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Abstract

Rapamycin (sirolimus) is a macrolide immunosuppressant that inhibits the mechanistic target of rapamycin (mTOR) protein kinase and extends lifespan in model organisms including mice. Although rapamycin is an FDA-approved drug for select indications, a diverse set of negative side effects may preclude its wide-scale deployment as an antiaging therapy. mTOR forms two different protein complexes, mTORC1 and mTORC2; the former is acutely sensitive to rapamycin whereas the latter is only chronically sensitive to rapamycin in vivo. Over the past decade, it has become clear that although genetic and pharmacological inhibition of mTORC1 extends lifespan and delays aging, inhibition of mTORC2 has negative effects on mammalian health and longevity and is responsible for many of the negative side effects of rapamycin. In this review, we discuss recent advances in understanding the molecular and physiological effects of rapamycin treatment, and we discuss how the use of alternative rapamycin treatment regimens or rapamycin analogs has the potential to mitigate the deleterious side effects of rapamycin treatment by more specifically targeting mTORC1. Although the side effects of rapamycin are still of significant concern, rapid progress is being made in realizing the revolutionary potential of rapamycin-based therapies for the treatment of diseases of aging.

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... The mammalian target of rapamycin (mTOR) is a serine/threonine kinase, which is a part of the phosphoinositide 3-kinase (PI3K)-related kinase family. mTOR functions as an intracellular energy sensor and a central regulator of growth, proliferation, metabolism and aging [154][155][156]. mTOR exists as two distinct protein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2) [154]. ...
... Although the underlying mechanisms are still not fully elucidated, the relationship between mTOR signaling and aging has been shown to be conserved from worms to mammals [161][162][163][164]. mTORC1 can be controlled by the tuberous sclerosis complex (TSC)-Rheb pathway and Ras-related GTP-binding protein (Rag) that mediates amino acid signaling [23]. Several studies have indicated that mTORC1 is sensitive to environmental factors such as oxygen, amino acids, growth factors and glucose, regulating many cell processes such as protein translation, autophagy and cell growth [156,165]. On the other hand, mTORC2 is an effector of insulin/insulinlike growth factor-1 (IGF-1) signaling (IIS) downstream of PI3K and is essential for the activation of many kinases. ...
... On the other hand, mTORC2 is an effector of insulin/insulinlike growth factor-1 (IGF-1) signaling (IIS) downstream of PI3K and is essential for the activation of many kinases. For instance, mTORC2 phosphorylates and activates Akt/PKB, which is a key regulator of cell survival [156,166]. According to the negative effects of mTOR on aging, it was plausible to hypothesize that the expression of mTOR would be increased in the elderly. ...
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Aging is a fundamental biological process accompanied by a general decline in tissue function. Indeed, as the lifespan increases, age-related dysfunction, such as cognitive impairment or dementia, will become a growing public health issue. Aging is also a great risk factor for many age-related diseases. Nowadays, people want not only to live longer but also healthier. Therefore, there is a critical need in understanding the underlying cellular and molecular mechanisms regulating aging that will allow us to modify the aging process for healthy aging and alleviate age-related disease. Here, we reviewed the recent breakthroughs in the mechanistic understanding of biological aging, focusing on the adenosine monophosphate-activated kinase (AMPK), Sirtuin 1 (SIRT1) and mammalian target of rapamycin (mTOR) pathways, which are currently considered critical for aging. We also discussed how these proteins and pathways may potentially interact with each other to regulate aging. We further described how the knowledge of these pathways may lead to new interventions for antiaging and against age-related disease.
... In particular, mTOR has been associated with an increased lifespan by modulating the processes of transcription, translation, autophagy, and metabolism [108]. mTOR was initially shown to be important in reducing aging effects with the observation that its inhibitor rapamycin could extend lifespan and delay aging in model organisms, including a murine in vivo model of mTOR inhibition [109]. The applicability of chronic mTOR attenuation in aging-associated diseases such as cerebrovascular dysfunction and cognitive decline has also been addressed, and significant benefits were shown in experimental animal models of cerebrovascular and neuronal dysfunction, thus suggesting that mTOR inhibitors such as rapamycin may decrease the risk of developing age-associated neurological disorders including vascular dementia and Alzheimer's disease [110]. ...
... mTOR forms two different protein complexes, mTORC1, which is acutely sensitive to rapamycin, and mTORC2, which is only sensitive to rapamycin when it is administered chronically in vivo. For this reason, the side effects seen with the chronic administration of rapamycin have been attributed to the inhibition of mTORC2, thus prompting the quest for mTORC1 specific analogs or the development of alternative treatment regimens aiming to selectively inhibit mTORC1, such as intermittent rapamycin administration [109,[111][112][113]. Recently, there have been interesting clinical results regarding the effect of rapamycin on the aging of the skin. ...
Article
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Aging results from the progressive dysregulation of several molecular pathways and mTOR and AMPK signaling have been suggested to play a role in the complex changes in key biological networks involved in cellular senescence. Moreover, multiple factors, including poor nutritional balance, drive immunosenescence progression, one of the meaningful aspects of aging. Unsurprisingly, nutraceutical and pharmacological interventions could help maintain an optimal biological response by providing essential bioactive micronutrients required for the development, maintenance, and the expression of the immune response at all stages of life. In this regard, many studies have provided evidence of potential antiaging properties of resveratrol, as well as rapamycin and metformin. Indeed, in vitro and in vivo models have demonstrated for these molecules a number of positive effects associated with healthy aging. The current review focuses on the mechanisms of action of these three important compounds and their suggested use for the clinical treatment of immunosenescence and aging.
... However, Palovarotene is teratogenic and prompts limb malformations in immature skeletons. Additionally, it may lead to pancreatitis, vision impairment, mucocutaneous ulcers and sensitivity to sunlight [44]. Μore studies are required to evaluate RARγ efficacy, due to its wide expression, on chondrocytes and chondrogenic cells, compared to RARa and RARß [13]. ...
... It blocks the mTOR pathway, preventing HIF-1α translation without affecting its transcription. Rapamycin also inhibits the hypoxia-induced expression of VEGF [44,45]. Despite its wide application on patients following kidney and liver transplant, its side effects are well-known, including hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, glucose intolerance, insulin resistance and diabetes, anemia, thrombocytopenia, dermatological and gastrointestinal disorders, sinusitis, respiratory and urinary infections, and testicular dysfunction [45]. ...
Article
The term neurogenic heterotopic ossification (NHO) is used to describe the pathological bone formation in soft tissues, due to spinal cord or brain injury. Commonly is presented with pain and stiffness of the affected joint. NHO affects the quality of life of these patients, delays their rehabilitation and therefore increases morbidity. The aim of this article is to emphasize pathophysiology mechanism and review new molecular treatments of heterotopic ossification (HO). It was demonstrated that potent treatment strategies are based on understanding the molecular mechanisms and aiming to inhibit the pathological process of the HO in various stages. New treatments are targeting several factors such as bone morphogenetic proteins (BMPs), retinoic acid receptors (RARs), hypoxic inhibitors (Hif1-inhibitors, rapamycin), free radical scavengers and immunological agents (imatinib). The endogenous pathways that lead to HO at molecular and cellular levels have been the aim of many studies in recent years. New treatment options for HO should be recommended due to the ineffectiveness of traditional older options, such as anti-inflammatory drugs and radiation, especially in the case of NHO.
... MTORC1 inhibition by the macrolide rapamycin and its analogs increase autophagy by blocking the inhibitory phosphorylation of these downstream targets. Although several studies confirmed that rapamycin treatment facilitates degradation of protein aggregates in various disease models through autophagy 9-14 and prolongs life-span in yeast and animal models 15-20 , its wider use as a drug is relatively limited due to strong anti-proliferative and immunosuppressive effects 21,22 . Instead, rapamycin emerged as a broadly used key tool compound for studying the effects of autophagy stimulation, together with other tool compounds which are inhibitors of the autophagosome-lysosome fusion. ...
... MTORC1 inhibition by the macrolide rapamycin and its analogs increase autophagy by blocking the inhibitory phosphorylation of these downstream targets. Although several studies confirmed that rapamycin treatment facilitates degradation of protein aggregates in various disease models through autophagy [9][10][11][12][13][14] and prolongs life-span in yeast and animal models [15][16][17][18][19][20] , its wider use as a drug is relatively limited due to strong anti-proliferative and immunosuppressive effects 21,22 . Instead, rapamycin emerged as a broadly used key tool compound for studying the effects of autophagy stimulation, together with other tool compounds which are inhibitors of the autophagosome-lysosome fusion. ...
Article
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Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.
... Providing the mTOR inhibitor rapamycin (Rap) in the diet can extend lifespan in mice and delay the onset of several age-related morbidities [12,14]. However, despite slowing aging and delaying the onset of select age-related pathologies, Rap also has several notable side effects, including hyperglycemia, new onset diabetes, and dyslipidemia [15]. The antidiabetic drug metformin (Met) can activate AMPK and, when added to Rap, extends lifespan to a greater extent than historical cohorts of mice treated with Met or Rap alone and helps mitigate Rap-induced glucose intolerance [16,17]. ...
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Background The objective of this study was to determine if mechanistic target of rapamycin (mTOR) inhibition with or without AMP-activated protein kinase (AMPK) activation can protect against primary, age-related OA. Design Dunkin-Hartley guinea pigs develop mild primary OA pathology by 5 months of age that progresses to moderate OA by 8 months of age. At 5 months, guinea pigs served as young control ( n = 3) or were fed either a control diet ( n = 8), a diet enriched with the mTOR-inhibitor rapamycin (Rap, 14 ppm, n = 8), or Rap with the AMPK-activator metformin (Rap+Met, 1000 ppm, n = 8) for 12 weeks. Knee joints were evaluated by OARSI scoring, micro-computed tomography, and immunohistochemistry. Glenohumeral articular cartilage was collected for western blotting. Results Rap- and Rap+Met-treated guinea pigs displayed lower body weight than control. Rap and Rap+Met inhibited articular cartilage mTORC1 but not mTORC2 signaling. Rap+Met, but not Rap alone, stimulated AMPK. Despite lower body weight and articular cartilage mTORC1 inhibition, Rap- and Rap+Met-treated guinea pigs had greater OA severity in the medial tibial plateau due to articular cartilage structural damage and/or proteoglycan loss. Rap and Rap+Met increased plasma glucose compared to control. Plasma glucose concentration was positively correlated with proteoglycan loss, suggesting hyperglycemic stress after Rap treatment was related to worsened OA. Conclusions This is the first study to show that Rap induced increase in plasma glucose was associated with greater OA severity. Further, articular cartilage mTORC1 inhibition and bodyweight reduction by dietary Rap and Rap+Met did not appear to protect against primary OA during the prevailing hyperglycemia.
