Targeting tissue-specific metabolic signaling pathways in aging: the promise and limitations

It has been well established that most of the age-related diseases such as insulin resistance, type 2 diabetes, hypertension, cardiovascular disease, osteoporosis, and atherosclerosis are all closely related to metabolic dysfunction. On the other hand, interventions on metabolism such as calorie restriction or genetic manipulations of key metabolic signaling pathways such as the insulin and mTOR signaling pathways slow down the aging process and improve healthy aging. These findings raise an important question as to whether improving energy homeostasis by targeting certain metabolic signaling pathways in specific tissues could be an effective anti-aging strategy. With a more comprehensive understanding of the tissue-specific roles of distinct metabolic signaling pathways controlling energy homeostasis and the cross-talks between these pathways during aging may lead to the development of more effective therapeutic interventions not only for metabolic dysfunction but also for aging.     

Induction of oxidative stress causes functional alterations in mouse urothelium via a TRPM8‐mediated mechanism: implications for aging

The incidence of bladder conditions such as overactive bladder syndrome and its associated urinary incontinence is highly prevalent in the elderly. However, the mechanisms underlying these disorders are unclear. Studies suggest that the urothelium forms a ‘sensory network’ with the underlying innervation, alterations in which, could compromise bladder function. As the accumulation of reactive oxygen species can cause functional alterations with age, the aim of this study was to investigate whether oxidative stress alters urothelial sensory signalling and whether the mechanism underlying the effect of oxidative stress on the urothelium plays a role in aging. Five‐month‐old(young) and 24‐month‐old (aged) mice were used. H₂O₂, used to induce oxidative stress, resulted in an increase in bladder afferent nerve activity and urothelial intracellular calcium in preparations from young mice. These functional changes were concurrent with upregulation of TRPM8 in the urothelium. Moreover, application of a TRPM8 antagonist significantly attenuated the H₂O₂‐induced calcium responses. Interestingly, an upregulation of TRPM8 was also found in the urothelium from aged mice, where high oxidative stress levels were observed, together with a greater calcium response to the TRPM8 agonist WS12. Furthermore, these calcium responses were attenuated by pretreatment with the antioxidant N‐acetyl‐cysteine. This study shows that oxidative stress affects urothelial function involving a TRPM8‐mediated mechanism and these effects may have important implications for aging. These data provide an insight into the possible mechanisms by which oxidative stress causes physiological alterations in the bladder, which may also occur in other organs susceptible to aging.     

Rotifers as experimental tools for investigating aging

Comparative biogerontology has much to contribute to the study of aging. A broad range of aging rates have evolved to meet environmental challenges, and understanding these adaptations can produce valuable insights into aging. The supra Phylum Lophotrochozoa is particularly understudied and has several groups that have intriguing patterns of aging. Members of the Lophotrochozoan phylum Rotifera are particularly useful for aging studies because cohort life tables can be conducted with them easily, and biochemical and genomic tools are available for examining aging mechanisms. This paper reviews a variety of caloric restriction (CR) regimens, small molecule inhibitors, and dietary supplements that extend rotifer lifespan, as well as important interactions between CR and genotype, antioxidant supplements, and TOR and jun-N-terminal kinase (JNK) pathways, and the use of RNAi to identify key genes involved in modulating the aging response. Examples of how rapamycin and JNK inhibitor exposure keeps mortality rates low during the reproductive phase of the life cycle are presented, and the ease of conducting life table experiments to screen natural products from red algae for life extending effects is illustrated. Finally, experimental evolution to produce longer-lived rotifer individuals is demonstrated, and future directions to determine the genetic basis of aging are discussed.     

