mTOR Function Research Articles

Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition.

The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5′ untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.

Distinct roles for the mTOR pathway in postnatal morphogenesis, maturation and function of pancreatic islets

While much is known about the molecular pathways that regulate embryonic development and adult homeostasis of the endocrine pancreas, little is known about what regulates early postnatal development and maturation of islets. Given that birth marks the first exposure to enteral nutrition, we investigated how nutrient-regulated signaling pathways influence postnatal islet development in mice. We performed loss-of-function studies of mechanistic target of rapamycin (mTOR), a highly conserved kinase within a nutrient-sensing pathway known to regulate cellular growth, morphogenesis and metabolism. Deletion of Mtor in pancreatic endocrine cells had no significant effect on their embryonic development. However, within the first 2 weeks after birth, mTOR-deficient islets became dysmorphic, β-cell maturation and function were impaired, and animals lost islet mass. Moreover, we discovered that these distinct functions of mTOR are mediated by separate downstream branches of the pathway, in that mTORC1 (with adaptor protein Raptor) is the main complex mediating the maturation and function of islets, whereas mTORC2 (with adaptor protein Rictor) impacts islet mass and architecture. Taken together, these findings suggest that nutrient sensing may be an essential trigger for postnatal β-cell maturation and islet development.

Role of mammalian target of rapamycin signaling in autophagy and the neurodegenerative process using a senescence accelerated mouse-prone 8 model.

The mammalian target of rapamycin (mTOR) kinase is an inhibitor of autophagy, which is an intracellular system involved in the degradation of long-lived proteins and organelles in lysosomes. Recent evidence suggests that the steady incline in mTOR function during aging may be associated with the cognitive decline related to aging and may also promote development of Tau pathology. At present, the senescence accelerated mouse prone 8 (SAMP8) is an experimental model that has been proposed for the study of age-related neurodegenerative changes associated with aging. In the present study, mTOR signaling in the hippocampus of SAMP8 newborn mice and in the control-strain SAMR1 mice was investigated. Consequently, hyper phosphorylated Tau (pS199 or pS396) and upregulated mTOR activity were observed in SAMP8 when compared with SAMR1; however, 0.5 µM rapamycin administration significantly reduced the levels of phosphorylated Tau and p70S6K (pT389) in SAMP8 mice. Related to these findings, SAMP8 exhibited an increase in the neuronal loss of hippocampus that was associated with lower levels of anti-apoptotic proteins. These results indicate that mTOR signaling participates in the neurodegenerative process and rapamycin administration may protect neurons of SAMP8 mice and may have a potential role in curing cognitive decline.

The PI3K/Akt/mTOR pathway is involved in CVB3-induced autophagy of HeLa cells.

Recent studies have found that viral myocarditis (VMC) associated with coxsackievirus B3 (CVB3) causes autophagy activation after infection, but the specific mechanism is not clear. The present study demonstrated that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/mammalian target of rapamycin (mTOR) signaling pathway participates in CVB3-induced autophagy. We found that the light chain 3 (LC3)-II/LC3-I ratio was increased and p62 and p-mTOR were altered at different times during CVB3 infection. To further assess the effects of this signaling pathway on CVB3 infection and viral replication, we selected 24 h post-inoculation (h.p.i.) as our research time point to conduct our next study. We inhibited the function of PI3K, Akt1 and mTOR. The outcome showed that inhibition of PI3K with ZSTK474 alleviated autophagy and decreased CVB3 mRNA replication and VP1 expression. Inhibition of mTOR with rapamycin promoted autophagy and viral mRNA replication but did not impact VP1 expression. Inhibition of Akt with MK2206 aggravated autophagy induced by viral infection. In our research, p62 exhibited a decrease at the beginning of infection but then increased as infection time increased. This finding may serve as a clue to elucidate the function of autophagy at different times of infection. However, the details merit further study. In conclusion, our findings suggest that the PI3K/Akt/mTOR signaling pathway participates in the process of autophagy induced by CVB3 infection. This finding may provide a new perspective of CVB3-induced autophagy.

Regulation of endothelial cell proliferation of infantile hemangioma through mTORC2 and NDRG1 signaling pathways

Infantile hemangioma (IH), the most common vascular tumor in infants, is characterized by during the first weeks to months of a child's life, followed by a spontaneous involution over 10 years. Although benign, some IHs cause deformation and destruction of features or endanger life. Propranolol, a nonselective β-adrenergic receptor (βAR) has proved efficacious for problematic IH and can lower cAMP levels and activate the MAPK pathway downstream of βARs. Recent in vitro and in vivo studies show that rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, reduces the self-renewal capacity of the hemangioma stem cells, diminishes differentiation potential, and inhibits the vasculogenic activity of these cells. mTOR functions in two distinct complexes, mTORC1 and mTORC2, and little is known regarding the relative contribution of mTORC1 versus mTORC2 in vascular endothelial cells. The aim of this study was to assess the role of rapamycin-insensitive companion of mTOR (Rictor) in control of infantile hemangioma proliferation and cell signaling mechanism in human umbilical vein endothelial cells. We found that Rictor ablation inhibited VEGF-induced endothelial cell proliferation in vitro. The loss of Rictor reduced the phosphorylation of PKCα and NDRG1 without affecting the mTORC1 pathway. Our results showed that loss of Rictor down-regulated phosphorylation of S6 by which IH cell growth and proliferation were greatly suppressed. The role and mechanism of Rictor in IH were assessed by western blotting and real time PCR. Taken together, these data indicate that mTORC2 is significant for VEGF-mediated IH cell proliferation through the regulation of PKCα and NDRG1.

