Autophagy and cardiovascular aging: Lesson learned from rapamycin

The biological aging process is commonly associated with increased risk of cardiovascular diseases. Several theories have been put forward for aging-associated deterioration in ventricular function, including attenuation of growth hormone (insulin-like growth factors and insulin) signaling, loss of DNA replication and repair, histone acetylation and accumulation of reactive oxygen species. Recent evidence has depicted a rather unique role of autophagy as another important pathway in the regulation of longevity and senescence. Autophagy is a predominant cytoprotective (rather than self-destructive) process. It carries a prominent role in determination of lifespan. Reduced autophagy has been associated with aging, leading to accumulation of dysfunctional or damaged proteins and organelles. To the contrary, measures such as caloric restriction and exercise may promote autophagy to delay aging and associated comorbidities. Stimulation of autophagy using rapamycin may represent a novel strategy to prolong lifespan and combat aging-associated diseases. Rapamycin regulates autophagy through inhibition of the nutrient-sensing molecule mammalian target of rapamycin (mTOR). Inhibition of mTOR through rapamycin and caloric restriction promotes longevity. The purpose of this review is to recapitulate some of the recent advances in an effort to better understand the interplay between rapamycin-induced autophagy and decelerating cardiovascular aging.     

Comparative idiosyncrasies in life extension by reduced mTOR signalling and its distinctiveness from dietary restriction

Reduced mechanistic target of rapamycin (mTOR) signalling extends lifespan in yeast, nematodes, fruit flies and mice, highlighting a physiological pathway that could modulate aging in evolutionarily divergent organisms. This signalling system is also hypothesized to play a central role in lifespan extension via dietary restriction. By collating data from 48 available published studies examining lifespan with reduced mTOR signalling, we show that reduced mTOR signalling provides similar increases in median lifespan across species, with genetic mTOR manipulations consistently providing greater life extension than pharmacological treatment with rapamycin. In contrast to the consistency in changes in median lifespan, however, the demographic causes for life extension are highly species specific. Reduced mTOR signalling extends lifespan in nematodes by strongly reducing the degree to which mortality rates increase with age (aging rate). By contrast, life extension in mice and yeast occurs largely by pushing back the onset of aging, but not altering the shape of the mortality curve once aging starts. Importantly, in mice, the altered pattern of mortality induced by reduced mTOR signalling is different to that induced by dietary restriction, which reduces the rate of aging. Effects of mTOR signalling were also sex dependent, but only within mice, and not within flies, thus again species specific. An alleviation of age‐associated mortality is not a shared feature of reduced mTOR signalling across model organisms and does not replicate the established age‐related survival benefits of dietary restriction.     

Autophagy and aging

Genetic inhibition of autophagy induces degenerative changes in mammalian tissues that resemble those associated with aging, and normal and pathological aging are often associated with a reduced autophagic potential. Pharmacological or genetic manipulations that increase life span in model organisms often stimulate autophagy, and its inhibition compromises the longevity-promoting effects of caloric restriction, Sirtuin 1 activation, inhibition of insulin/insulin growth factor signaling, or the administration of rapamycin, resveratrol, or spermidine. Here, we discuss the probable cause and effect relationship between perturbed autophagy and aging, as well as possible molecular mechanisms that may mediate the anti-aging effects of autophagy.     

mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies

Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence that reducing mTOR signaling increases lifespan and healthspan, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide preclinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies.     

