Increased gene dosage of Ink4/Arf and p53 delays age‐associated central nervous system functional decline

The impairment of the activity of the brain is a major feature of aging, which coincides with a decrease in the function of neural stem cells. We have previously shown that an extra copy of regulated Ink4/Arf and p53 activity, in s‐Ink4/Arf/p53 mice, elongates lifespan and delays aging. In this work, we examined the physiology of the s‐Ink4/Arf/p53 brain with aging, focusing on the neural stem cell (NSC) population. We show that cells derived from old s‐Ink4/Arf/p53 mice display enhanced neurosphere formation and self‐renewal activity compared with wt controls. This correlates with augmented expression of Sox2, Sox9, Glast, Ascl1, and Ars2 NSC markers in the subventricular zone (SVZ) and in the subgranular zone of the dentate gyrus (DG) niches. Furthermore, aged s‐Ink4/Arf/p53 mice express higher levels of Doublecortin and PSA‐NCAM (neuroblasts) and NeuN (neurons) in the olfactory bulbs (OB) and DG, indicating increased neurogenesis in vivo. Finally, aged s‐Ink4/Arf/p53 mice present enhanced behavioral and neuromuscular coordination activity. Together, these findings demonstrate that increased but regulated Ink4/Arf and p53 activity ameliorates age‐related deterioration of the central nervous system activity required to maintain the stem cell pool, providing a mechanism not only for the extended lifespan but also for the health span of these mice.     

Anti-aging activity of the Ink4/Arf locus

The proteins encoded by the Ink4/Arf locus, p16^Ink4a, p19^Arf and p15^Ink4b are major tumour suppressors that oppose aberrant mitogenic signals. The expression levels of the locus are progressively increased during aging and genome-wide association studies have linked the locus to a number of aging-associated diseases and frailty in humans. However, direct measurement of the global impact of the Ink4/Arf locus on organismal aging and longevity was lacking. In this work, we have examined the fertility, cancer susceptibility, aging and longevity of mice genetically modified to carry one ( Ink4/Arf -tg) or two ( Ink4/Arf -tg/tg) intact additional copies of the locus. First, increased gene dosage of Ink4/Arf impairs the production of male germ cells, and in the case of Ink4/Arf -tg/tg mice results in a Sertoli cell-only-like syndrome and a complete absence of sperm. Regarding cancer, there is a lower incidence of aging-associated cancer proportional to the Ink4/Arf gene dosage. Interestingly, increased Ink4/Arf gene dosage resulted in lower scores in aging markers and in extended median longevity. The increased survival was also observed in cancer-free mice indicating that cancer protection and delayed aging are separable activities of the Ink4/Arf locus. In contrast to these results, mice carrying one or two additional copies of the p53 gene ( p53 -tg and p53 -tg/tg) had a normal longevity despite their increased cancer protection. We conclude that the Ink4/Arf locus has a global anti-aging effect, probably by favouring quiescence and preventing unnecessary proliferation.     

Autophagy in stem cell aging

Aging is responsible for changes in mammalian tissues that result in an imbalance to tissue homeostasis and a decline in the regeneration capacity of organs due to stem cell exhaustion. Autophagy is a constitutive pathway necessary to degrade damaged organelles and protein aggregates. Autophagy is one of the hallmarks of aging, which involves a decline in the number and functionality of stem cells. Recent studies show that stem cells require autophagy to get rid of cellular waste produced during the quiescent stage. In particular, two independent studies in muscle and hematopoietic stem cells demonstrate the relevance of the autophagy impairment for stem cell exhaustion and aging. In this review, we summarize the main results of these works, which helped to elucidate the impact of autophagy in stem cell activity as well as in age‐associated diseases.     

