS/LCSTie2Cre+/tgand LmnaLCS/LCSSM22αCre+/tg mice, which express progerin specifically in endothelial cells (ECs) and in vascular smooth muscle cells (VSMCs), respectively, to determine the specific contribution of each cell type to vascular pathology. We found vessel stiffness and inward remodeling in arteries of LmnaG609G/G609G and LmnaLCS/LCSSM22αCre+/tg, but not in those from LmnaLCS/LCSTie2Cre+/tgmice. Structural alterations in aortas of progeroid mice were associated with decreased smooth muscle tissue content, increased collagen deposition, and decreased transverse waving of elastin layers in the media. Functional studies identified collagen (unlike elastin and the cytoskeleton) as an underlying cause of aortic stiffness in progeroid mice. Consistent with this, we found increased deposition of collagens III, IV, V, and XII in the media of progeroid aortas. Vessel stiffness and inward remodeling in progeroid mice were prevented by adding sodium nitrite in drinking water. In conclusion, LmnaG609G/G609G arteries exhibit stiffness and inward remodeling, mainly due to progerin‐induced damage to VSMCs, which causes increased deposition of medial collagen and a secondary alteration in elastin structure. Treatment with nitrites prevents vascular stiffness in progeria.

Measuring biological age in mice using differential mass spectrometry.

Aging is an ill-defined process that increases the risk of morbidity and mortality. Aging is also heterogeneous meaning that biological and chronological age can differ. Here, we used unbiased differential mass spectrometry to quantify thousands of proteins in mouse liver and select those that that consistently change in expression as mice age. A panel of 14 proteins from inbred C57BL/6 mice was used to equate chronological and biological age in this reference population, against which other mice could be compared. This “biological age calculator” identified two strains of f1 hybrid mice as biologically younger than inbred mice and progeroid mice as being biologically older. In an independent validation experiment, the calculator identified mice treated with rapamycin, known to extend lifespan of mice, as 18% younger than mice fed a placebo diet. This demonstrates that it is possible to measure subtle changes in biologic age in mammals using a proteomics approach.

SFRP3 inhibition improves age-related cellular changes in BubR1 progeroid mice.

Wnt signaling is a well‐known molecular pathway in age‐related pathogenesis and therapy of disease. While prior studies have mainly focused on Wnt ligands or Wnt activators, the in vivo functions of naturally secreted Wnt inhibitors are not clear, especially in brain aging. Using BubR1 H/H mice as a novel mouse model of accelerated aging, we report that genetic inhibition of sFRP3 restores the reduced body and brain size observed in BubR1 H/H mice. Furthermore, sFRP3 inhibition ameliorates hypomyelination in the corpus callosum and rescues neural progenitor proliferation in the hippocampal dentate gyrus of BubR1 H/H mice. Taken together, our study identifies sFRP3 as a new molecular factor that cooperates with BubR1 function to regulate brain development, myelination, and hippocampal neurogenesis.

Impaired immune surveillance accelerates accumulation of senescent cells and aging

Cellular senescence is a stress response that imposes stable cell-cycle arrest in damaged cells, preventing their propagation in tissues. However, senescent cells accumulate in tissues in advanced age, where they might promote tissue degeneration and malignant transformation. The extent of immune-system involvement in regulating age-related accumulation of senescent cells, and its consequences, are unknown. Here we show that Prf1−/− mice with impaired cell cytotoxicity exhibit both higher senescent-cell tissue burden and chronic inflammation. They suffer from multiple age-related disorders and lower survival. Strikingly, pharmacological elimination of senescent-cells by ABT-737 partially alleviates accelerated aging phenotype in these mice. In LMNA+/G609G progeroid mice, impaired cell cytotoxicity further promotes senescent-cell accumulation and shortens lifespan. ABT-737 administration during the second half of life of these progeroid mice abrogates senescence signature and increases median survival. Our findings shed new light on mechanisms governing senescent-cell presence in aging, and could motivate new strategies for regenerative medicine.