Dietary restriction affects lifespan but not cognitive aging in Drosophila melanogaster

Dietary restriction extends lifespan in a wide variety of animals, including Drosophila, but its relationship to functional and cognitive aging is unclear. Here, we study the effects of dietary yeast content on fly performance in an aversive learning task (association between odor and mechanical shock). Learning performance declined at old age, but 50‐day‐old dietary‐restricted flies learned as poorly as equal‐aged flies maintained on yeast‐rich diet, even though the former lived on average 9 days (14%) longer. Furthermore, at the middle age of 21 days, flies on low‐yeast diets showed poorer short‐term (5 min) memory than flies on rich diet. In contrast, dietary restriction enhanced 60‐min memory of young (5 days old) flies. Thus, while dietary restriction had complex effects on learning performance in young to middle‐aged flies, it did not attenuate aging‐related decline of aversive learning performance. These results are consistent with the hypothesis that, in Drosophila, dietary restriction reduces mortality and thus leads to lifespan extension, but does not affect the rate with which somatic damage relevant for cognitive performance accumulates with age.     

Autophagy and aging: Maintaining the proteome through exercise and caloric restriction

Accumulation of dysfunctional and damaged cellular proteins and organelles occurs during aging, resulting in a disruption of cellular homeostasis and progressive degeneration and increases the risk of cell death. Moderating the accrual of these defunct components is likely a key in the promotion of longevity. While exercise is known to promote healthy aging and mitigate age‐related pathologies, the molecular underpinnings of this phenomenon remain largely unclear. However, recent evidences suggest that exercise modulates the proteome. Similarly, caloric restriction (CR), a known promoter of lifespan, is understood to augment intracellular protein quality. Autophagy is an evolutionary conserved recycling pathway responsible for the degradation, then turnover of cellular proteins and organelles. This housekeeping system has been reliably linked to the aging process. Moreover, autophagic activity declines during aging. The target of rapamycin complex 1 (TORC1), a central kinase involved in protein translation, is a negative regulator of autophagy, and inhibition of TORC1 enhances lifespan. Inhibition of TORC1 may reduce the production of cellular proteins which may otherwise contribute to the deleterious accumulation observed in aging. TORC1 may also exert its effects in an autophagy‐dependent manner. Exercise and CR result in a concomitant downregulation of TORC1 activity and upregulation of autophagy in a number of tissues. Moreover, exercise‐induced TORC1 and autophagy signaling share common pathways with that of CR. Therefore, the longevity effects of exercise and CR may stem from the maintenance of the proteome by balancing the synthesis and recycling of intracellular proteins and thus may represent practical means to promote longevity.     

Global remodeling of the mouse DNA methylome during aging and in response to calorie restriction

Aging is characterized by numerous molecular changes, such as accumulation of molecular damage and altered gene expression, many of which are linked to DNA methylation. Here, we characterize the blood DNA methylome across 16 age groups of mice and report numerous global, region‐ and site‐specific features, as well as the associated dynamics of methylation changes. Transition of the methylome throughout lifespan was not uniform, with many sites showing accelerated changes in late life. The associated genes and promoters were enriched for aging‐related pathways, pointing to a fundamental link between DNA methylation and control of the aging process. Calorie restriction both shifted the overall methylation pattern and was accompanied by its gradual age‐related remodeling, the latter contributing to the lifespan‐extending effect. With age, both highly and poorly methylated sites trended toward intermediate levels, and aging was accompanied by an accelerated increase in entropy, consistent with damage accumulation. However, the entropy effects differed for the sites that increased, decreased and did not change methylation with age. Many sites trailed behind, whereas some followed or even exceeded the entropy trajectory and altered the developmental DNA methylation pattern. The patterns we observed in certain genomic regions were conserved between humans and mice, suggesting common principles of functional DNA methylome remodeling and its critical role in aging. The highly resolved DNA methylome remodeling provides an excellent model for understanding systemic changes that characterize the aging process.     

SIRT1 but not its increased expression is essential for lifespan extension in caloric‐restricted mice

The SIRT1 deacetylase is one of the best-studied putative mediators of some of the anti-aging effects of calorie restriction (CR), but its role in CR-dependent lifespan extension has not been demonstrated. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild-type mice on an ad libitum diet. Here, we report that median lifespan extension in CR heterozygote SIRT1^+/− mice was identical (51%) to that observed in wild-type mice, but SIRT1^+/− mice displayed a higher frequency of certain pathologies. Although larger studies in additional genetic backgrounds are needed, these results provide strong initial evidence for the requirement of SIRT1 for the lifespan extension effects of CR, but suggest that its high expression is not required for CR-induced lifespan extension.     

