The m-TORC1 inhibitor Sirolimus increases the effectiveness of Photodynamic therapy in the treatment of cutaneous Squamous Cell Carcinoma, impairing NRF2 antioxidant signaling.

Squamous Cell Carcinoma (SCC) is a subtype of Non-Melanoma Skin Cancer, the most common group of malignancies worldwide. Photodynamic therapy (PDT) is a non-invasive treatment approved for specific subtypes of SCC. Some malignancies resist PDT, forming more aggressive tumors and multiple relapses. Thus, new approaches aimed at optimizing the response to PDT are needed. The mTORC1 inhibitor rapamycin, also known as Sirolimus (SRL), interferes with protein synthesis and cell metabolism. The use of SRL as an immunosuppressant is associated to lower rates of SCC in kidney-transplanted patients, which are frequently affected by this pathology. We have evaluated SRL pre-treatment efficacy to enhance the damage induced by PDT with Methyl 5-aminolevulinate in two different cutaneous SCC established cell lines (SCC13 and A431) in vitro and therapy sensitization in PDT-resistant cell lines. We tested for the first time the SRL + PDT combination in a SKH-1 mouse model of photocarcinogenesis, diminishing the frequency of lesions and restraining tumor growth. Molecular studies revealed that protoporphyrin IX and reactive oxygen species production induced by PDT were promoted by SRL pre-treatment. Lastly, SRL modifies the expression and intracellular location of NRF2, interfering with the downstream antioxidant response modulated by NQO1 and HO-1. In conclusion, we propose SRL as a potential adjuvant to enhance PDT efficacy for SCC treatment.

Post-Transcriptional Induction of the Antiviral Host Factor GILT/IFI30 by Interferon Gamma.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays pivotal roles in both adaptive and innate immunities. GILT exhibits constitutive expression within antigen-presenting cells, whereas in other cell types, its expression is induced by interferon gamma (IFN-γ). Gaining insights into the precise molecular mechanism governing the induction of GILT protein by IFN-γ is of paramount importance for adaptive and innate immunities. In this study, we found that the 5' segment of GILT mRNA inhibited GILT protein expression regardless of the presence of IFN-γ. Conversely, the 3' segment of GILT mRNA suppressed GILT protein expression in the absence of IFN-γ, but it loses this inhibitory effect in its presence. Although the mTOR inhibitor rapamycin suppressed the induction of GILT protein expression by IFN-γ, the expression from luciferase sequence containing the 3' segment of GILT mRNA was resistant to rapamycin in the presence of IFN-γ, but not in its absence. Collectively, this study elucidates the mechanism behind GILT induction by IFN-γ: in the absence of IFN-γ, GILT mRNA is constitutively transcribed, but the translation process is hindered by both the 5' and 3' segments. Upon exposure to IFN-γ, a translation inhibitor bound to the 3' segment is liberated, and a translation activator interacts with the 3' segment to trigger the initiation of GILT translation.

Preclinical evaluation of the functionality of a polymer-coated sirolimuseluting stent in pigs.

PURPOSE: To compare the endothelial coverage of different stents in porcine carotid arteries. Research problem: How effective are polyurethane stents (PU) and PU + rapamycin (PU + RAPA) compared to bare-metal stents on endothelial coverage by neointima in pigs after 28 days? METHODS: The methodology had two phases for an interventional, experimental, prospective study, with three Moura pigs, 12 weeks old and weighing between 19 and 22.5 kg. In phase I, eight stents were implanted in carotid arteries; three stents coated with PU, three coated with PU + RAPA, and two without coating. After 28 days, phase II was carried out, consisting of euthanasia, removal of the stents, to evaluate the exposed area of the stent struts, and the percentage of endothelialization through optical microscopy and scanning electron microscopy. RESULTS: The eight stents implanted with ultrasound sizing and post-dilation with a larger diameter balloon were analyzed by Doppler ultrasound, intravascular ultrasound, and angiography after 28 days. CONCLUSIONS: This study showed complete endothelial coverage by the endoluminal neointima of the stent struts, good integration and coverage with the arterial wall, with no exposed struts showing the presence of intimal hyperplasia (whitish tissue).

mTORC1 pathway activity biases cell fate choice.