Hyperactive S6K1 mediates oxidative stress and endothelial dysfunction in aging: inhibition by resveratrol

Mammalian target of rapamycin (mTOR)/S6K1 signalling emerges as a critical regulator of aging. Yet, a role of mTOR/S6K1 in aging-associated vascular endothelial dysfunction remains unknown. In this study, we investigated the role of S6K1 in aging-associated endothelial dysfunction and effects of the polyphenol resveratrol on S6K1 in aging endothelial cells. We show here that senescent endothelial cells displayed higher S6K1 activity, increased superoxide production and decreased bioactive nitric oxide (NO) levels than young endothelial cells, which is contributed by eNOS uncoupling. Silencing S6K1 in senescent cells reduced superoxide generation and enhanced NO production. Conversely, over-expression of a constitutively active S6K1 mutant in young endothelial cells mimicked endothelial dysfunction of the senescent cells through eNOS uncoupling and induced premature cellular senescence. Like the mTOR/S6K1 inhibitor rapamycin, resveratrol inhibited S6K1 signalling, resulting in decreased superoxide generation and enhanced NO levels in the senescent cells. Consistent with the data from cultured cells, an enhanced S6K1 activity, increased superoxide generation, and decreased bioactive NO levels associated with eNOS uncoupling were also detected in aortas of old WKY rats (aged 20-24 months) as compared to the young animals (1-3 months). Treatment of aortas of old rats with resveratrol or rapamycin inhibited S6K1 activity, oxidative stress, and improved endothelial NO production. Our data demonstrate a causal role of the hyperactive S6K1 in eNOS uncoupling leading to endothelial dysfunction and vascular aging. Resveratrol improves endothelial function in aging, at least in part, through inhibition of S6K1. Targeting S6K1 may thus represent a novel therapeutic approach for aging-associated vascular disease.     

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.     

Depletion of NBR1 in urothelial carcinoma cells enhances rapamycin-induced apoptosis through impaired autophagy and mitochondrial dysfunction

Rapamycin is well-recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1-knockdown URCa cells. NBR1-knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment-induced autophagy by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in NBR1-knockdown cells, it did not process the conjugated form of LC3B-II after activation by unc-51 like autophagy-activating kinase 1 (ULK1). NBR1-knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.     

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD⁺ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.     

Combinatorial Approach Using Caenorhabditis elegans and Mammalian Systems for Aging Research

Aging is associated with functional and structural declines in organisms over time. Organisms as diverse as the nematode Caenorhabditis elegans and mammals share signaling pathways that regulate aging and lifespan. In this review, we discuss recent combinatorial approach to aging research employing C. elegans and mammalian systems that have contributed to our understanding of evolutionarily conserved aging-regulating pathways. The topics covered here include insulin/IGF-1, mechanistic target of rapamycin (mTOR), and sirtuin signaling pathways; dietary restriction; autophagy; mitochondria; and the nervous system. A combinatorial approach employing high-throughput, rapid C. elegans systems, and human model mammalian systems is likely to continue providing mechanistic insights into aging biology and will help develop therapeutics against age-associated disorders.     

Aging and Immortality: Quasi-Programmed Senescence and Its Pharmacologic Inhibition

While ruling out programmed aging, evolutionary theory predicts a quasi-program for aging, a continuation of the developmental program that is not turned off, is constantly on, becoming hyper-functional and damaging, causing diseases of aging. Could it be switched off pharmacologically? This would require identification of a molecular target involved in cell senescence, organism aging and diseases of aging. Notably, cell senescence is associated with activation of the TOR (target of rapamycin) nutrient- and mitogen-sensing pathway, which promotes cell growth, even though cell cycle is blocked. Is TOR involved in organism aging? In fact, in yeast (where the cell is the organism), caloric restriction, rapamycin and mutations that inhibit TOR all slow down aging. In animals from worms to mammals caloric restrictions, life-extending agents, and numerous mutations that increase longevity all converge on the TOR pathway. And, in humans, cell hypertrophy, hyper-function and hyperplasia, typically associated with activation of TOR, contribute to diseases of aging. Theoretical and clinical considerations suggest that rapamycin may be effective against atherosclerosis, hypertension and hyper-coagulation (thus, preventing myocardial infarction and stroke), osteoporosis, cancer, autoimmune diseases and arthritis, obesity, diabetes, macula-degeneration, Alzheimer’s and Parkinson’s diseases. Finally, I discuss that extended life span will reveal new causes for aging (e.g., ROS, ‘wear and tear’, Hayflick limit, stem cell exhaustion) that play a limited role now, when quasi-programmed senescence kills us first.     