... Rapamycin is a macrolide antibiotic that is produced by Streptomyces hygroscopicus and is known to have immunosuppressive effects and anti-proliferative properties, which have been shown to inhibit the effects on fibrosis and are used in anti-cancer and anti-fibrotic therapies. 16 Studies have found that rapamycin can reduce TGF-β1 expression in animal models of renal fibrosis 17 and prevent TGF-β1 induced pulmonary fibrosis 18 , but the mechanism of action remains unclear. Rapamycin can bind to FK-binding protein 12 and inhibit the function of the Mammalian target of rapamycin (mTOR), which is a key protein of mTOR complex 1 (mTORC1) that can inhibit the downstream signaling factors of the mTOR signaling pathway. ...
Article
Background: Rapamycin was shown to reduce transforming growth factor β1 (TGF-β1) expression, inhibit the Mammalian target of rapamycin function, and prevent TGF-β1-induced pulmonary fibrosis. Rapamycin-eluting stents (RES) were successfully used to prevent coronary artery restenosis. Urethral stricture is one of the most challenging problems in urology. Thus, combining the pharmacological effects of rapamycin and the mechanical support of the stent on the urethra may prevent urethral stricture formation. However, the use of RES for urethral stricture treatment has not been studied. Aims: To observe the effects of RES in urethral stricture in a rabbit model. Study design: Animal experimentation. Methods: Twenty adult male New Zealand rabbits were randomly divided into control, urethral stricture model, bare-metal stent, and RES groups. The rabbits in the control group underwent urethroscopy alone without electrocoagulation. The rabbit model of urethral stricture was established by electrocoagulation using a self-made electrocoagulation device under direct vision using ureteroscopy. After model establishment, the rabbits in the bare-metal stent and RES groups received stent placement by ureteroscopy. On day 30, retrograde urethrography was performed to assess urethral stricture formation, ureteroscopy to remove the stents, and histological examinations to assess the degree of fibrosis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to evaluate the expression levels of TGF-β1, Smad3, and matrix metalloproteinase 1 (MMP1). Results: Urethral stricture formation was seen in the model group, whereas not in the bare-metal stent group. The bare-metal stents did not displace but were difficult to remove. In the RES group, RES was dislodged in itself at postoperative day 27 in one rabbit, whereas successfully removed by ureteroscopy in the remaining four rabbits, and urethral stricture formation was not seen on retrograde urethrography after stent removal. Histological examination revealed a large number of dense fibroblasts and blue-stained collagen fibers in the bare-metal stent group, whereas the number of fibroblasts and collagen fibers under the mucosa was reduced in the RES group. RT-qPCR and Western blot analyses showed that the messenger ribonucleic acid (mRNA) and protein expression of TGF-β1and Smad3 was significantly decreased, and mRNA and protein expression of MMP1 was significantly increased in the RES group than that in the model ((P < 0.001) and bare-metal stent groups (P < 0.001). Conclusion: RES can effectively prevent electrocoagulation-induced urethral stricture in rabbits. The mechanism may be related to the effect of rapamycin on inhibiting TGF-β1 and Smad3 expression and promoting MMP1 expression in urethral tissues.
... For example, the mTOR inhibitor rapamycin is a widely used autophagy activator and showed protective effect against H 2 O 2 -induced oxidative damage in RPE cells [8]. However, a variety of side effects of rapamycin, such as respiratory and urinary infections, glucose intolerance, diabetes, and thrombocytopenia, have been reported [33,34], emphasizing the importance to develop a new drugs. ...
Article
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Age-related macular degeneration (AMD) is a major cause of severe and irreversible vision loss with limited effective therapies. Diminished autophagy and increased oxidative damage caused by ROS in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of AMD, and strategies aimed at enhancing autophagy are likely to protect these cells from oxidative damage. We have previously shown that berberine (BBR), an isoquinoline alkaloid isolated from Chinese herbs, was able to protect human RPE cells from H2O2-induced oxidative damage through AMPK activation. However, the precise mechanisms behind this protective effect remain unclear. Given the essential role of AMPK in autophagy activation, we postulated that BBR may confer protection against H2O2-induced oxidative damage by stimulating AMPK-dependent autophagy. Our results showed that BBR was able to induce autophagy in D407 cells, whereas autophagy inhibitor PIKIII or silencing of LC3B blocked the protective effect of BBR. Further analysis showed that BBR activated the AMPK/mTOR/ULK1 signaling pathways and that both pharmacological and genetic inhibitions of the AMPK pathway abolished the autophagy-stimulating effect of BBR. Similar results were obtained in primary cultured human RPE cells. Taken together, these results demonstrate that BBR is able to stimulate autophagy in D407 cells via the activation of AMPK pathway and that its protective effect against H2O2-induced oxidative damage relies on its autophagy-modulatory effect. Our findings also provide evidence to support the potential application of BBR in preventing and treating AMD.
... fenotiplerinin veya belirli salgı fenotipi aracılarının ekspresyonunu bloke ederek modüle etmek (senomorfik) için yaşlanan hücrelerin seçici ölümünü içerebilir. Bu yaklaşımlara uygun olarak çeşitli in-vivo ve in-vitro çalışmalar yapılmaktadır (43)(44)(45)(46) 4. SONUÇ Hücresel yaşlanma, yaşlanmayla insidansı artan nörodejeneratif hastalıklarda önemli roller üstlenir. Yapılan araştırmalarda, astrositleri, mikrogliaları, oligodendrosit progenitörlerini ve nöral kök hücreleri etkileyen hücresel yaşlanma kanıtları giderek artmaktadır. ...
... fenotiplerinin veya belirli salgı fenotipi aracılarının ekspresyonunu bloke ederek modüle etmek (senomorfik) için yaşlanan hücrelerin seçici ölümünü içerebilir. Bu yaklaşımlara uygun olarak çeşitli in-vivo ve in-vitro çalışmalar yapılmaktadır (43)(44)(45)(46) 4. SONUÇ Hücresel yaşlanma, yaşlanmayla insidansı artan nörodejeneratif hastalıklarda önemli roller üstlenir. Yapılan araştırmalarda, astrositleri, mikrogliaları, oligodendrosit progenitörlerini ve nöral kök hücreleri etkileyen hücresel yaşlanma kanıtları giderek artmaktadır. ...
... In NSCLC, high expression of CircZFR activates the CircZFR/miR-944/LASP1 axis, resulting in DDP resistance in A549 and H1299 cell lines [11]. RAPA is a macrolide that specifically inhibits mTOR [72,73]. The mTOR signaling pathway is a major regulator of cell growth and metabolism, and its abnormal expression can induce a variety of human diseases, such as cancer, diabetes, and neurological diseases [74]. ...
Article
miRNA is a class of endogenous short-chain non-coding RNAs consisting of about 22 nucleotides. miR-944 is located in the fourth intron of the TP63 gene in the 3q28 region. miR-944 is abnormally expressed in cancers in multiple systems including neural, endocrine, respiratory, reproductive, and digestive systems. miR-944 can target at least 27 protein-coding genes. miR-944 can regulate a series of cell behaviors, such as cell cycle, proliferation, invasion and migration, EMT, apoptosis, etc. miR-944 participates in the networks of 11 ceRNAs, including six circRNAs and five lncRNAs. miR-944 is involved in three signaling pathways. The abnormal expression of miR-944 is closely related to the clinicopathological conditions of various cancer patients. Deregulated expression of miR-944 is significantly associated with clinicopathology and prognosis in cancer patients. In addition, miR-944 is also associated with the development of DDP, RAPA, DOX, and PTX resistance in cancer cells. miR-944 is involved in the anticancer molecular mechanisms of matrine and Rhenium-liposome drugs. In conclusion, this work systematically summarizes the related findings of miR-944, which will provide potential hints for follow-up research on miR-944.
... Rapamycin, an FDA-approved immune modulator, effectively suppresses the mTOR pathway and extends lifespan in model organisms (Arriola Apelo and Lamming, 2016). Rapamycin dampens the SASP through inhibition of IL1α, which in turn reduces the transcription of inflammatory genes regulated by NF-κB (Laberge et al., 2015;Mongelli et al., 2020). ...
Article
The pathogenic processes driving Alzheimer's disease (AD) are complex. An incomplete understanding of underlying disease mechanisms has presented insurmountable obstacles for developing effective disease-modifying therapies. Advanced chronological age is the greatest risk factor for developing AD. Intervening on biological aging may alter disease progression and represents a novel, complementary approach to current strategies. Toward this end, cellular senescence has emerged as a promising target. This complex stress response harbors damaged cells in a cell cycle arrested, apoptosis-resistant cell state. Senescent cells accumulate with age where they notoriously secrete molecules that contribute to chronic tissue dysfunction and disease. Thus, benefits of cell survival in a senescent fate are countered by their toxic secretome. The removal of senescent cells improves brain structure and function in rodent models at risk of developing AD, and in those with advanced Aβ and tau pathology. The present review describes the path to translating this promising treatment strategy to AD clinical trials. We review evidence for senescent cell accumulation in the human brain; considerations and strategies for senescence-targeting trials specific to AD; approaches to detect senescent brain cells in biofluids and summarize the goals of the first senolytic trials for the treatment of AD (NCT04063124 and NCT04685590).
... Metformin, which is known to activate AMPK by impairing mitochondrial ATP production, has been shown to cause an anti-aging effect via autophagy induction [168,169]. On the other hand, rapamycin, an mTOR inhibitor that induces autophagy, has also been shown to extend the lifespan of wild-type mice [170][171][172]. Rapamycin also protects against neuronal death when studied using in vitro and in vivo models of neurodegenerative diseases [173,174]. ...
Article
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With an increased life expectancy among humans, aging has recently emerged as a major focus in biomedical research. The lack of in vitro aging models—especially for neurological disorders, where access to human brain tissues is limited—has hampered the progress in studies on human brain aging and various age-associated neurodegenerative diseases at the cellular and molecular level. In this review, we provide an overview of age-related changes in the transcriptome, in signaling pathways, and in relation to epigenetic factors that occur in senescent neurons. Moreover, we explore the current cell models used to study neuronal aging in vitro, including immortalized cell lines, primary neuronal culture, neurons directly converted from fibroblasts (Fib-iNs), and iPSC-derived neurons (iPSC-iNs); we also discuss the advantages and limitations of these models. In addition, the key phenotypes associated with cellular senescence that have been observed by these models are compared. Finally, we focus on the potential of combining human iPSC-iNs with genome editing technology in order to further our understanding of brain aging and neurodegenerative diseases, and discuss the future directions and challenges in the field.
... Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 758763 variety of diseases including an ongoing clinical trial with the mTOR inhibitor, rapamycin in PAH (Houssaini et al., 2013;Arriola Apelo and Lamming, 2016). Prior studies have shown that mTORC1 inhibition with rapamycin attenuates pre-clinical PAH development (Houssaini et al., 2013;Goncharova et al., 2020). ...