Pluripotent stem cells can differentiate into distinct cell types but the intracellular pathways controlling cell fate choice are not well understood. The social amoeba Dictyostelium discoideum is a simplified system to study choice preference as proliferating amoebae enter a developmental cycle upon starvation and differentiate into two major cell types, stalk and spores, organised in a multicellular fruiting body. Factors such as acidic vesicle pH predispose amoebae to one fate. Here we show that the mechanistic target of rapamycin complex 1 (mTORC1) pathway has a role in cell fate bias in Dictyostelium. Inhibiting the mTORC1 pathway activity by disruption of Rheb (activator Ras homolog enriched in brain), or treatment with the mTORC1 inhibitor rapamycin prior to development, biases cells to a spore cell fate. Conversely activation of the pathway favours stalk cell differentiation. The Set1 histone methyltransferase, responsible for histone H3 lysine4 methylation, in Dictyostelium cells regulates transcription at the onset of development. Disruption of Set1 leads to high mTORC1 pathway activity and stalk cell predisposition. The ability of the mTORC1 pathway to regulate cell fate bias of cells undergoing differentiation offers a potential target to increase the efficiency of stem cell differentiation into a particular cell type.

Biomimetic nanoparticles with enhanced rapamycin delivery for autism spectrum disorder treatment via autophagy activation and oxidative stress modulation.

Rationale: Autism spectrum disorder (ASD) represents a complex neurodevelopmental condition lacking specific pharmacological interventions. Given the multifaced etiology of ASD, there exist no effective treatment for ASD. Rapamycin (RAPA) can activate autophagy by inhibiting the mTOR pathway and has exhibited promising effects in treating central nervous system disorders; however, its limited ability to cross the blood-brain barrier (BBB) has hindered its clinical efficacy, leading to substantial side effects. Methods: To address this challenge, we designed a drug delivery system utilizing red blood cell membrane (CM) vesicles modified with SS31 peptides to enhance the brain penetration of RAPA for the treatment of autism. Results: The fabricated SCM@RAPA nanoparticles, with an average diameter of 110 nm, exhibit rapid release of RAPA in a pathological environment characterized by oxidative stress. In vitro results demonstrate that SCM@RAPA effectively activate cellular autophagy, reduce intracellular ROS levels, improve mitochondrial function, thereby ameliorating neuronal damage. SS31 peptide modification significantly enhances the BBB penetration and rapid brain accumulation of SCM@RAPA. Notably, SCM@RAPA nanoparticles demonstrate the potential to ameliorate social deficits, improve cognitive function, and reverse neuronal impairments in valproic acid (VPA)-induced ASD models. Conclusions: The therapeutic potential of SCM@RAPA in managing ASD signifies a paradigm shift in autism drug treatment, holding promise for clinical interventions in diverse neurological conditions.

Evaluation of Anticancer Efficacy of D-α-Tocopheryl Polyethylene-Glycol Succinate and Soluplus® Mixed Micelles Loaded with Olaparib and Rapamycin Against Ovarian Cancer.

Purpose: Ovarian cancer has the highest mortality rate and lowest survival rate among female reproductive system malignancies. There are treatment options of surgery and chemotherapy, but both are limited. In this study, we developed and evaluated micelles composed of D-α-tocopheryl polyethylene-glycol (PEG) 1000 succinate (TPGS) and Soluplus® (SOL) loaded with olaparib (OLA), a poly(ADP-ribose)polymerase (PARP) inhibitor, and rapamycin (RAPA), a mammalian target of rapamycin (mTOR) inhibitor in ovarian cancer. Methods: We prepared micelles containing different molar ratios of OLA and RAPA embedded in different weight ratios of TPGS and SOL (OLA/RAPA-TPGS/SOL) were prepared and physicochemical characterized. Furthermore, we performed in vitro cytotoxicity experiments of OLA, RAPA, and OLA/RAPA-TPGS/SOL. In vivo toxicity and antitumor efficacy assays were also performed to assess the efficacy of the mixed micellar system. Results: OLA/RAPA-TPGS/SOL containing a 4:1 TPGS:SOL weight ratio and a 2:3 OLA:RAPA molar ratio showed synergistic effects and were optimized. The drug encapsulation efficiency of this formulation was >65%, and the physicochemical properties were sustained for 180 days. Moreover, the formulation had a high cell uptake rate and significantly inhibited cell migration (**p < 0.01). In the in vivo toxicity test, no toxicity was observed, with the exception of the high dose group. Furthermore, OLA/RAPA-TPGS/SOL markedly inhibited tumor spheroid and tumor growth in vivo. Conclusion: Compared to the control, OLA/RAPA-TPGS/SOL showed significant tumor inhibition. These findings lay a foundation for the use of TPGS/SOL mixed micelles loaded with OLA and RAPA in the treatment of ovarian cancer.