Rapamycin improves healthspan but not inflammaging in nfκb1−/− mice

Increased activation of the major pro‐inflammatory NF‐κB pathway leads to numerous age‐related diseases, including chronic liver disease (CLD). Rapamycin, an inhibitor of mTOR, extends lifespan and healthspan, potentially via suppression of inflammaging, a process which is partially dependent on NF‐κB signalling. However, it is unknown if rapamycin has beneficial effects in the context of compromised NF‐κB signalling, such as that which occurs in several age‐related chronic diseases. In this study, we investigated whether rapamycin could ameliorate age‐associated phenotypes in a mouse model of genetically enhanced NF‐κB activity (nfκb1^?/?) characterized by low‐grade chronic inflammation, accelerated aging and CLD. We found that, despite showing no beneficial effects in lifespan and inflammaging, rapamycin reduced frailty and improved long‐term memory, neuromuscular coordination and tissue architecture. Importantly, markers of cellular senescence, a known driver of age‐related pathology, were alleviated in rapamycin‐fed animals. Our results indicate that, in conditions of genetically enhanced NF‐κB, rapamycin delays aging phenotypes and improves healthspan uncoupled from its role as a suppressor of inflammation.     

Altered proteome turnover and remodeling by short‐term caloric restriction or rapamycin rejuvenate the aging heart

Chronic caloric restriction (CR) and rapamycin inhibit the mechanistic target of rapamycin (mTOR) signaling, thereby regulating metabolism and suppressing protein synthesis. Caloric restriction or rapamycin extends murine lifespan and ameliorates many aging‐associated disorders; however, the beneficial effects of shorter treatment on cardiac aging are not as well understood. Using a recently developed deuterated‐leucine labeling method, we investigated the effect of short‐term (10 weeks) CR or rapamycin on the proteomics turnover and remodeling of the aging mouse heart. Functionally, we observed that short‐term CR and rapamycin both reversed the pre‐existing age‐dependent cardiac hypertrophy and diastolic dysfunction. There was no significant change in the cardiac global proteome (823 proteins) turnover with age, with a median half‐life 9.1 days in the 5‐month‐old hearts and 8.8 days in the 27‐month‐old hearts. However, proteome half‐lives of old hearts significantly increased after short‐term CR (30%) or rapamycin (12%). This was accompanied by attenuation of age‐dependent protein oxidative damage and ubiquitination. Quantitative proteomics and pathway analysis revealed an age‐dependent decreased abundance of proteins involved in mitochondrial function, electron transport chain, citric acid cycle, and fatty acid metabolism as well as increased abundance of proteins involved in glycolysis and oxidative stress response. This age‐dependent cardiac proteome remodeling was significantly reversed by short‐term CR or rapamycin, demonstrating a concordance with the beneficial effect on cardiac physiology. The metabolic shift induced by rapamycin was confirmed by metabolomic analysis.     

Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN‐deficient human fibroblasts

Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double‐strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short‐term rapamycin treatment. Long‐term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near‐normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.     

mTORC1 activation decreases autophagy in aging and idiopathic pulmonary fibrosis and contributes to apoptosis resistance in IPF fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal disease associated with aging. However, the molecular mechanisms of the aging process that contribute to the pathogenesis of IPF have not been elucidated. IPF is characterized by abundant foci of highly active fibroblasts and myofibroblasts resistant to apoptosis. Remarkably, the role of aging in the autophagy activity of lung fibroblasts and its relationship with apoptosis, as adaptive responses, has not been evaluated previously in this disease. In the present study, we analyzed the dynamics of autophagy in primary lung fibroblasts from IPF compared to young and age‐matched normal lung fibroblasts. Our results showed that aging contributes for a lower induction of autophagy on basal conditions and under starvation which is mediated by mTOR pathway activation. Treatment with rapamycin and PP242, that target the PI3K/AKT/mTOR signaling pathway, modified starvation‐induced autophagy and apoptosis in IPF fibroblasts. Interestingly, we found a persistent activation of this pathway under starvation that contributes to the apoptosis resistance in IPF fibroblasts. These findings indicate that aging affects adaptive responses to stress decreasing autophagy through activation of mTORC1 in lung fibroblasts. The activation of this pathway also contributes to the resistance to cell death in IPF lung fibroblasts.     