Adult Sox2+ stem cell exhaustion in mice results in cellular senescence and premature aging

Aging is characterized by a gradual functional decline of tissues with age. Adult stem and progenitor cells are responsible for tissue maintenance, repair, and regeneration, but during aging, this population of cells is decreased or its activity is reduced, compromising tissue integrity and causing pathologies that increase vulnerability, and ultimately lead to death. The causes of stem cell exhaustion during aging are not clear, and whether a reduction in stem cell function is a cause or a consequence of aging remains unresolved. Here, we took advantage of a mouse model of induced adult Sox2+ stem cell depletion to address whether accelerated stem cell depletion can promote premature aging. After a short period of partial repetitive depletion of this adult stem cell population in mice, we observed increased kyphosis and hair graying, and reduced fat mass, all of them signs of premature aging. It is interesting that cellular senescence was identified in kidney after this partial repetitive Sox2+ cell depletion. To confirm these observations, we performed a prolonged protocol of partial repetitive depletion of Sox2+ cells, forcing regeneration from the remaining Sox2+ cells, thereby causing their exhaustion. Senescence specific staining and the analysis of the expression of genetic markers clearly corroborated that adult stem cell exhaustion can lead to cellular senescence induction and premature aging.     

Altered mammary gland development in the p53+/m mouse, a model of accelerated aging

The tumor suppressor p53 is important for inhibiting the development of breast carcinomas. However, little is known about the effects of increased p53 activity on mammary gland development. Therefore, the effect of p53 dosage on mammary gland development was examined by utilizing the p53+/m mouse, a p53 mutant which exhibits increased wild-type p53 activity, increased tumor resistance, a shortened longevity, and a variety of accelerated aging phenotypes. Here we report that p53+/m virgin mice exhibit a defect in mammary gland ductal morphogenesis. Transplants of mammary epithelium into p53+/m recipient mice demonstrate decreased outgrowth of wild-type and p53+/m donor epithelium, suggesting systemic or stromal alterations in the p53+/m mouse. Supporting these data, p53+/m mice display decreased levels of serum IGF-1 and reduced IGF-1 signaling in virgin glands. The induction of pregnancy or treatment of p53+/m mice with estrogen, progesterone, estrogen and progesterone in combination, or IGF-1 stimulates ductal outgrowth, rescuing the p53+/m mammary phenotype. Serial mammary epithelium transplants demonstrate that p53+/m epithelium exhibits decreased transplant capabilities, suggesting early stem cell exhaustion. These data indicate that appropriate levels of p53 activity are important in regulating mammary gland ductal morphogenesis, in part through regulation of the IGF-1 pathway.     

p19Arf-p53 tumor suppressor pathway regulates cell motility by suppression of phosphoinositide 3-kinase and Rac1 GTPase activities

The p19(Arf)-p53 tumor suppressor pathway plays a critical role in cell-cycle checkpoint control and apoptosis, whereas Rho family small GTPases are key regulators of actin structure and cell motility. By using primary mouse embryonic fibroblasts that lack Arf, p53, or both, we studied the involvement of the p19(Arf)-p53 pathway in the regulation of cell motility and its relationship with Rho GTPases. Deletion of Arf and/or p53 led to actin cytoskeleton reorganization and a significant increase in cell motility. The endogenous phosphoinositide (PI) 3- kinase and Rac1 activities were elevated in Arf(-/-) and p53(-/-) cells, and these activities are required for p19(Arf)- and p53-regulated migration. Reintroduction of the wild type Arf or p53 genes into Arf(-/-) or p53(-/-) cells reversed the PI 3-kinase and Rho GTPase activities as well as the migration phenotype. These results suggest a functional relationship between an established tumor suppressor pathway and a signaling module that controls actin structure and cell motility and show that p19(Arf) and p53 negatively regulate cell migration by suppression of PI 3-kinase and Rac1 activities.     

p53: The barrier to cancer stem cell formation

The role of p53 as the “guardian of the genome” in differentiated somatic cells, triggering various biological processes, is well established. Recent studies in the stem cell field have highlighted a profound role of p53 in stem cell biology as well. These studies, combined with basic data obtained 20 years ago, provide insight into how p53 governs the quantity and quality of various stem cells, ensuring a sufficient repertoire of normal stem cells to enable proper development, tissue regeneration and a cancer free life. In this review we address the role of p53 in genomically stable embryonic stem cells, a unique predisposed cancer stem cell model and adult stem cells, its role in the generation of induced pluripotent stem cells, as well as its role as the barrier to cancer stem cell formation.     