Rapamycin-Induced Translocation of Meiotic Nuclear Proteins in Saccharomyces cerevisiae.

Conditional depletion of proteins is a potential strategy to elucidate protein function, especially in complex cellular processes like meiosis. Several methods are available to effectively deplete a protein in a conditional manner. Conditional loss of a protein function can be achieved by depleting it from its region of action by degrading it. A conditional loss of protein function can also be achieved by sequestering it to a functionally unavailable compartment inside the cell. This chapter describes anchor away, a conditional depletion tool that can deplete a protein both temporally and spatially by translocation. It utilizes the affinity of FRB to bind FKBP12 in the presence of rapamycin for a quick and efficient translocation of the protein to a designated location. Anchor away is a reliable tool for the study of meiotic proteins, as only small quantities of rapamycin are required to efficiently and rapidly translocate the protein of interest without compromising meiotic progression.

Calorie restriction and rapamycin distinctly mitigate aging-associated protein phosphorylation changes in mouse muscles.

Calorie restriction (CR) and treatment with rapamycin (RM), an inhibitor of the mTORC1 growth-promoting signaling pathway, are known to slow aging and promote health from worms to humans. At the transcriptome and proteome levels, long-term CR and RM treatments have partially overlapping effects, while their impact on protein phosphorylation within cellular signaling pathways have not been compared. Here we measured the phosphoproteomes of soleus, tibialis anterior, triceps brachii and gastrocnemius muscles from adult (10 months) and 30-month-old (aged) mice receiving either a control, a calorie restricted or an RM containing diet from 15 months of age. We reproducibly detected and extensively analyzed a total of 6960 phosphosites, 1415 of which are not represented in standard repositories. We reveal the effect of these interventions on known mTORC1 pathway substrates, with CR displaying greater between-muscle variation than RM. Overall, CR and RM have largely consistent, but quantitatively distinct long-term effects on the phosphoproteome, mitigating age-related changes to different degrees. Our data expands the catalog of protein phosphorylation sites in the mouse, providing important information regarding their tissue-specificity, and revealing the impact of long-term nutrient-sensing pathway inhibition on mouse skeletal muscle.

A structured biomimetic nanoparticle as inflammatory factor sponge and autophagy-regulatory agent against intervertebral disc degeneration and discogenic pain.

Lower back pain (LBP) is a common condition closely associated with intervertebral disc degeneration (IDD), causing a significant socioeconomic burden. Inflammatory activation in degenerated discs involves pro-inflammatory cytokines, dysregulated regulatory cytokines, and increased levels of nerve growth factor (NGF), leading to further intervertebral disc destruction and pain sensitization. Macrophage polarization is closely related to autophagy. Based on these pathological features, a structured biomimetic nanoparticle coated with TrkA-overexpressing macrophage membranes (TMNP@SR) with a rapamycin-loaded mesoporous silica core is developed. TMNP@SR acted like sponges to adsorbe inflammatory cytokines and NGF and delivers the autophagy regulator rapamycin (RAPA) into macrophages through homologous targeting effects of the outer engineered cell membrane. By regulating autophagy activation, TMNP@SR promoted the M1-to-M2 switch of macrophages to avoid continuous activation of inflammation within the degenerated disc, which prevented the apoptosis of nucleus pulposus cells. In addition, TMNP@SR relieved mechanical and thermal hyperalgesia, reduced calcitonin gene-related peptide (CGRP) and substance P (SP) expression in the dorsal root ganglion, and downregulated GFAP and c-FOS signaling in the spinal cord in the rat IDD model. In summary, TMNP@SR spontaneously inhibits the aggravation of disc inflammation to alleviate disc degeneration and reduce the ingress of sensory nerves, presenting a promising treatment strategy for LBP induced by disc degeneration.