Novel compounds for the modulation of mTOR and autophagy to treat neurodegenerative diseases

Most neurodegenerative diseases show a disruption of autophagic function and display abnormal accumulation of toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons clear abnormal protein aggregates and survive. The kinase mammalian target of rapamycin (mTOR) is a major regulator of the autophagic process and is regulated by starvation, growth factors, and cellular stressors. The phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway, which promotes cellular survival, is the main modulator upstream of mTOR, and alterations in this pathway are common in neurodegenerative diseases, e.g. Alzheimer’s disease (AD) and Parkinson’s disease (PD). In the present work we revised mammalian target of rapamycin complex 1 (mTORC1) pathway and mTORC2 as a complementary an important element in mTORC1 signaling. In addition, we revised the extracellular signal regulated kinase (ERK) pathway, which has become relevant in the regulation of the autophagic process and cellular survival through mTORC2 signaling. Finally, we summarize novel compounds that promote autophagy and neuronal protection in the last five years.     

Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy

Caloric restriction and autophagy-inducing pharmacological agents can prolong lifespan in model organisms including mice, flies, and nematodes. In this study, we show that transgenic expression of Sirtuin-1 induces autophagy in human cells in vitro and in Caenorhabditis elegans in vivo. The knockdown or knockout of Sirtuin-1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in C. elegans. Conversely, Sirtuin-1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in C. elegans. The knockdown or pharmacological inhibition of Sirtuin-1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. Along similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy-proficient nematodes, whereas these beneficial effects on longevity were abolished by the knockdown of the essential autophagic modulator Beclin-1. We conclude that autophagy is universally required for the lifespan-prolonging effects of caloric restriction and pharmacological Sirtuin-1 activators.     

The role of mitochondria in mTOR‐regulated longevity

Several unbiased genome‐wide RNA interference (RNAi) screens have pointed to mitochondrial metabolism as the major factor for lifespan regulation. However, conflicting data remain to be clarified concerning the mitochondrial free radical theory of aging (MFRTA). Recently, mTOR (mechanistic target of rapamycin) has been proposed to be the central regulator of aging although how mTOR modulates lifespan is poorly understood. Interestingly, mTOR has been shown to regulate many aspects of mitochondrial function, such as mitochondrial biogenesis, apoptosis, mitophagy and mitochondrial hormesis (mitohormesis) including the retrograde response and mitochondrial unfolded protein response (mito‐UPR). Here we discuss the data linking mitochondrial metabolism to mTOR regulation of lifespan, suggesting that hormetic effects may be key to explaining some controversial results regarding the MFRTA. We also discuss the possibility that dysfunction of mitochondrial adaptive responses rather than free radicals per se contributes to the aging process.     

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration

Disruption of autophagy plays an import role in neurodegenerative disorders, where deficient elimination of abnormal and toxic protein aggregates promotes cellular stress, failure and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons clear abnormal protein aggregates and survive. The kinase mammalian target of rapamycin (mTOR) is a major regulator of the autophagic process and is regulated by starvation, growth factors, and cellular stressors. Upstream of mTOR the survival PI3K/AKT pathway modulates mTOR activity that is also altered in neurodegenerative diseases of Alzheimer and Parkinson. Nevertheless, the interplay between the PI3K/AKT/mTOR pathway and the autophagic process is complex and a more detailed examination of tissue from patients suffering neurodegenerative diseases and of animal and cellular models is needed. In the present work we review the recent findings on the role of the PI3K/AKT/mTOR pathway in the modulation of the autophagic process in neuronal protection.