Accelerating aging by mouse reverse genetics: a rational approach to understanding longevity

Investigating the molecular basis of aging has been difficult, primarily owing to the pleiotropic and segmental nature of the aging phenotype. There are many often interacting symptoms of aging, some of which are obvious and appear to be common to every aged individual, whereas others affect only a subset of the elderly population. Although at first sight this would suggest multiple molecular mechanisms of aging, there now appears to be almost universal consensus that aging is ultimately the result of the accumulation of somatic damage in cellular macromolecules, with reactive oxygen species likely to be the main damage-inducing agent. What remains significant is unravelling how such damage can give rise to the large variety of aging symptoms and how these can be controlled. Although humans, with over a century of clinical observations, remain the obvious target of study, the mouse, with a relatively short lifespan, easy genetic accessibility and close relatedness to humans, is the tool par excellence to model aging-related phenotypes and test strategies of intervention. Here we present the argument that mouse models with engineered defects in genome maintenance systems are especially important because they often exhibit a premature appearance of aging symptoms. Confirming studies on human segmental progeroid syndromes, most of which are based on heritable mutations in genes involved in genome maintenance, the results thus far obtained with mouse models strongly suggest that lifespan and onset of aging are directly related to the quality of DNA metabolism. This may be in keeping with the recent discovery of a possible 'universal survival' pathway that improves antioxidant defence and genome maintenance and simultaneously extends lifespan in the mouse and several invertebrate species.     

Noncoding variations in Cyp24a1 gene are associated with Klotho‐mediated aging phenotypes in different strains of mice

In mutant mice, reduced levels of Klotho promoted high levels of active vitamin D in the serum. Genetic or dietary manipulations that diminished active vitamin D alleviated aging‐related phenotypes caused by Klotho down‐regulation. The hypomorphic Klotho [kl/kl] allele that decreases Klotho expression in C3H, BALB/c, 129, and C57BL/6 genetic backgrounds substantially increases 1,25(OH)2D3 levels in the sera of susceptible C3H, BALB/c, and 129, but not C57BL/6 mice. This may be attributed to increased basal expression of Cyp24a1 in C57BL/6 mice, which promotes inactivation of 1,25(OH)2D3. Decreased expression of Cyp24a1 in susceptible strains was associated with genetic alterations in noncoding regions of Cyp24a1 gene, which were strongly reminiscent of super‐enhancers that regulate gene expression. These observations suggest that higher basal expression of an enzyme required for catabolizing vitamin D renders B6‐kl/kl mice less susceptible to changes in Klotho expression, providing a plausible explanation for the lack of aging phenotypes on C57BL/6 strain.     

Lifespan‐extending caloric restriction or mTOR inhibition impair adaptive immunity of old mice by distinct mechanisms

Aging of the world population and a concomitant increase in age‐related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age‐related decline of the immune system, infectious diseases remain among the top 5–10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T‐cell development in the thymus, peripheral T‐cell maintenance, T‐cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.     

Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging

Female reproductive capacity declines dramatically in the fourth decade of life as a result of an age‐related decrease in oocyte quality and quantity. The primary causes of reproductive aging and the molecular factors responsible for decreased oocyte quality remain elusive. Here, we show that aging of the female germ line is accompanied by mitochondrial dysfunction associated with decreased oxidative phosphorylation and reduced Adenosine tri‐phosphate (ATP) level. Diminished expression of the enzymes responsible for CoQ production, Pdss2 and Coq6, was observed in oocytes of older females in both mouse and human. The age‐related decline in oocyte quality and quantity could be reversed by the administration of CoQ10. Oocyte‐specific disruption of Pdss2 recapitulated many of the mitochondrial and reproductive phenotypes observed in the old females including reduced ATP production and increased meiotic spindle abnormalities, resulting in infertility. Ovarian reserve in the oocyte‐specific Pdss2‐deficient animals was diminished, leading to premature ovarian failure which could be prevented by maternal dietary administration of CoQ10. We conclude that impaired mitochondrial performance created by suboptimal CoQ10 availability can drive age‐associated oocyte deficits causing infertility.     