Neohesperidin exerts antidepressant-like effect via the mechanistic target of rapamycin complex 1 in the medial prefrontal cortex in male mice.

Neohesperidin, a citrus flavonoid, shows potential for activating the mechanistic target of rapamycin complex 1 (mTORC1). Here, the antidepressant-like effect of neohesperidin was examined in male ICR mice (naïve mice and mice treated repeatedly with prednisolone, a synthetic glucocorticoid, which induces depression-like behavior). Oral neohesperidin administration exerted an antidepressant-like effect in the forced swim test 1 h post-treatment, in naïve mice; this effect was no longer observed at 24 h. Neohesperidin also reversed prednisolone-induced depression-like behavior. This effect was blocked by infusing rapamycin, an mTORC1 inhibitor, into the medial prefrontal cortex. Neohesperidin may rapidly produce an antidepressant-like effect.

Topical calcineurin and mammalian target of rapamycin inhibitors in inflammatory dermatoses: Current challenges and nanotechnology­based prospects (Review).

Topical therapy remains a critical component in the management of immune­mediated inflammatory dermatoses such as psoriasis and atopic dermatitis. In this field, macrolactam immunomodulators, including calcineurin and mammalian target of rapamycin inhibitors, can offer steroid­free therapeutic alternatives. Despite their potential for skin­selective treatment compared with topical corticosteroids, the physicochemical properties of these compounds, such as high lipophilicity and large molecular size, do not meet the criteria for efficient penetration into the skin, especially with conventional topical vehicles. Thus, more sophisticated approaches are needed to address the pharmacokinetic limitations of traditional formulations. In this regard, interest has increasingly focused on nanoparticulate systems to optimize penetration kinetics and enhance the efficacy and safety of topical calcineurin and mTOR inhibitors in inflamed skin. Several types of nanovectors have been explored as topical carriers to deliver tacrolimus in both psoriatic and atopic skin, while preclinical data on nanocarrier­based delivery of topical sirolimus in inflamed skin are also emerging. Given the promising preliminary outcomes and the complexities of drug delivery across inflamed skin, further research is required to translate these nanotherapeutics into clinical settings for inflammatory skin diseases. The present review outlined the dermatokinetic profiles of topical calcineurin and mTOR inhibitors, particularly tacrolimus, pimecrolimus and sirolimus, focusing on their penetration kinetics in psoriatic and atopic skin. It also summarizes the potential anti­inflammatory benefits of topical sirolimus and explores novel preclinical studies investigating dermally applied nanovehicles to evaluate and optimize the skin delivery, efficacy and safety of these 'hard­to­formulate' macromolecules in the context of psoriasis and atopic dermatitis.

Evaluation of suitable reference genes for qPCR normalisation of gene expression in a Achilles tendon injury model.

Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.

Targeted co-delivery of rapamycin and oxaliplatin by liposomes suppresses tumor growth and metastasis of colorectal cancer.

The activation of tumor cell immunogenicity through oxaliplatin (OXP)-induced immunogenic cell death (ICD) has significant implications in cancer treatment. However, the anti-tumor effect of OXP monotherapy still has many shortcomings, and the systemic administration of OXP leads to low drug concentration at the tumor site, which is susceptible to systemic toxic side effects. In this study, a combined therapeutic strategy using folate-modified nanoliposomes co-delivered with rapamycin (Rapa) and OXP (abbreviated as FA@R/O Lps) is proposed for the treatment of colorectal cancer (CRC). Rapa and OXP can directly inhibit tumor cell proliferation and induce apoptosis. OXP induces ICD by triggering the release of danger signals, such as HMGB1, ATP, and calreticulin. FA@R/O Lps with a particle size of about 134.1±1.8 nm and a small dispersion were successfully prepared. This novel liposomal system can be used to target and increase drug accumulation in tumors. In-vivo experiments showed that FA@R/O Lps successfully inhibit CRC growth and liver metastasis, and simultaneously reduce off-target toxicity. In particular, FA@R/O Lps showed greater therapeutic effects than free Rapa/OXP and R/O Lps. Taken together, this study provides a novel combination of Rapa and OXP, and a nano-delivery system for enhanced anti-CRC efficacy. The results suggest that FA@R/O Lps could be a promising strategy for the treatment of CRC.