Article
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Rationale: Enhanced proliferation and distal migration of human pulmonary arterial smooth muscle cells (hPASMCs) both contribute to the progressive increases in pulmonary vascular remodeling and resistance in pulmonary arterial hypertension (PAH). Our previous studies revealed that Rictor deletion, to disrupt mTOR Complex 2 (mTORC2), over longer periods result in a paradoxical rise in platelet-derived growth factor receptor (PDGFR) expression in PASMCs. Thus, the purpose of this study was to evaluate the role of combination therapy targeting both mTOR signaling with PDGFR inhibition to attenuate the development and progression of PAH. Methods and Results: Immunoblotting analyses revealed that short-term exposure to rapamycin (6h) significantly reduced phosphorylation of p70S6K (mTORC1-specific) in hPASMCs but had no effect on the phosphorylation of AKT (p-AKT S473, considered mTORC2-specific). In contrast, longer rapamycin exposure (>24 h), resulted in differential AKT (T308) and AKT (S473) phosphorylation with increases in phosphorylation of AKT at T308 and decreased phosphorylation at S473. Phosphorylation of both PDGFRα and PDGFRβ was increased in hPASMCs after treatment with rapamycin for 48 and 72 h. Based on co-immunoprecipitation studies, longer exposure to rapamycin (24–72 h) significantly inhibited the binding of mTOR to Rictor, mechanistically suggesting mTORC2 inhibition by rapamycin. Combined exposure of rapamycin with the PDGFR inhibitor, imatinib significantly reduced the proliferation and migration of hPASMCs compared to either agent alone. Pre-clinical studies validated increased therapeutic efficacy of rapamycin combined with imatinib in attenuating PAH over either drug alone. Specifically, combination therapy further attenuated the development of monocrotaline (MCT)- or Hypoxia/Sugen-induced pulmonary hypertension (PH) in rats as demonstrated by further reductions in the Fulton index, right ventricular systolic pressure (RVSP), pulmonary vascular wall thickness and vessel muscularization, and decreased proliferating cell nuclear antigen (PCNA) staining in PASMCs. Conclusion: Prolonged rapamycin treatment activates PDGFR signaling, in part, via mTORC2 inhibition. Combination therapy with rapamycin and imatinib may be a more effective strategy for the treatment of PAH.
... Importantly, we have also identified Pol III as a downstream effector of TORC1 (Filer et al., 2017). Rapamycin is a macrolide compound which inhibits activity of the TOR kinase and rapamycin treatment extends lifespan in a range of organisms (Bjedov et al., 2010;Arriola Apelo and Lamming, 2016), with rapamycin treatment in flies decreasing pre-tRNAs in both whole flies and within the intestine (Filer et al., 2017). Indeed, we found that whilst both rapamycin and intestine-or ISC-specific Pol III knockdown extended lifespan to a similar extent, these treatments were not additive (Filer et al., 2017). ...
Article
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Transcription in eukaryotic cells is performed by three RNA polymerases. RNA polymerase I synthesises most rRNAs, whilst RNA polymerase II transcribes all mRNAs and many non-coding RNAs. The largest of the three polymerases is RNA polymerase III (Pol III) which transcribes a variety of short non-coding RNAs including tRNAs and the 5S rRNA, in addition to other small RNAs such as snRNAs, snoRNAs, SINEs, 7SL RNA, Y RNA, and U6 spilceosomal RNA. Pol III-mediated transcription is highly dynamic and regulated in response to changes in cell growth, cell proliferation and stress. Pol III-generated transcripts are involved in a wide variety of cellular processes, including translation, genome and transcriptome regulation and RNA processing, with Pol III dys-regulation implicated in diseases including leukodystrophy, Alzheimer’s, Fragile X-syndrome and various cancers. More recently, Pol III was identified as an evolutionarily conserved determinant of organismal lifespan acting downstream of mTORC1. Pol III inhibition extends lifespan in yeast, worms and flies, and in worms and flies acts from the intestine and intestinal stem cells respectively to achieve this. Intriguingly, Pol III activation achieved through impairment of its master repressor, Maf1, has also been shown to promote longevity in model organisms, including mice. In this review we introduce the Pol III transcription apparatus and review the current understanding of RNA Pol III’s role in ageing and lifespan in different model organisms. We then discuss the potential of Pol III as a therapeutic target to improve age-related health in humans.
... Rapamycin is a macrolide antibiotic that is produced by Streptomyces hygroscopicus, a soil bacterium of the family Actinomycetaceae that was isolated on Easter Island [9]. Rapamycin has been shown to also have immunosuppressive activity, and therefore, it is now used to prevent organ transplant rejection. ...
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Background Although retinitis pigmentosa (RP) is most frequently studied in mouse models, rats, rabbits, and pigs are also used as animal models of RP. However, no studies have reported postnatal photoreceptor cell loss before complete development in these models. Here, we generated a transgenic rat strain, named the P347L rat, in which proline at position 347 in the rhodopsin protein was replaced with leucine. Results A pathological analysis of photoreceptor cells in the P347L rat model was performed, and drugs with potential use as therapeutic agents against RP were investigated. The data clearly showed rapid degeneration and elimination of the outer nuclear layer even before the photoreceptor cells were fully established in P347L rats. To test the usefulness of the P347L rat in the search for new therapeutic agents against RP, the effects of rapamycin on RP were investigated in this rat strain. The findings suggest that rapamycin promotes autophagy and autophagosomal uptake of the rhodopsin that has accumulated abnormally in the cytoplasm, thereby alleviating stress and delaying photoreceptor cell death. Conclusions In this RP model, the time to onset of retinal degeneration was less than that of previously reported RP models with other rhodopsin mutations, enabling quicker in vivo evaluation of drug efficacy. Administration of rapamycin delayed the photoreceptor cell degeneration by approximately 1 day.
... 6 Rapamycin, implicated as an mTORC1 inhibitor, inhibits the proliferation of several cell types including lymphocytes, vascular smooth muscle cells, and endothelial cells. 7 Our preliminary results showed that rapamycin could inhibit mesangial cell proliferation and ameliorate renal fibrosis and hypofunction in a model of rat IgA nephropathy. 8 However, many clinical and experimental studies have shown that rapamycin and its analogs activate Akt and ERK1/2. ...
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Glomerular mesangial cell (MC) proliferation is one of the causative factors of glomerular diseases and one of their prominent pathological features. Rapamycin can inhibit MC proliferation and slow the progression to chronic renal fibrosis. The present study was designed to observe the role of rapamycin in MC proliferation and to explore the mechanism by which rapamycin acts on Akt and MAPK/ERK1/2 pathways in mesangial cells. MTT assay and flow cytometry were used to evaluate the proliferation and the cell cycle phase of glomerular mesangial cells respectively. The mRNA expression level of p70S6K was detected by RT-qPCR. Western blotting was performed to determine p70S6K, PI3K/Akt, and PI3K/MAPK protein expression. We found that rapamycin could reduce mesangial cell proliferation and arrest the cell cycle in the G1 phase, however the inhibition effect of 1000 nmol/L rapamycin was not higher than that in the 100 nmol/L group. The results of western blotting showed that 1000 nmol/L rapamycin more significantly inhibited the phosphorylation of p70S6K than 100 nmol/L, suggesting there should be another signaling pathway that activates the proliferation of MCs. Moreover, our results revealed that 1000 nmol/L rapamycin led to Raf1-MEK1/2-ERK pathway activation through a p70S6K-PI3K-mediated feedback loop in MCs. This study demonstrated that high-dose rapamycin leads to ERK1/2 activation through a p70S6K/PI3K/MAPK feedback loop in rat MCs, thus reducing the inhibitory effect of rapamycin on MC proliferation.
... The initial studies on mTOR were conducted in invertebrates (reviewed in 65 ), but in 2009 it was found that rapamycin extends lifespan in aged mice 66 . Since then several independent laboratories have confirmed the ability of rapamycin to prolong lifespan in multiple strains of mice, even when treatment is intermittent or conducted for only a short period 67,68 (reviewed in 69 ). As summarized in Supplementary information 2, rapamycin and DR have similar positive effects on many age-related diseases in mice, and in a recent small study, rapamycin even improved diastolic and systolic cardiac function in middle-aged dogs 70 . ...
Article
Dietary restriction with adequate nutrition is the gold standard for delaying ageing and extending healthspan and lifespan in diverse species, including rodents and non-human primates. In this Review, we discuss the effects of dietary restriction in these mammalian model organisms and discuss accumulating data that suggest that dietary restriction results in many of the same physiological, metabolic and molecular changes responsible for the prevention of multiple ageing-associated diseases in humans. We further discuss how different forms of fasting, protein restriction and specific reductions in the levels of essential amino acids such as methionine and the branched-chain amino acids selectively impact the activity of AKT, FOXO, mTOR, nicotinamide adenine dinucleotide (NAD+), AMP-activated protein kinase (AMPK) and fibroblast growth factor 21 (FGF21), which are key components of some of the most important nutrient-sensing geroprotective signalling pathways that promote healthy longevity. Dietary restriction in rodents and non-human primates affects key nutrient-sensing signalling pathways to increase healthspan and lifespan. This Review discusses these geroprotective mechanisms and recent insights suggesting that dietary restriction results in similar molecular and metabolic changes in humans, contributing to the prevention of ageing-associated diseases.
... August 2021 | Volume 2 | Article 707372 aging effects in model organisms and in humans. Importantly, rapamycin and its analogs are currently being used in the clinic against various cancers and neurological disorders (Benjamin et al., 2011;Li et al., 2014;Arriola Apelo and Lamming, 2016). Rapamycin acts as a specific allosteric inhibitor of mTORC1, the master regulator of cellular and organismal physiology. ...
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The mechanistic Target of Rapamycin (mTOR) is a growth-related kinase that, in the context of the mTOR complex 1 (mTORC1), touches upon most fundamental cellular processes. Consequently, its activity is a critical determinant for cellular and organismal physiology, while its dysregulation is commonly linked to human aging and age-related disease. Presumably the most important stimulus that regulates mTORC1 activity is nutrient sufficiency, whereby amino acids play a predominant role. In fact, mTORC1 functions as a molecular sensor for amino acids, linking the cellular demand to the nutritional supply. Notably, dietary restriction (DR), a nutritional regimen that has been shown to extend lifespan and improve healthspan in a broad spectrum of organisms, works via limiting nutrient uptake and changes in mTORC1 activity. Furthermore, pharmacological inhibition of mTORC1, using rapamycin or its analogs (rapalogs), can mimic the pro-longevity effects of DR. Conversely, nutritional amino acid overload has been tightly linked to aging and diseases, such as cancer, type 2 diabetes and obesity. Similar effects can also be recapitulated by mutations in upstream mTORC1 regulators, thus establishing a tight connection between mTORC1 signaling and aging. Although the role of growth factor signaling upstream of mTORC1 in aging has been investigated extensively, the involvement of signaling components participating in the nutrient sensing branch is less well understood. In this review, we provide a comprehensive overview of the molecular and cellular mechanisms that signal nutrient availability to mTORC1, and summarize the role that nutrients, nutrient sensors, and other components of the nutrient sensing machinery play in cellular and organismal aging.