The yin and yang of the Cdkn2a locus in senescence and aging

Senescence of cultured cells involves activation of the p19Arf-p53 and the p16Ink4a-Rb tumor suppressor pathways. This, together with the observation that p19Arf and p16Ink4a expression increases with age in many tissues of humans and rodents, led to the speculation that these pathways drive in vivo senescence and natural aging. However, it has been difficult to test this hypothesis using a mammalian model system because inactivation of either of these pathways results in early death from tumors. One approach to bypass this problem would be to inactivate these pathways in a murine segmental progeria model such as mice that express low amounts of the mitotic checkpoint protein BubR1 (BubR1 hypomorphic mice). These mice have a five-fold reduced lifespan and develop a variety of early-aging associated phenotypes including cachetic dwarfism, skeletal muscle degeneration, cataracts, arterial stiffening, (subcutaneous) fat loss, reduced stress tolerance and impaired wound healing. Importantly, BubR1 hypomorphism elevates both p16Ink4a and p19Arf expression in skeletal muscle and fat. Inactivation of p16Ink4a in BubR1 mutant mice delays both cellular senescence and aging specifically in these tissues. Surprisingly, however, inactivation of p19Arf has the opposite effect; it exacerbates in vivo senescence and aging in skeletal muscle and fat. These mouse studies suggest that p16Ink4a is indeed an effector of aging and in vivo senescence, but p19Arf an attenuator. Thus, the role of the p19Arf-p53 pathway in aging and in vivo senescence seems far more complex than previously anticipated.     

Insulin‐like growth factor‐1 regulates the SIRT1‐p53 pathway in cellular senescence

Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF‐1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF‐1‐SIRT1‐p53 signaling in cellular senescence and aging.     

Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

Dysregulation of the Bmi‐1/p16Ink4a pathway provokes an aging‐associated decline of submandibular gland function

Bmi‐1 prevents stem cell aging, at least partly, by blocking expression of the cyclin‐dependent kinase inhibitor p16^Ink4a. Therefore, dysregulation of the Bmi‐1/p16^Ink4a pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi‐1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi‐1/p16^Ink4a pathway occurs during aging in vivo. Using real‐time in vivo imaging of p16^Ink4a expression in Bmi‐1‐KO mice, we uncovered a novel function of the Bmi‐1/p16^Ink4a pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16^Ink4a expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging‐related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.     

Intrinsically unstructured domains of Arf and Hdm2 form bimolecular oligomeric structures in vitro and in vivo

Arf, Hdm2, and p53 regulate the tumor-suppressor pathway that is most frequently disrupted in human cancer. In the absence of tumorigenic stress, Hdm2 actively attenuates p53-dependent cell cycle arrest and apoptosis by mediating ubiquitination-dependent degradation of p53. Mitogenic stress activates Arf, which indirectly activates p53 by binding to and nullifying the anti-p53 activities of Hdm2. Small conserved domains within Arf and Hdm2 mediate their direct interaction. Individually, these domains are intrinsically unstructured and, when combined in vitro, cofold into bimolecular oligomeric structures that resemble amyloid fibrils in some features. Detailed structural characterization of Hdm2/Arf complexes has previously been hampered by their heterogeneity and large size. Here, we report that a nine-residue fragment of the N-terminus of mouse Arf (termed "A1-mini") cofolds specifically with the Arf-binding domain of Hdm2 to form bimolecular oligomers. We characterized these unprecedented structures using analytical ultracentrifugation and NMR spectroscopy, providing insights into their structural organization. The A1-mini peptide not only binds specifically to Hdm2 in vitro but also recapitulates the nucleolar localization features of full-length Arf in cells. Furthermore, larger fragments of Arf that contain the A1-mini segment have previously been shown to activate p53 in mouse and human cells. Our studies provide the first insights into the molecular basis through which Arf nullifies the p53-inhibiting activity of Hdm2, indirectly activating the tumor-suppressor function of p53 in mammalian cells.     

Neuronal p38α mediates age‐associated neural stem cell exhaustion and cognitive decline

Neuronal activity regulates cognition and neural stem cell (NSC) function. The molecular pathways limiting neuronal activity during aging remain largely unknown. In this work, we show that p38MAPK activity increases in neurons with age. By using mice expressing p38α‐lox and CamkII‐Cre alleles (p38α?‐N), we demonstrate that genetic deletion of p38α in neurons suffices to reduce age‐associated elevation of p38MAPK activity, neuronal loss and cognitive decline. Moreover, aged p38α?‐N mice present elevated numbers of NSCs in the hippocampus and the subventricular zone. These results reveal novel roles for neuronal p38MAPK in age‐associated NSC exhaustion and cognitive decline.