Characterization of Effects of mTOR Inhibitors on Aging in Caenorhabditis elegans.

Pharmacological inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway with rapamycin can extend lifespan in several organisms. Although this includes the nematode Caenorhabditis elegans, effects in this species are relatively weak and sometimes difficult to reproduce. Here we test effects of drug dosage and timing of delivery to establish the upper limits of its capacity to extend life, and investigate drug effects on age-related pathology and causes of mortality. Liposome-mediated rapamycin treatment throughout adulthood showed a dose-dependent effect, causing a maximal 21.9% increase in mean lifespan, but shortening of lifespan at the highest dose, suggesting drug toxicity. Rapamycin treatment of larvae delayed development, weakly reduced fertility and modestly extended lifespan. By contrast, treatment initiated later in life robustly increased lifespan, even from Day 16 (or ~70 years in human terms). The rapalog temsirolimus extended lifespan similarly to rapamycin, but effects of everolimus were weaker. As in mouse, rapamycin had mixed effects on age-related pathologies, inhibiting one (uterine tumor growth) but not several others, suggesting a segmental antigeroid effect. These findings should usefully inform future experimental studies with rapamycin and rapalogs in C. elegans.

Rationale and design of INFINITY-SWEDEHEART: A registry-based randomized clinical trial comparing clinical outcomes of the sirolimus-eluting DynamX bioadaptor to the zotarolimus-eluting Resolute Onyx stent.

BACKGROUND: Modern drug-eluting stents have seen significant improvements, yet still create a rigid cage within the coronary artery. There is a 2% to 4% annual incidence of target lesion failure (TLF) beyond 1 year, and half of the patients experience angina after 5 years. The DynamX bioadaptor is a sirolimus-eluting, thin (71 µm) cobalt-chromium platform with helical strands that unlock and separate after in vivo degradation of the bioresorbable polymer coating. This allows the vessel to return to normal physiological function and motion, along with compensatory adaptive remodeling, which may reduce the need for reintervention and alleviate angina following percutaneous coronary intervention (PCI). METHODS: The INFINITY-SWEDEHEART trial is a single-blind, registry-based randomized clinical trial (R-RCT) to evaluate the safety and effectiveness of the DynamX bioadaptor compared to the Resolute Onyx stent in the treatment of patients with ischemic heart disease with de novo native coronary artery lesions. The R-RCT framework allows for recruitment, randomization, and pragmatic data collection of baseline demographics, medications, and clinical outcomes using existing national clinical registries integrated with the trial database. The primary objective is to demonstrate noninferiority in terms of freedom from TLF (cardiovascular death, target vessel myocardial infarction, or ischemia-driven target lesion revascularization) at 1 year. Powered secondary endpoints will be tested sequentially for superiority from 6 months to the end of follow-up (5 years) for the following: 1) TLF in all subjects, 2) target vessel failure in all subjects, and 3) TLF in subjects with acute coronary syndrome (ACS). Subsequent superiority testing will be performed at a time determined depending on the number of events, ensuring sufficient statistical power. Change in angina-related symptoms, function and quality of life will be assessed using the Seattle Angina Questionnaire-short version. Predefined sub-groups will be analyzed. In total, 2400 patients have been randomized at 20 sites in Sweden. Available baseline characteristic reveal relatively old age (68 years) and a large proportion of ACS patients including 25% STEMI and 37% NSTEMI patients. SUMMARY: The INFINITY-SWEDEHEART study is designed to evaluate the long-term safety and efficacy of the DynamX bioadaptor compared to the Resolute Onyx stent in a general PCI patient population.