... mTOR complex 1 (mTORC1) and AMPK are now recognized as the two central metabolic sensors, meeting on the surface of the lysosome to determine cellular longevity and youthfulness versus senescence [106,107]. Pharmacologic inhibition of mTORC1 is currently being explored as a therapeutic option to extend lifespan [108]. Increased activity of mTORC1 is a distinguishing feature of CD4 + T cells in both GCA and TAK (Fig. 6) [109], suggesting that these important regulators of granuloma formation have features of senescent T cells. ...
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Two vasculitides, giant cell arteritis (GCA) and Takayasu arteritis (TAK), are recognized as autoimmune and autoinflammatory diseases that manifest exclusively within the aorta and its large branches. In both entities, the age of the affected host is a critical risk factor. TAK manifests during the 2nd–4th decade of life, occurring while the immune system is at its height of performance. GCA is a disease of older individuals, with infrequent cases during the 6th decade and peak incidence during the 8th decade of life. In both vasculitides, macrophages and T cells infiltrate into the adventitia and media of affected vessels, induce granulomatous inflammation, cause vessel wall destruction, and reprogram vascular cells to drive adventitial and neointimal expansion. In GCA, abnormal immunity originates in an aged immune system and evolves within the aged vascular microenvironment. One hallmark of the aging immune system is the preferential loss of CD8 ⁺ T cell function. Accordingly, in GCA but not in TAK, CD8 ⁺ effector T cells play a negligible role and anti-inflammatory CD8 ⁺ T regulatory cells are selectively impaired. Here, we review current evidence of how the process of immunosenescence impacts the risk for GCA and how fundamental differences in the age of the immune system translate into differences in the granulomatous immunopathology of TAK versus GCA.
... Wu et al. found that metformin may fight cancer and aging by restricting transit of RagC GTPase through the nuclear pore complex [51,52]. Rapamycin is able to inhibit aging by inhibiting mTOR but has some negative side effects [53,54]. Julie Chao et al. revealed that kallistatin regulates aging and cancer by affecting the expression levels of miR-34a and miR-21 [55]. ...
Article
Aging has a significant role in the proliferation and development of cancers. This study explored the expression profiles, prognostic value, and potential roles of aging-related genes in gliomas. We designed risk score and cluster models based on aging-related genes and glioma cases using LASSO Cox regression analysis, consensus clustering analysis and univariate cox regression analyses. High risk score was related to malignant clinical features and poor prognosis based on 10 datasets, 2953 cases altogether. Genetic alterations analysis revealed that high risk scores were associated with genomic aberrations of aging-related oncogenes. GSVA analysis exhibited the potential function of the aging-related genes. More immune cell infiltration was found in high-risk group cases, and glioma patients in high-risk group may be more responsive to immunotherapy. Knock-down of CTSC, an aging-related gene, can inhibit cell cycle progression, colony formation, cell proliferation and increase cell senescence in glioma cell lines in vitro. Indeed, high expression of CTSC was associated with poor prognosis in glioma cases. In conclusion, this study revealed that aging-related genes have prognostic potential for glioma patients and further identified potential mechanisms for aging-related genes in tumorigenesis and progression in gliomas.
... It is utilized as an immunosuppressant after organ transplantation. Current studies explore potential effects for different types of cancer (122,123). For neurodegenerative diseases, rapamycin is also a potential candidate after preclinical studies showed neuroprotective effects in animals with decreased dopaminergic cell death in the substantia nigra in a model of Parkinson's disease and improvement of cognitive deficits in models of Alzheimer's dementia (124). ...
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Neurodegeneration with Brain Iron Accumulation (NBIA) is a heterogeneous group of progressive neurodegenerative diseases characterized by iron deposition in the globus pallidus and the substantia nigra. As of today, 15 distinct monogenetic disease entities have been identified. The four most common forms are pantothenate kinase-associated neurodegeneration (PKAN), phospholipase A2 group VI (PLA2G6)-associated neurodegeneration (PLAN), beta-propeller protein-associated neurodegeneration (BPAN) and mitochondrial membrane protein-associated neurodegeneration (MPAN). Neurodegeneration with Brain Iron Accumulation disorders present with a wide spectrum of clinical symptoms such as movement disorder signs (dystonia, parkinsonism, chorea), pyramidal involvement (e.g., spasticity), speech disorders, cognitive decline, psychomotor retardation, and ocular abnormalities. Treatment remains largely symptomatic but new drugs are in the pipeline. In this review, we discuss the rationale of new compounds, summarize results from clinical trials, provide an overview of important results in cell lines and animal models and discuss the future development of disease-modifying therapies for NBIA disorders. A general mechanistic approach for treatment of NBIA disorders is with iron chelators which bind and remove iron. Few studies investigated the effect of deferiprone in PKAN, including a recent placebo-controlled double-blind multicenter trial, demonstrating radiological improvement with reduction of iron load in the basal ganglia and a trend to slowing of disease progression. Disease-modifying strategies address the specific metabolic pathways of the affected enzyme. Such tailor-made approaches include provision of an alternative substrate (e.g., fosmetpantotenate or 4′-phosphopantetheine for PKAN) in order to bypass the defective enzyme. A recent randomized controlled trial of fosmetpantotenate, however, did not show any significant benefit of the drug as compared to placebo, leading to early termination of the trials' extension phase. 4′-phosphopantetheine showed promising results in animal models and a clinical study in patients is currently underway. Another approach is the activation of other enzyme isoforms using small molecules (e.g., PZ-2891 in PKAN). There are also compounds which counteract downstream cellular effects. For example, deuterated polyunsaturated fatty acids (D-PUFA) may reduce mitochondrial lipid peroxidation in PLAN. In infantile neuroaxonal dystrophy (a subtype of PLAN), desipramine may be repurposed as it blocks ceramide accumulation. Gene replacement therapy is still in a preclinical stage.
... SIRT1 is considered as aging-related because it was found to play an important role in improving oxidative stress resistance of cells and inhibiting cell death [49], and hence is involved in many age-related diseases [50]. mTOR is considered aging-related because it functions as a sensor of intracellular energy and a central regulator of biological processes, including aging [51,52]. • PI3K-AKT pathway is considered as aging-related because it was identified as the signaling pathway of several aging-related diseases: diabetic encephalopathy [53] and cancer [54]. ...
Preprint
Identification of human genes involved in the aging process is critical due to the incidence of many diseases with age. A state-of-the-art approach for this purpose infers a weighted dynamic aging-specific subnetwork by mapping gene expression (GE) levels at different ages onto the protein-protein interaction network (PPIN). Then, it analyzes this subnetwork in a supervised manner by training a predictive model to learn how network topologies of known aging- vs. non-aging-related genes change across ages. Finally, it uses the trained model to predict novel aging-related genes. However, the best current subnetwork resulting from this approach still yields suboptimal prediction accuracy. This could be because it was inferred using outdated GE and PPIN data. Here, we evaluate whether analyzing a weighted dynamic aging-specific subnetwork inferred from newer GE and PPIN data improves prediction accuracy upon analyzing the best current subnetwork inferred from outdated data. Unexpectedly, we find that not to be the case. To understand this, we perform aging-related pathway and Gene Ontology (GO) term enrichment analyses. We find that the suboptimal prediction accuracy, regardless of which GE or PPIN data is used, may be caused by the current knowledge about which genes are aging-related being incomplete, or by the current methods for inferring or analyzing an aging-specific subnetwork being unable to capture all of the aging-related knowledge. These findings can potentially guide future directions towards improving supervised prediction of aging-related genes via -omics data integration.
... Besides enhancing autophagy, mTORC1 inhibition blocks protein and nucleotide synthesis, inhibits cell proliferation, and inhibits metabolism [170]. Longterm rapamycin administration may cause immunosuppression, and glucose intolerance due to mTORC1 is acutely sensitive to rapamycin whereas mTORC2 chronically sensitive to rapamycin in vivo [171]. In addition, recent studies mainly focused on the effect of autophagy regulation on neuronal cell damage but less on cell growth and secretion in ischemic stroke. ...
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Stroke is one of the leading causes of death and disability worldwide. Autophagy is a conserved cellular catabolic pathway that maintains cellular homeostasis by removal of damaged proteins and organelles, which is critical for the maintenance of energy and function homeostasis of cells. Accumulating evidence demonstrates that autophagy plays important roles in pathophysiological mechanisms under ischemic stroke. Previous investigations show that autophagy serves as a “double-edged sword” in ischemic stroke as it can either promote the survival of neuronal cells or induce cell death in special conditions. Following ischemic stroke, autophagy is activated or inhibited in several cell types in brain, including neurons, astrocytes, and microglia, as well as microvascular endothelial cells, which involves in inflammatory activation, modulation of microglial phenotypes, and blood-brain barrier permeability. However, the exact mechanisms of underlying the role of autophagy in ischemic stroke are not fully understood. This review focuses on the recent advances regarding potential molecular mechanisms of autophagy in different cell types. The focus is also on discussing the “double-edged sword” effect of autophagy in ischemic stroke and its possible underlying mechanisms. In addition, potential therapeutic strategies for ischemic stroke targeting autophagy are also reviewed.
... Rapamycin is another potential drug that could target ageing. Discovered in the soil on Easter Island more than half a century ago [25], initial studies identified its capacity to inhibit cancer cell proliferation in mouse models, while parallel studies explored the potential of rapamycin as an immunosuppressant for organ transplants [26]. But more recent experiments have found that consuming rapamycin can extend lifespan, including in mammals. ...
Article
The year 2021 marks the 50th anniversary of the National Cancer Act of 1971 and President Richard Nixon’s declaration of a “war on cancer”. In 1971 cancer was the second leading cause of death in the USA, and today it is still the second leading cause of death with an estimated 606,520 Americans dying of cancer in the year 2020. The half a century campaign to eliminate cancer reveals at least two important public health lessons that must be heeded for the next 50 years of the war against the disease—(1) recognizing the limits of behaviour control and (2) recognizing the significance of rate (of ageing) control. These two lessons result in a somewhat paradoxical conclusion in that we must have both humility and ambition in our attitudes towards future preventative medicine for the world’s ageing populations. Geroscience must become an integral part of public health if serious headway is to be made preventing not only cancer but most of the other chronic conditions of late life.