Crystal structure of a fully glycosylated HIV‐1 gp120 core reveals a stabilizing role for the glycan at Asn262

The crystal structure of a fully glycosylated HIV‐1 gp120 core in complex with CD4 receptor and Fab 17b at 4.5‐Å resolution reveals 9 of the 15 N‐linked glycans of core gp120 to be partially ordered. The glycan at position Asn262 had the most extensive and well‐ordered electron density, and a GlcNAc₂Man₇ was modeled. The GlcNAc stem of this glycan is largely buried in a cleft in gp120, suggesting a role in gp120 folding and stability. Its arms interact with the stems of neighboring glycans from the oligomannose patch, which is a major target for broadly neutralizing antibodies. Proteins 2015; 83:590–596. © 2014 Wiley Periodicals, Inc.     

Accelerated hematopoietic stem cell aging in a mouse model of dyskeratosis congenita responds to antioxidant treatment

Mutations in DKC1, encoding telomerase associated protein dyskerin, cause X-linked dyskeratosis congenita (DC), a bone marrow (BM) failure, and cancer susceptibility syndrome. Decreased accumulation of telomerase RNA resulting in excessive telomere shortening and premature cellular senescence is thought to be the primary cause of disease in X-linked DC. Affected tissues are those that require constant renewal by stem cell activity. We previously showed that in Dkc1(Δ15) mice, which contain a mutation that is a copy of a human mutation causing DC, mutant cells have a telomerase-dependent proliferative defect and increased accumulation of DNA damage in the first generation before the telomeres are short. We now demonstrate the presence of the growth defect in Dkc1(Δ15) mouse embryonic fibroblasts in vitro and show that accumulation of DNA damage and levels of reactive oxygen species increase with increasing population doublings. Treatment with the antioxidant, N-acetyl cysteine (NAC), partially rescued the growth disadvantage of mutant cells in vitro and in vivo. Competitive BM repopulation experiments showed that the Dkc1(Δ15) mutation is associated with a functional stem cell defect that becomes more severe with increasing age, consistent with accelerated senescence, a hallmark of DC hematopoiesis. This stem cell phenotype was partially corrected by NAC treatment. These results suggest that a pathogenic Dkc1 mutation accelerates stem cell aging, that increased oxidative stress might play a role in the pathogenesis of X-linked DC, and that some manifestations of DC may be prevented or delayed by antioxidant treatment.     

Caloric restriction impacts plasma microRNAs in rhesus monkeys

Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Identification of factors involved in CR brings a promise of translatability to human health and aging. Here, we show that CR induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR's mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR‐regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identify a role in signaling downstream of the insulin receptor. The highly abundant miR‐125a‐5p correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR. Putative target pathways of CR‐associated miRNAs were highly enriched for growth and insulin signaling that have previously been implicated in delayed aging. Clustering analysis further pointed to CR‐induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA‐based homeostatic mechanisms to coordinate a program of delayed aging.     

Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction

Rapamycin, an inhibitor of mTOR kinase, increased median lifespan of genetically heterogeneous mice by 23% (males) to 26% (females) when tested at a dose threefold higher than that used in our previous studies; maximal longevity was also increased in both sexes. Rapamycin increased lifespan more in females than in males at each dose evaluated, perhaps reflecting sexual dimorphism in blood levels of this drug. Some of the endocrine and metabolic changes seen in diet‐restricted mice are not seen in mice exposed to rapamycin, and the pattern of expression of hepatic genes involved in xenobiotic metabolism is also quite distinct in rapamycin‐treated and diet‐restricted mice, suggesting that these two interventions for extending mouse lifespan differ in many respects.