Hsp70 Negatively Regulates Autophagy via Governing AMPK Activation, and Dual Hsp70-Autophagy Inhibition Induces Synergetic Cell Death in NSCLC Cells.

Proteostasis mechanisms, such as proteotoxic-stress response and autophagy, are increasingly recognized for their roles in influencing various cancer hallmarks such as tumorigenesis, drug resistance, and recurrence. However, the precise mechanisms underlying their coordination remain not fully elucidated. The aim of this study is to investigate the molecular interplay between Hsp70 and autophagy in lung adenocarcinoma cells and elucidate its impact on the outcomes of anticancer therapies in vitro. For this purpose, we utilized the human lung adenocarcinoma A549 cell line and genetically modified it by knockdown of Hsp70 or HSF1, and the H1299 cell line with knockdown or overexpression of Hsp70. In addition, several treatments were employed, including treatment with Hsp70 inhibitors (VER-155008 and JG-98), HSF1 activator ML-346, or autophagy modulators (SAR405 and Rapamycin). Using immunoblotting, we found that Hsp70 negatively regulates autophagy by directly influencing AMPK activation, uncovering a novel regulatory mechanism of autophagy by Hsp70. Genetic or chemical Hsp70 overexpression was associated with the suppression of AMPK and autophagy. Conversely, the inhibition of Hsp70, genetically or chemically, resulted in the upregulation of AMPK-mediated autophagy. We further investigated whether Hsp70 suppression-mediated autophagy exhibits pro-survival- or pro-death-inducing effects via MTT test, colony formation, CellTiter-Glo 3D-Spheroid viability assay, and Annexin/PI apoptosis assay. Our results show that combined inhibition of Hsp70 and autophagy, along with cisplatin treatment, synergistically reduces tumor cell metabolic activity, growth, and viability in 2D and 3D tumor cell models. These cytotoxic effects were exerted by substantially potentiating apoptosis, while activating autophagy via rapamycin slightly rescued tumor cells from apoptosis. Therefore, our findings demonstrate that the combined inhibition of Hsp70 and autophagy represents a novel and promising therapeutic approach that may disrupt the capacity of refractory tumor cells to withstand conventional therapies in NSCLC.

Sex, tissue, and mitochondrial interactions modify the transcriptional response to rapamycin in Drosophila.

BACKGROUND: Many common diseases exhibit uncontrolled mTOR signaling, prompting considerable interest in the therapeutic potential of mTOR inhibitors, such as rapamycin, to treat a range of conditions, including cancer, aging-related pathologies, and neurological disorders. Despite encouraging preclinical results, the success of mTOR interventions in the clinic has been limited by off-target side effects and dose-limiting toxicities. Improving clinical efficacy and mitigating side effects require a better understanding of the influence of key clinical factors, such as sex, tissue, and genomic background, on the outcomes of mTOR-targeting therapies. RESULTS: We assayed gene expression with and without rapamycin exposure across three distinct body parts (head, thorax, abdomen) of D. melanogaster flies, bearing either their native melanogaster mitochondrial genome or the mitochondrial genome from a related species, D. simulans. The fully factorial RNA-seq study design revealed a large number of genes that responded to the rapamycin treatment in a sex-dependent and tissue-dependent manner, and relatively few genes with the transcriptional response to rapamycin affected by the mitochondrial background. Reanalysis of an earlier study confirmed that mitochondria can have a temporal influence on rapamycin response. CONCLUSIONS: We found significant and wide-ranging effects of sex and body part, alongside a subtle, potentially time-dependent, influence of mitochondria on the transcriptional response to rapamycin. Our findings suggest a number of pathways that could be crucial for predicting potential side effects of mTOR inhibition in a particular sex or tissue. Further studies of the temporal response to rapamycin are necessary to elucidate the effects of the mitochondrial background on mTOR and its inhibition.

mTORC1 activation in presumed classical monocytes: observed correlation with human size variation and neuropsychiatric disease.