... However, this has the adverse effect of increasing the risk of type 2 diabetes (165). The effect of sirolimus is complex, even though many studies have indicated that sirolimus can enhance mouse lifespan in a sexspecific manner (166). Temsirolimus, a derivative and prodrug of sirolimus, is converted to sirolimus (rapamycin) in vivo. ...
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Glioblastoma is the most common primary malignant tumor in the brain and has a dismal prognosis despite patients accepting standard therapies. Alternation of genes and deregulation of proteins, such as receptor tyrosine kinase, PI3K/Akt, PKC, Ras/Raf/MEK, histone deacetylases, poly (ADP-ribose) polymerase (PARP), CDK4/6, branched-chain amino acid transaminase 1 (BCAT1), and Isocitrate dehydrogenase (IDH), play pivotal roles in the pathogenesis and progression of glioma. Simultaneously, the abnormalities change the cellular biological behavior and microenvironment of tumor cells. The differences between tumor cells and normal tissue become the vulnerability of tumor, which can be taken advantage of using targeted therapies. Small molecule inhibitors, as an important part of modern treatment for cancers, have shown significant efficacy in hematologic cancers and some solid tumors. To date, in glioblastoma, there have been more than 200 clinical trials completed or ongoing in which trial designers used small molecules as monotherapy or combination regimens to correct the abnormalities. In this review, we summarize the dysfunctional molecular mechanisms and highlight the outcomes of relevant clinical trials associated with small-molecule targeted therapies. Based on the outcomes, the main findings were that small-molecule inhibitors did not bring more benefit to newly diagnosed glioblastoma, but the clinical studies involving progressive glioblastoma usually claimed “noninferiority” compared with historical results. However, as to the clinical inferiority trial, similar dosing regimens should be avoided in future clinical trials.
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Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. Here we show that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally, through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organization, induces intestinal autophagy through transcriptional regulation, prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription , and alters chromatin organisation in the small intestine, suggesting the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.
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The battle between pathogens and hosts is an ongoing phenomenon that has been studied for centuries. Although multicellular organisms have developed sophisticated immune systems to defend against pathogens, pathogens are often able to successfully infect their hosts. To enhance their chance of survival, pathogens can hijack the host's metabolism, which serves as a critical control point that can potentially impact infections. Mammalian target of rapamycin (mTOR) is the nutrient sensor and central regulator of metabolism in innate and adaptive immune cells. Rapamycin, a drug targeting the mTOR pathway, has been widely used in clinical practice for decades and is relatively safe. However, some unexpected infections occur after long-term administration of rapamycin, indicating the presence of a close relationship between mTOR signaling and the infection process. In this manuscript, we briefly review the mTOR pathway and the current clinical use of rapamycin; we also summarize the role of the mTOR pathway in immune cell death, including apoptosis, necroptosis, pyroptosis, and ferroptosis, and its impact on anti-infection immunity. Finally, potential application of mTOR inhibitor in coronavirus disease 2019 clinical therapy is discussed.
Preprint
Inhibition of mTORC1 (mechanistic Target Of Rapamycin Complex 1) signaling promotes health and longevity in diverse model organisms. Over the past decade, excitement has built over the possibility that treatment with the mTORC1 inhibitor rapamycin can be utilized to treat or prevent age-related disease in humans. However, concerns over the side effects of rapamycin on immunity and metabolism have precluded the routine use of rapamycin as a geroprotective therapy. Here, we discuss the evidence that these negative side effects of rapamycin are largely mediated by off-target inhibition of a second mTOR Complex (mTORC2). Further, we discuss how intermittent treatment with rapamycin, specific dietary regimens, and new molecules may provide routes to the safer and more selective inhibition of mTORC1. We conclude that the time is ripe for the development of therapies based on the safe and selective inhibition of mTORC1 for the treatment or prevention of diseases of aging.
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The mammalian target of rapamycin complex 1 (mTORC1) is a crucial regulator of adipogenesis and systemic energy metabolism. Its dysregulation leads to a diversity of metabolic diseases, including obesity and type 2 diabetes. DEP-domain containing 5 (DEPDC5) is a critical component of GATOR1 complex that functions as a key inhibitor of mTORC1. So far, its function in adipose tissue remains largely unknown. Herein we evaluated how persistent mTORC1 activation in adipocyte via Depdc5 knockout modulates adiposity in vivo. Our data indicated that adipocyte-specific knockout of Depdc5 in aged mice led to reduced visceral fat, aggravated insulin resistance and enhanced adipose tissue inflammation. Moreover, we found that Depdc5 ablation resulted in upregulation of adipose triglyceride lipase (ATGL) in adipocytes and elevated levels of serum free fatty acids (FFAs). Intriguingly, rapamycin treatment did not reverse insulin resistance but alleviated adipose tissue inflammation caused by Depdc5 deletion. Taken together, our findings revealed that mTORC1 activation caused by Depdc5 deletion promotes lipolysis process and further exacerbates insulin resistance and adipose tissue inflammation in mice.
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Objective: This study investigates the synergistic effects of Gleevec (imatinib) and rapamycin on the proliferative and angiogenic properties of mouse bone marrow-derived endothelial progenitor cells (EPCs). Materials and methods: EPCs were isolated from mouse bone marrow and treated with different concentrations of Gleevec or rapamycin individually or in combination. The cell viability and proliferation were examined using the MTT assay. An analysis of cell cycle and apoptosis was performed using flow cytometry. Formation of capillary-like tubes was examined in vitro, and the protein expression of cell differentiation markers was determined using Western blot analysis. Results: Gleevec significantly reduced cell viability, cell proliferation, and induced cell apoptosis in EPCs. Rapamycin had similar effects on EPCs, but it did not induce cell apoptosis. The combination of Gleevec and rapamycin reduced the cell proliferation but increased cell apoptosis. Although rapamycin had no demonstratable effect on tube formation, the combined therapy of Gleevec and rapamycin significantly reduced tube formation when compared with Gleevec alone. Mechanistically, Gleevec, but not rapamycin, induced a significant elevation in caspase-3 activity in EPCs, and it attenuated the expression of the endothelial protein marker platelet-derived growth factor receptor α. Functionally, rapamycin, but not Gleevec, significantly enhanced the expression of endothelial differentiation marker proteins, while attenuating the expression of mammalian target of rapamycin signaling-related proteins. Conclusions: Gleevec and rapamycin synergistically suppress cell proliferation and tube formation of EPCs by inducing cell apoptosis and endothelial differentiation. Mechanistically, it is likely that rapamycin enhances the proapoptotic and antiangiogenic effects of Gleevec by promoting the endothelial differentiation of EPCs. Given that EPCs are involved in the pathogenesis of some cardiovascular diseases and critical to angiogenesis, pharmacological inhibition of EPC proliferation by combined Gleevec and rapamycin therapy may be a promising approach for suppressing cardiovascular disease pathologies associated with angiogenesis.
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Recent epidemiological studies in Sweden, a country with traditionally high milk consumption, revealed that the intake of non-fermented pasteurized milk increased all-cause mortality in a dose-dependent manner. In contrast, the majority of epidemiological and clinical studies report beneficial health effects of fermented milk products, especially of yogurt. It is the intention of this review to delineate potential molecular aging mechanisms related to the intake of non-fermented milk versus yogurt on the basis of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Non-fermented pasteurized milk via its high bioavailability of insulinotropic branched-chain amino acids (BCAAs), abundance of lactose (glucosyl-galactose) and bioactive exosomal microRNAs (miRs) enhances mTORC1 signaling, which shortens lifespan and increases all-cause mortality. In contrast, fermentation-associated lactic acid bacteria metabolize BCAAs and degrade galactose and milk exosomes including their mTORC1-activating microRNAs. The Industrial Revolution, with the introduction of pasteurization and refrigeration of milk, restricted the action of beneficial milk-fermenting bacteria, which degrade milk´s BCAAs, galactose and bioactive miRs that synergistically activate mTORC1. This unrecognized behavior change in humans after the Neolithic revolution increased aging-related over-activation of mTORC1 signaling in humans, who persistently consume large quantities of non-fermented pasteurized cow´s milk, a potential risk factor for aging and all-cause mortality.
Chapter
Modern medicine takes advantage of sophisticated knowledge of physiological and pathological mechanisms and uses equally stylish methodologies, instruments, techniques and drugs.
Chapter
Aging is a biological process that affects cells, organs and organisms, mainly characterized by a time-related deterioration of the physiological functions necessary for fertility and survival. Aging affects all the individuals of a species at variable rates and is unavoidable. Therefore, it is important to elucidate the mechanisms underlying this process. When lay audiences refer to aging, they usually refer to senior people and the highlighted features are typically: a decline in motor abilities, cognitive performance and “external” visual signs of age, such as wrinkles. However, aging is much more complex than that and engages the accumulation of detrimental modifications in deoxyribonucleic acid (DNA), proteins and lipids, as well as the appearance of cellular protein aggregates and lysosomal lipofuscin, a specific hallmark of senescent, post-mitotic cells. Generally, this process is accompanied by a progressive decrease in the capacity to properly react to stress conditions, which occurs in association with the homeostatic failure and accumulation of molecular damage.
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Autophagy, an essential cellular process that mediates degradation of proteins and organelles in lysosomes, has been tightly linked to cellular quality control for its role as part of the proteostasis network. The current interest in identifying the cellular and molecular determinants of aging, has highlighted the important contribution of malfunctioning of autophagy with age to the loss of proteostasis that characterizes all old organisms. However, the diversity of cellular functions of the different types of autophagy and the often reciprocal interactions of autophagy with other determinants of aging, is placing autophagy at the center of the aging process. In this work, we summarize evidence for the contribution of autophagy to health- and lifespan and provide examples of the bidirectional interplay between autophagic pathways and several of the so-called hallmarks of aging. This central role of autophagy in aging, and the dependence on autophagy of many geroprotective interventions, has motivated a search for direct modulators of autophagy that could be used to slow aging and extend healthspan. Here, we review some of those ongoing therapeutic efforts and comment on the potential of targeting autophagy in aging.
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Alzheimer's disease (AD) is one of the biggest human health threats due to increases in aging of the global population. Unfortunately, drugs for treating AD have been largely ineffective. Interestingly, downregulation of macroautophagy (autophagy) plays an essential role in AD pathogenesis. Therefore, targeting autophagy has drawn considerable attention as a therapeutic approach for the treatment of AD. However, developing new therapeutics is time-consuming and requires huge investments. One of the strategies currently under consideration for many diseases is “drug repositioning” or “drug repurposing”. In this comprehensive review, we have provided an overview of the impact of autophagy on AD pathophysiology, reviewed the therapeutics that upregulate autophagy and are currently used in the treatment of other diseases, including cancers, and evaluated their repurposing as a possible treatment option for AD. In addition, we discussed the potential of applying nano-drug delivery to neurodegenerative diseases, such as AD, to overcome the challenge of crossing the blood brain barrier and specifically target molecules/pathways of interest with minimal side effects.