BACKGROUND: Gain of function disturbances in nutrient sensing are likely the largest component in human age-related disease. Mammalian target of rapamycin complex 1 (mTORC1) activity affects health span and longevity. The drugs ketamine and rapamycin are effective against chronic pain and depression, and both affect mTORC1 activity. Our objective was to measure phosphorylated p70S6K, a marker for mTORC1 activity, in individuals with psychiatric disease to determine whether phosphorylated p70S6K could predict medication response. METHODS: Twenty-seven females provided blood samples in which p70S6K and phosphorylated p70S6K were analyzed. Chart review gathered biometric measurements, clinical phenotypes, and medication response. Questionnaires assessed anxiety, depression, autism traits, and mitochondrial dysfunction, to determine neuropsychiatric disease profiles. Univariate and multivariate statistical analyses were used to identify predictors of medication response. RESULTS: mTORC1 activity correlated highly with both classical biometrics (height, macrocephaly, pupil distance) and specific neuropsychiatric disease profiles (anxiety and autism). Across all cases, phosphorylated p70S6K was the best predictor for ketamine response, and also the best predictor for rapamycin response in a single instance. CONCLUSIONS: The data illustrate the importance of mTORC1 activity in both observable body structure and medication response. This report suggests that a simple assay may allow cost-effective prediction of medication response.

Effect of mTORC Agonism via MHY1485 with and without Rapamycin on C2C12 Myotube Metabolism.

The mechanistic target of rapamycin complex (mTORC) regulates protein synthesis and can be activated by branched-chain amino acids (BCAAs). mTORC has also been implicated in the regulation of mitochondrial metabolism and BCAA catabolism. Some speculate that mTORC overactivation by BCAAs may contribute to insulin resistance. The present experiments assessed the effect of mTORC activation on myotube metabolism and insulin sensitivity using the mTORC agonist MHY1485, which does not share structural similarities with BCAAs. METHODS: C2C12 myotubes were treated with MHY1485 or DMSO control both with and without rapamycin. Gene expression was assessed using qRT-PCR and insulin sensitivity and protein expression by western blot. Glycolytic and mitochondrial metabolism were measured by extracellular acidification rate and oxygen consumption. Mitochondrial and lipid content were analyzed by fluorescent staining. Liquid chromatography-mass spectrometry was used to assess extracellular BCAAs. RESULTS: Rapamycin reduced p-mTORC expression, mitochondrial content, and mitochondrial function. Surprisingly, MHY1485 did not alter p-mTORC expression or cell metabolism. Neither treatment altered indicators of BCAA metabolism or extracellular BCAA content. CONCLUSION: Collectively, inhibition of mTORC via rapamycin reduces myotube metabolism and mitochondrial content but not BCAA metabolism. The lack of p-mTORC activation by MHY1485 is a limitation of these experiments and warrants additional investigation.

The role and mechanism of action of miR­92a in endothelial cell autophagy.

Although microRNAs (miRNAs/miRs) serve a significant role in the autophagy of vascular endothelial cells (ECs), the effect of miR­92a on the autophagy of ECs is currently unclear. Therefore, the present study aimed to investigate the impact of miR­92a on autophagy in ECs and the underlying molecular processes that control this biological activity. Firstly, an autophagy model of EA.hy926 cells was generated via treatment with the autophagy inducer rapamycin (rapa­EA.hy926 cells). The expression levels of miR­92a were then detected by reverse transcription­quantitative PCR, and the effect of miR­92a expression on the autophagic activity of rapa­EA.hy926 cells was studied by overexpressing or inhibiting miR­92a. The level of autophagy was evaluated by western blot analysis, immunofluorescence staining and transmission electron microscopy. Dual­luciferase reporter assays were used to confirm the interaction between miR­92a and FOXO3. The results demonstrated that the expression levels of miR­92a were decreased in the rapa­EA.hy926 cell autophagy model. Furthermore, overexpression and inhibition of miR­92a revealed that upregulation of miR­92a in these cells inhibited autophagy, whereas miR­92a knockdown promoted it. It was also confirmed that miR­92a directly bound to the 3'­untranslated region of the autophagy­related gene FOXO3 and reduced its expression. In conclusion, the present study suggested that miR­92a inhibits autophagy activity in EA.hy926 cells by targeting FOXO3.