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Rapamycin treatment has positive and negative effects on progression of type 2 diabetes (T2D) in a recombinant inbred polygenic mouse model, male NONcNZO10/LtJ (NcZ10). Here, we show that combination treatment with metformin ameliorates negative effects of rapamycin while maintaining its benefits. From 12 to 30 weeks of age, NcZ10 males were fed a control diet or diets supplemented with rapamycin, metformin, or a combination of both. Rapamycin alone reduced weight gain, adiposity, HOMA-IR, and inflammation, and prevented hyperinsulinemia and pre-steatotic hepatic lipidosis, but exacerbated hyperglycemia, hypertriglyceridemia, and pancreatic islet degranulation. Metformin alone reduced hyperinsulinemia and circulating c-reactive protein, but exacerbated nephropathy. Combination treatment retained the benefits of both while preventing many of the deleterious effects. Importantly, the combination treatment reversed effects of rapamycin on markers of hepatic insulin resistance and normalized systemic insulin sensitivity in this inherently insulin-resistant model. In adipose tissue, rapamycin attenuated the expression of genes associated with adipose tissue expansion (Mest, Gpam), inflammation (Itgam, Itgax, Hmox1, Lbp), and cell senescence (Serpine1). In liver, the addition of metformin counteracted rapamycin-induced alterations of G6pc, Ppara, and Ldlr expressions that promote hyperglycemia and hypertriglyceridemia. Both rapamycin and metformin treatment reduced hepatic Fasn expression, potentially preventing lipidosis. These results delineate a state of "insulin signaling restriction" that withdraws endocrine support for further adipogenesis, progression of the metabolic syndrome, and the development of its comorbidities. Our results are relevant for the treatment of T2D, the optimization of current rapamycin-based treatments for posttransplant rejection and various cancers, and for the development of treatments for healthy aging.
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The gut microbiome is undoubtedly a key modulator of human health, which can promote or impair homeostasis throughout life. This is even more relevant in old age, when there is a gradual loss of function in multiple organ systems, related to growth, metabolism, and immunity. Several studies have described changes in the gut microbiome across age groups up to the extreme limits of lifespan, including maladaptations that occur in the context of age-related conditions, such as frailty, neurodegenerative diseases, and cardiometabolic diseases. The gut microbiome can also interact bi-directionally with anti-age-related disease therapies, being affected and in turn influencing their efficacy. In this framework, the development of integrated microbiome-based intervention strategies, aimed at favoring a eubiotic configuration and trajectory, could therefore represent an innovative approach for the promotion of healthy aging and the achievement of longevity.
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Ageing is accompanied by alterations in several biochemical processes, highly influenced by its environment. It is controlled by the interactions at various levels of biological hierarchy. To maintain homeostasis, a number of nutrient sensors respond to the nutritional status of the cell and control its energy metabolism. Mitochondrial physiology is influenced by the energy status of the cell. The alterations in mitochondrial physiology and the network of nutrient sensors result in mitochondrial damage leading to age related metabolic degeneration and diseases. Calorie restriction (CR) has proved to be as the most successful intervention to achieve the goal of longevity and healthspan. CR elicits a hormetic response and regulates metabolism by modulating these networks. In this review, the authors summarize the interdependent relationship between mitochondrial physiology and nutrient sensors during the ageing process and their role in regulating metabolism.
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Although aging is the biggest risk factor for human chronic (cancer, diabetic, cardiovascular, and neurodegenerative) diseases, few interventions are known besides caloric restriction and a small number of drugs (with substantial side effects) that directly address aging. Thus, there is an urgent need for new options that can generally delay aging processes and prevent age-related diseases. Cellular aging is at the basis of aging processes. Chronological lifespan (CLS) of yeast Saccharomyces cerevisiae is the well-established model system for investigating the interventions of human post-mitotic cellular aging. CLS is defined as the number of days cells remain viable in a stationary phase. We developed a new, cheap, and fast quantitative method for measuring CLS in cell cultures incubated together with various chemical agents and controls on 96-well plates. Our PICLS protocol with (1) the use of propidium iodide for fluorescent-based cell survival reading in a microplate reader and (2) total cell count measurement via OD600nm absorption from the same plate provides real high-throughput capacity. Depending on logistics, large numbers of plates can be processed in parallel so that the screening of thousands of compounds becomes feasible in a short time. The method was validated by measuring the effect of rapamycin and calorie restriction on yeast CLS. We utilized this approach for chemical agent screening. We discovered the anti-aging/geroprotective potential of 2,5-anhydro-D-mannitol (2,5-AM) and suggest its usage individually or in combination with other anti-aging interventions.
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Human longevity has increased dramatically during the past century. More than 20% of the 9 billion population of the world will exceed the age of 60 in 2050. Since the last three decades, some interventions and many preclinical studies have been found to show slowing aging and increasing the healthy lifespan of organisms from yeast, flies, rodents to nonhuman primates. The interventions are classified into two groups: lifestyle modifications and pharmacological/genetic manipulations. Some genetic pathways have been characterized to have a specific role in controlling aging and lifespan. Thus, all genes in the pathways are potential antiaging targets. Currently, many antiaging compounds target the calorie-restriction mimetic, autophagy induction, and putative enhancement of cell regeneration, epigenetic modulation of gene activity such as inhibition of histone deacetylases and DNA methyltransferases, are under development. It appears evident that the exploration of new targets for these antiaging agents based on biogerontological research provides an incredible opportunity for the healthcare and pharmaceutical industries. The present review focus on the properties of slow aging and healthy life span extension of natural products from various biological resources, endogenous substances, drugs, and synthetic compounds, as well as the mechanisms of targets for antiaging evaluation. These bioactive compounds that could benefit healthy aging and the potential role of life span extension are discussed.
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Adult neurogenesis, the process by which neurons are generated in certain areas of the adult brain, declines in an age-dependent manner and is one potential target for extending cognitive healthspan. Aging is a major risk factor for neurodegenerative diseases and, as lifespans are increasing, these health challenges are becoming more prevalent. An age-associated loss in neural stem cell number and/or activity could cause this decline in brain function, so interventions that reverse aging in stem cells might increase the human cognitive healthspan. In this review, we describe the involvement of adult neurogenesis in neurodegenerative diseases and address the molecular mechanistic aspects of neurogenesis that involve some of the key aggregation-prone proteins in the brain (i.e., tau, Aβ, α-synuclein, …). We summarize the research pertaining to interventions that increase neurogenesis and regulate known targets in aging research, such as mTOR and sirtuins. Lastly, we share our outlook on restoring the levels of neurogenesis to physiological levels in elderly individuals and those with neurodegeneration. We suggest that modulating neurogenesis represents a potential target for interventions that could help in the fight against neurodegeneration and cognitive decline.
Chapter
The development of therapeutic strategies aimed at the aging process of cells has attracted increasing attention in recent decades due to the involvement of this process in the development of many chronic and age-related diseases. Interestingly, preclinical studies have shown the success of a number of anti-aging approaches in the treatment of a range of chronic diseases. These approaches are directed against aging processes such as oxidative stress, telomerase shortening, inflammation, and deficient autophagy. Many strategies has been shown to be effective in delaying aging, including antiaging strategies based on establishing healthy lifestyle habits and pharmacological interventions aimed at disrupting senescent cells and senescent-associated secretory phenotype. Caloric restriction and intermittent fasting were reported to activate autophagy and reduce inflammation. In turn, immune-based strategies, senolytic agents, and senomorphics mediate their effects either by eliminating senescent cells through inducing apoptosis or by disrupting pathways by which senescent cells mediate their detrimental effects. In addition, given the association of the decline in the regenerative potential of stem cells with aging, many experimental and clinical studies indicate the effectiveness of stem cell transplantation in preventing or slowing the progress of age-related diseases by enhancing the repairing mechanisms and the secretion of many growth factors and cytokines.
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Nutrition affects all physiological processes including those linked to the development and function of our immune system. Here, we discuss recent evidence and emerging concepts supporting the idea that our newfound relationship with nutrition in industrialized countries has fundamentally altered the way in which our immune system is wired. This will be examined through the lens of studies showing that mild or transient reductions in dietary intake can enhance protective immunity while also limiting aberrant inflammatory responses. We will further discuss how trade-offs and priorities begin to emerge in the context of severe nutritional stress. In those settings, specific immunological functions are heightened to re-enforce processes and tissue sites most critical to survival. Altogether, these examples will emphasize the profound influence nutrition has over the immune system and highlight how a mechanistic exploration of this cross talk could ultimately lead to the design of novel therapeutic approaches that prevent and treat disease.
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Sirolimus is a potent macrolide immunosuppressant, with a long half-life, a narrow therapeutic window, and large individual variations in pharmacokinetics. In this study, an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold-based immunochromatographic strip were developed to establish a rapid and efficient clinical screening of sirolimus. The hapten for sirolimus was synthesized by the oximation reaction and then coupled to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare the immunogen and coating antigen. Based on highly specific monoclonal antibodies (mAbs), the 50% inhibitory concentration (IC50) of the optimized ic-ELISA was 1.54 ng mL-1 and the limit of detection (LOD) was 0.19 ng mL-1 in whole blood samples. Good specificity was revealed by its low cross-reactivity (CR) with several analogs of sirolimus. The visual limit of detection (vLOD) and cut-off values of the strip were 20 and 100 ng mL-1 in whole blood samples, as assessed by the naked eye. Therefore, both methods could be used as potentially rapid monitoring approaches for the detection of sirolimus in human whole blood.
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Rapid insulin release plays an essential role in maintaining blood‐glucose homeostasis in mammalians. Patients diagnosed with type‐I diabetes mellitus experience chronic and remarkably high blood‐sugar levels, and require lifelong insulin injection therapy, so there is a need for more convenient and less invasive insulin delivery systems to increase patients’ compliance and also to enhance their quality of life. Here, an endoplasmic‐reticulum‐localized split sec‐tobacco etch virus protease (TEVp)‐based rapamycin‐actuated protein‐induction device (RAPID) is engineered, which is composed of the rapamycin‐inducible dimerization domains FK506 binding protein (FKBP) and FKBP‐rapamycin binding protein fused with modified split sec‐TEVp components. Insulin accumulation inside the endoplasmic reticulum (ER) is achieved through tagging its C‐terminus with KDEL, an ER‐retention signal, spaced by a TEVp cleavage site. In the presence of rapamycin, the split sec‐TEVp‐based RAPID components dimerize, regain their proteolytic activity, and remove the KDEL retention signal from insulin. This leads to rapid secretion of accumulated insulin from cells within few minutes. Using liver hydrodynamic transfection methodology, it is shown that RAPID quickly restores glucose homeostasis in type‐1‐diabetic (T1DM) mice treated with an oral dose of clinically licensed rapamycin. This rapid‐release technology may become the foundation for other cell‐based therapies requiring instantaneous biopharmaceutical availability.
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The Streptococcus mitis group constitutes a part of the oral flora in humans and has been reported to cause infective endocarditis, brain abscesses, sepsis, pneumonia, and peritonitis. However, the S. mitis group rarely causes meningitis in children. We experienced a case of bacterial meningitis due to the S. mitis group in a 14-year-old girl with Gorham-Stout disease undergoing treatment with sirolimus for skull base osteolysis and cerebrospinal fluid (CSF) leak. Antibiotic treatment was initiated with linezolid and levofloxacin due to allergies against β-lactam antibiotics. On the third treatment day, antibiotics were switched to penicillin G according to CSF culture results, which were positive for penicillin-susceptible S. mitis group. Antibiotic therapy was successfully completed after 14 days without any neurological sequelae. There have apparently been no reports of S. mitis meningitis in pediatric patients with skull base osteolysis and CSF leak as in our case. Our findings suggest that clinicians should be aware of the possibility of S. mitis meningitis for patients with skull base osteolysis and/or CSF leakage.
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Skin aging caused by UV radiation is called photoaging is characterized by skin roughness and dryness accompanied by a significant reduction of dermal collagen. Rapamycin is a macrolide immunosuppressant which has been shown to exhibit “anti-aging” effects in cells and organisms, however, its roles in the skin photoaging remains unclear. Here, we investigate the role of rapamycin and HSP27, which we have previously identified as an inhibitor of UV-induced apoptosis and senescence in HaCat cells, in a UVA-induced photoaging model of primary human dermal fibroblasts (HDFs). Results from senescence-associated beta-galactosidase (SA-β-gal) staining revealed that rapamycin significantly reduced senescence in UVA-treated HDFs. In addition, treatment with rapamycin significantly increased cell autophagy levels, decreased the expression of p53 and phosphorylated HSP27, and reduced genotoxic and oxidative cellular stress levels in UVA-induced HDFs. Knockdown of HSP27 resulted in a significant increase of MMP-1 and MMP-3 as well as a decrease in type I collagen expression. Rapamycin mitigated these effects by activation of the classical TGF-β/Smad signaling pathway and increasing the transcriptional activity of MAPK/AP-1. Taken together, these results suggest that rapamycin may potentially serve as a preventive and therapeutic agent for UVA-induced photoaging of the skin.
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Adipose tissue de novo lipogenesis (DNL) positively influences insulin sensitivity, is reduced in obesity, and predicts insulin resistance. Therefore, elucidating mechanisms controlling adipose tissue DNL could lead to therapies for type 2 diabetes. Here, we report that mechanistic target of rapamycin complex 2 (mTORC2) functions in white adipose tissue (WAT) to control expression of the lipogenic transcription factor ChREBPβ. Conditionally deleting the essential mTORC2 subunit Rictor in mature adipocytes decreases ChREBPβ expression, which reduces DNL in WAT, and impairs hepatic insulin sensitivity. Mechanistically, Rictor/mTORC2 promotes ChREBPβ expression in part by controlling glucose uptake, but without impairing pan-AKT signalling. High-fat diet also rapidly decreases adipose tissue ChREBPβ expression and insulin sensitivity in wild-type mice, and does not further exacerbate insulin resistance in adipose tissue Rictor knockout mice, implicating adipose tissue DNL as an early target in diet-induced insulin resistance. These data suggest mTORC2 functions in WAT as part of an extra-hepatic nutrient-sensing mechanism to control glucose homeostasis.
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Apolipoprotein E ɛ4 allele is a common susceptibility gene for late-onset Alzheimer's disease. Brain vascular and metabolic deficits can occur in cognitively normal apolipoprotein E ɛ4 carriers decades before the onset of Alzheimer's disease. The goal of this study was to determine whether early intervention using rapamycin could restore neurovascular and neurometabolic functions, and thus impede pathological progression of Alzheimer's disease-like symptoms in pre-symptomatic Apolipoprotein E ɛ4 transgenic mice. Using in vivo, multimodal neuroimaging, we found that apolipoprotein E ɛ4 mice treated with rapamycin had restored cerebral blood flow, blood-brain barrier integrity and glucose metabolism, compared to age- and gender-matched wild-type controls. The preserved vasculature and metabolism were associated with amelioration of incipient learning deficits. We also found that rapamycin restored the levels of the proinflammatory cyclophilin A in vasculature, which may contribute to the preservation of cerebrovascular function in the apolipoprotein E ɛ4 transgenics. Our results show that rapamycin improves functional outcomes in this mouse model and may have potential as an effective intervention to block progression of vascular, metabolic and early cognitive deficits in human Apolipoprotein E ɛ4 carriers. As rapamycin is FDA-approved and neuroimaging is readily used in humans, the results of the present study may provide the basis for future Alzheimer's disease intervention studies in human subjects.
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Inhibition of the mTOR (mechanistic Target Of Rapamycin) signaling pathway robustly extends the lifespan of model organisms including mice. The precise molecular mechanisms and physiological effects that underlie the beneficial effects of rapamycin are an exciting area of research. Surprisingly, while some data suggest that mTOR signaling normally increases with age in mice, the effect of age on mTOR signaling has never been comprehensively assessed. Here, we determine the age-associated changes in mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) signaling in the liver, muscle, adipose, and heart of C57BL/6J.Nia mice, the lifespan of which can be extended by rapamycin treatment. We find that the effect of age on several different readouts of mTORC1 and mTORC2 activity varies by tissue and sex in C57BL/6J.Nia mice. Intriguingly, we observed increased mTORC1 activity in the liver and heart tissue of young female mice compared to male mice of the same age. Tissue and substrate-specific results were observed in the livers of HET3 and DBA/2 mouse strains, and in liver, muscle and adipose tissue of F344 rats. Our results demonstrate that aging does not result in increased mTOR signaling in most tissues and suggest that rapamycin does not promote lifespan by reversing or blunting such an effect.
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Rapamycin inhibits the nutrient-sensing TOR pathway and extends life span in a wide range of organisms. Although life-span extension usually differs between the sexes, the reason for this is poorly understood. Because TOR influences growth, rapamycin likely affects life-history traits such as growth and reproduction. Sexes have different life-history strategies, and theory predicts that they will resolve the tradeoffs between growth, reproduction, and life span differently. Specifically, in taxa with female-biased sexual size dimorphism, reduced growth may have smaller effects on male fitness. We investigated the effects of juvenile, adult, or life-long rapamycin treatment on growth, reproduction, life span, and individual fitness in the outcrossing nematode Caenorhabditis remanei. Life-long exposure to rapamycin always resulted in the strongest response, whereas postreproductive exposure did not affect life span. Although rapamycin resulted in longer life span and smaller size in males, male individual fitness was not affected. In contrast, size and fitness were negatively affected in females, whereas life span was only extended under high rapamycin concentrations. Our results support the hypothesis that rapamycin affects key life-history traits in a sex-specific manner. We argue that the fitness cost of life-span extension will be sex specific and propose that the smaller sex generally pay less while enjoying stronger life-span increase.
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Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA-approved drug rapamycin has been shown to promote lifespan and delay age-related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long-term prophylactic use of rapamycin as a therapy for age-related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR-containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA-approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA-approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs.
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Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila.
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Synucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy. We determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches. After 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal. Taken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy.
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The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age-related debilities including increasing antigen-specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T- and B-lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer- and infection-prone mice supporting disease mitigation as a mechanism for mTOR suppression-mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
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Rapamycin extends lifespan and attenuates age-related pathologies in mice when administered through diet at 14 parts per million (PPM). Recently, we reported that daily intraperitoneal injection of rapamycin at 8 mg/kg attenuates mitochondrial disease symptoms and progression in the Ndufs4 knockout mouse model of Leigh Syndrome. Although rapamycin is a widely used pharmaceutical agent dosage has not been rigorously examined and no dose-response profile has been established. Given these observations we sought to determine if increased doses of oral rapamycin would result in more robust impact on mTOR driven parameters. To test this hypothesis, we compared the effects of dietary rapamycin at doses ranging from 14 to 378 PPM on developmental weight in control and Ndufs4 knockout mice and on health and survival in the Ndufs4 knockout model. High dose rapamycin was well tolerated, dramatically reduced weight gain during development, and overcame gender differences. The highest oral dose, approximately 27-times the dose shown to extend murine lifespan, increased survival in Ndufs4 knockout mice similarly to daily rapamycin injection without observable adverse effects. These findings have broad implications for the effective use of rapamycin in murine studies and for the translational potential of rapamycin in the treatment of mitochondrial disease. This data, further supported by a comparison of available literature, suggests that 14 PPM dietary rapamycin is a sub-optimal dose for targeting mTOR systemically in mice. Our findings suggest that the role of mTOR in mammalian biology may be broadly underestimated when determined through treatment with rapamycin at commonly used doses.
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The synthesis of lipids in response to food intake represents a key advantage that allows organisms to survive when energy availability is limited. In mammals, circulating levels of insulin and nutrients, which fluctuate between fasting and feeding, dictate whether lipids are synthesized or catabolized by tissues. The mechanistic target of rapamycin (mTOR), a kinase that is activated by anabolic signals, plays fundamental roles in regulating lipid biosynthesis and metabolism in response to nutrition. The mTOR kinase nucleates two large protein complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Following their activation, these complexes facilitate the accumulation of triglycerides by promoting adipogenesis and lipogenesis and by shutting down catabolic processes such as lipolysis and β-oxidation. Here, we review and discuss the roles of mTOR complexes in various aspects of lipid metabolism in mammals. We also use this opportunity to discuss the implication of these relations to the maintenance of systemic lipid homeostasis.
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Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is a key downstream effector of mTOR complex 1 (mTORC1) that represses cap-dependent mRNA translation initiation by sequestering the translation initiation factor eIF4E. Reduced mTORC1 signaling is associated with life span extension and improved metabolic homeostasis, yet the downstream targets that mediate these benefits are unclear. Here, we demonstrated that enhanced 4E-BP1 activity in mouse skeletal muscle protects against age- and diet-induced insulin resistance and metabolic rate decline. Transgenic animals displayed increased energy expenditure; altered adipose tissue distribution, including reduced white adipose accumulation and preserved brown adipose mass; and were protected from hepatic steatosis. Skeletal muscle-specific 4E-BP1 mediated metabolic protection directly through increased translation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and enhanced respiratory function. Non-cell autonomous protection was through preservation of brown adipose tissue metabolism, which was increased in 4E-BP1 transgenic animals during normal aging and in a response to diet-induced type 2 diabetes. Adipose phenotypes may derive from enhanced skeletal muscle expression and secretion of the known myokine FGF21. Unlike skeletal muscle, enhanced adipose-specific 4E-BP1 activity was not protective but instead was deleterious in response to the same challenges. These findings indicate that regulation of 4E-BP1 in skeletal muscle may serve as an important conduit through which mTORC1 controls metabolism.
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A fundamental goal of research into the basic mechanisms of aging is to develop translational strategies that improve human health by delaying the onset and progression of age-related pathology. Several interventions have been discovered that increase life span in invertebrate organisms, some of which have similar effects in mice. These include dietary restriction and inhibition of the mechanistic target of rapamycin by treatment with rapamycin. Key challenges moving forward will be to assess the extent to which these and other interventions improve healthy longevity and increase life span in mice and to develop practical strategies for extending this work to the clinic. Companion animals may provide an optimal intermediate between laboratory models and humans. By improving healthy longevity in companion animals, important insights will be gained regarding human aging while improving the quality of life for people and their pets. © The Author(s) 2015.
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The mechanism by which the drug rapamycin inhibits the mechanistic target of rapamycin (mTOR) is of intense interest because of its likely relevance in cancer biology, aging, and other age-related diseases. While rapamycin acutely and directly inhibits mTORC1, only chronic administration of rapamycin can inhibit mTORC2 in some, but not all, cell lines or tissues. The mechanism leading to cell specificity of mTORC2 inhibition by rapamycin is not understood and is especially important because many of the negative metabolic side effects of rapamycin, reported in mouse studies and human clinical trials, have been attributed recently to mTORC2 inhibition. Here, we identify the expression level of different FK506-binding proteins (FKBPs), primarily FKBP12 and FKBP51, as the key determinants for rapamycin-mediated inhibition of mTORC2. In support, enforced reduction of FKBP12 completely converts a cell line that is sensitive to mTORC2 inhibition to an insensitive cell line, and increased expression can enhance mTORC2 inhibition. Further reduction of FKBP12 in cell lines with already low FKBP12 levels completely blocks mTORC1 inhibition by rapamycin, indicating that relative FKBP12 levels are critical for both mTORC1 and mTORC2 inhibition, but at different levels. In contrast, reduction of FKBP51 renders cells more sensitive to mTORC2 inhibition. Our findings reveal that the expression of FKBP12 and FKBP51 is the rate limiting factor that determines the responsiveness of a cell line or tissue to rapamycin. These findings have implications for treating specific diseases, including neurodegeneration and cancer, as well as targeting aging in general. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
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Since the initial observation that a calorie-restricted (CR) diet can extend rodent lifespan, many genetic and pharmaceutical interventions that also extend lifespan in mammals have been discovered. The mechanism by which CR and these other interventions extend lifespan is the subject of significant debate and research. One proposed mechanism is that CR promotes longevity by increasing insulin sensitivity, but recent findings that dissociate longevity and insulin sensitivity cast doubt on this hypothesis. These findings can be reconciled if longevity is promoted not via increased insulin sensitivity, but instead via decreased PI3K/Akt/mTOR pathway signaling. This review presents a unifying hypothesis that explains the lifespan-extending effects of a variety of genetic mutations and pharmaceutical interventions and points towards new molecular pathways which may also be leveraged to promote healthy aging.
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The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway. Copyright © 2015, American Association for the Advancement of Science.
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Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
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Inhibition of the mammalian target of rapamycin (mTOR) pathway extends life span in all species studied to date, and in mice delays the onset of age-related diseases and comorbidities. However, it is unknown if mTOR inhibition affects aging or its consequences in humans. To begin to assess the effects of mTOR inhibition on human aging-related conditions, we evaluated whether the mTOR inhibitor RAD001 ameliorated immunosenescence (the decline in immune function during aging) in elderly volunteers, as assessed by their response to influenza vaccination. RAD001 enhanced the response to the influenza vaccine by about 20% at doses that were relatively well tolerated. RAD001 also reduced the percentage of CD4 and CD8 T lymphocytes expressing the programmed death-1 (PD-1) receptor, which inhibits T cell signaling and is more highly expressed with age. These results raise the possibility that mTOR inhibition may have beneficial effects on immunosenescence in the elderly. Copyright © 2014, American Association for the Advancement of Science.
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The inhibition of mTOR (mechanistic target of rapamycin) by the macrolide rapamycin has many beneficial effects in mice, including extension of lifespan and reduction or prevention of several age-related diseases. At the same time, chronic rapamycin treatment causes impairments in glucose metabolism including hyperglycemia, glucose intolerance and insulin resistance. It is unknown whether these metabolic effects of rapamycin are permanent or whether they can be alleviated. Here, we confirmed that rapamycin causes glucose intolerance and insulin resistance in both inbred and genetically heterogeneous mice fed either low fat or high fat diets, suggesting that these effects of rapamycin are independent of genetic background. Importantly, we also found that these effects were almost completely lost within a few weeks of cessation of treatment, showing that chronic rapamycin treatment does not induce permanent impairment of glucose metabolism. Somewhat surprisingly, chronic rapamycin also promoted increased accumulation of adipose tissue in high fat fed mice. However, this effect too was lost when rapamycin treatment was ended suggesting that this effect of rapamycin is also not permanent. The reversible nature of rapamycin's alterations of metabolic function suggests that these potentially detrimental side-effects might be managed through alternative dosing strategies or concurrent treatment options.
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DEP domain-containing mTOR-interacting protein (DEPTOR) has been recently discovered as an endogenous regulator of the mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2. mTORC1 is present in the brain and there is growing evidence that its dysregulation contributes to several brain alterations. This suggests the involvement of mTOR signaling and its modulators in neurobiological controls. Here, we characterized and mapped the expression of DEPTOR in the rat brain. We showed that DEPTOR was widely expressed from the forebrain to hindbrain, including the hippocampus, the mediobasal hypothalamus, and the circumventricular organs (CVOs). In the hippocampus, DEPTOR protein and Deptor mRNA were highly expressed in the dendate gyrus and CA3 field. In the CVOs, DEPTOR was expressed in the subfornical organ, in the median eminence as well as in the area postrema. In the mediobasal hypothalamus, DEPTOR was expressed in neurons of the ventromedial nucleus (VMH) and colocalized with proopiomelanocortin in the arcuate nucleus (ARC). The hypothalamic distribution suggested a role for DEPTOR in energy balance. Supporting this possibility, we observed that Deptor hypothalamic expression was modulated by the nutritional status in a context of diet-induced and genetic obesity; food deprivation increased Deptor mRNA in both the ARC and VMH of obese rats. In conclusion, the present results illustrate the presence of DEPTOR in the rat brain and suggest a role for DEPTOR in the hypothalamic regulation of energy balance, which further supports the role of mTOR in energy homeostasis. J. Comp. Neurol., 2014. © 2014 Wiley Periodicals, Inc.
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Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR), robustly extends the lifespan of model organisms including mice. We recently found that chronic treatment with rapamycin not only inhibits mTOR complex 1 (mTORC1), the canonical target of rapamycin, but also inhibits mTOR complex 2 (mTORC2) in vivo. While genetic evidence strongly suggests that inhibition of mTORC1 is sufficient to promote longevity, the impact of mTORC2 inhibition on mammalian longevity has not been assessed. RICTOR is a protein component of mTORC2 that is essential for its activity. We examined three different mouse models of Rictor loss: mice heterozygous for Rictor, mice lacking hepatic Rictor, and mice in which Rictor was inducibly deleted throughout the body in adult animals. Surprisingly, we find that depletion of RICTOR significantly decreases male, but not female, lifespan. While the mechanism by which RICTOR loss impairs male survival remains obscure, we find that the effect of RICTOR depletion on lifespan is independent of the role of hepatic mTORC2 in promoting glucose tolerance. Our results suggest that inhibition of mTORC2 signaling is detrimental to males, which may explain in part why interventions that decrease mTOR signaling show greater efficacy in females.
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The effect of acute inhibition of both mTORC1 and mTORC2 on metabolism is unknown. A single injection of the mTOR kinase inhibitor, AZD8055, induced a transient, yet marked increase in fat oxidation and insulin resistance in mice, whereas the mTORC1 inhibitor rapamycin had no effect. AZD8055, but not rapamycin reduced insulin-stimulated glucose uptake into incubated muscles, despite normal GLUT4 translocation in muscle cells. AZD8055 inhibited glycolysis in MEF cells. Abrogation of mTORC2 activity by SIN1 deletion impaired glycolysis and AZD8055 had no effect in SIN1 KO MEFs. Re-expression of wildtype SIN1 rescued glycolysis. Glucose intolerance following AZD8055 administration was absent in mice lacking the mTORC2 subunit Rictor in muscle, and in vivo glucose uptake into Rictor-deficient muscle was reduced despite normal Akt activity. Taken together, acute mTOR inhibition is detrimental to glucose homeostasis in part by blocking muscle mTORC2, indicating its importance in muscle metabolism in vivo.
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The mammalian target of rapamycin inhibitor everolimus (RAD001) is a successfully used immunosuppressant in solid organ transplantation. Several studies have already used RAD001 in combination with calcineurin inhibitors after hematopoietic stem cell transplantation (HSCT). We investigated a calcineurin inhibitor free pre- and post-transplant immunosuppression of RAD001 combined with mycophenolate mofetil (MMF) in a non-myeloablative HSCT setting. After non-myeloablative conditioning by 2 Gy total body irradiation eight dogs received HSCT from dog leukocyte antigen-identical siblings. Immunosuppressives were given at doses of 1.5 mg RAD001 twice daily from day -1 to +49 then tapered until day +56 and 20 mg/kg MMF from day 0 to +28 then tapered until day +42. Historical ciclosporin A (CsA)/MMF regimen was used as control group. All dogs engrafted. Median platelet nadir amounted in all dogs to 0 * 109/l (median day +10, duration < 50 * 109/l: 22 days) and median leukocyte nadir was 1.0 * 109/l (range 0.1-2.5 * 109/l, median day +13). Eventually five of eight (63 %) animals rejected their grafts. Two dogs died of infections on day +19 and +25. Pharmacokinetics of RAD001 and MMF showed median trough levels of 19.1 (10.5-43.2) μg/l and 0.3 (0.1-1.3) mg/l, respectively. The median area under the curve was 325 (178-593) μg/l*h for RAD001 and 29.6 (7.9-40.5) ng/l*h for MMF. All dogs developed clinically mucosal viral infections during the clinical course. Compared to the control group, the level of toxicities for RAD001/MMF increased in all qualities. Combined immunosuppression of RAD001 and MMF after non-myeloablative HSCT is associated with significant toxicities including a prolonged platelet recovery time as well as increased infections compared to the CsA/MMF regimen.
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