Escaping mTOR inhibition for cancer therapy: Tumor suppressor functions of mTOR

ABSTRACTA master promoter of cell growth, mammalian target of rapamycin (mTOR) is upregulated in a large percentage of cancer cells. Still, targeting mTOR using rapamycin has a limited outcome in patients. Our recent results highlight the additional role of mTOR as a tumor suppressor, explaining these modest results in the clinic.

Enhanced hepatic insulin signaling in the livers of high altitude native rats under basal conditions and in the livers of low altitude native rats under insulin stimulation: a mechanistic study

This study was designed to investigate the role of the liver in lowering fasting blood glucose levels (FBG) in rats native to high (HA) and low altitude (LA) areas. As compared with LA natives, besides the improved insulin and glucose tolerance, HA native rats had lower FBG, at least mediated by inhibition of hepatic gluconeogenesis and activation of glycogen synthesis. An effect that is mediated by the enhancement of hepatic insulin signaling mediated by the decreased phosphorylation of TSC induced inhibition of mTOR function. Such effect was independent of activation of AMPK nor stabilization of HIF1α, but most probably due to oxidative stress induced REDD1 expression. However, under insulin stimulation, and in spite of the less activated mTOR function in HA native rats, LA native rats had higher glycogen content and reduced levels of gluconeogenic enzymes with a more enhanced insulin signaling, mainly due to higher levels of p-IRS1 (tyr612).

MTORC1 signalling mediates PI3K-dependent large lipid droplet accumulation in Drosophila ovarian nurse cells.

ABSTRACTInsulin and insulin-like growth factor signalling (IIS), which is primarily mediated by the PI3-kinase (PI3K)/PTEN/Akt kinase signalling cassette, is a highly evolutionarily conserved pathway involved in co-ordinating growth, development, ageing and nutrient homeostasis with dietary intake. It controls transcriptional regulators, in addition to promoting signalling by mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), which stimulates biosynthesis of proteins and other macromolecules, and drives organismal growth. Previous studies in nutrient-storing germline nurse cells of the Drosophila ovary showed that a cytoplasmic pool of activated phosphorylated Akt (pAkt) controlled by Pten, an antagonist of IIS, cell-autonomously regulates accumulation of large lipid droplets in these cells at late stages of oogenesis. Here, we show that the large lipid droplet phenotype induced by Pten mutation is strongly suppressed when mTor function is removed. Furthermore, nurse cells lacking either Tsc1 or Tsc2, which negatively regulate mTORC1 activity, also accumulate large lipid droplets via a mechanism involving Rheb, the downstream G-protein target of TSC2, which positively regulates mTORC1. We conclude that elevated IIS/mTORC1 signalling is both necessary and sufficient to induce large lipid droplet formation in late-stage nurse cells, suggesting roles for this pathway in aspects of lipid droplet biogenesis, in addition to control of lipid metabolism.

Distinct roles for the mTOR pathway in postnatal morphogenesis, maturation and function of pancreatic islets.

While much is known about the molecular pathways that regulate embryonic development and adult homeostasis of the endocrine pancreas, little is known about what regulates early postnatal development and maturation of islets. Given that birth marks the first exposure to enteral nutrition, we investigated how nutrient-regulated signaling pathways influence postnatal islet development in mice. We performed loss-of-function studies of mechanistic target of rapamycin (mTOR), a highly conserved kinase within a nutrient-sensing pathway known to regulate cellular growth, morphogenesis and metabolism. Deletion of Mtor in pancreatic endocrine cells had no significant effect on their embryonic development. However, within the first 2 weeks after birth, mTOR-deficient islets became dysmorphic, β-cell maturation and function were impaired, and animals lost islet mass. Moreover, we discovered that these distinct functions of mTOR are mediated by separate downstream branches of the pathway, in that mTORC1 (with adaptor protein Raptor) is the main complex mediating the maturation and function of islets, whereas mTORC2 (with adaptor protein Rictor) impacts islet mass and architecture. Taken together, these findings suggest that nutrient sensing may be an essential trigger for postnatal β-cell maturation and islet development.

Gerosuppression by pan-mTOR inhibitors.

Rapamycin slows organismal aging and delays age-related diseases, extending lifespan in numerous species. In cells, rapamycin and other rapalogs such as everolimus suppress geroconversion from quiescence to senescence. Rapamycin inhibits some, but not all, activities of mTOR. Recently we and others demonstrated that pan-mTOR inhibitors, known also as dual mTORC1/C2 inhibitors, suppress senescent phenotype. As a continuation of these studies, here we investigated in detail a panel of pan-mTOR inhibitors, to determine their optimal gerosuppressive concentrations. During geroconversion, cells become hypertrophic and flat, accumulate lysosomes (SA-beta-Gal staining) and lipids (Oil Red staining) and lose their re-proliferative potential (RPP). We determined optimal gerosuppressive concentrations: Torin1 (30 nM), Torin 2 (30 nM), AZD8055 (100 nM), PP242 (300 nM), both KU-006379 and GSK1059615 (1000 nM). These agents decreased senescence-associated hypertrophy with IC50s: 20, 18, 15, 200 and 400 nM, respectively. Preservation of RPP by pan-mTOR inhibitors was associated with inhibition of the pS6K/pS6 axis. Inhibition of rapamycin-insensitive functions of mTOR further contributed to anti-hypertrophic and cytostatic effects. Torin 1 and PP242 were more “rapamycin-like” than Torin 2 and AZD8055. Pan-mTOR inhibitors were superior to rapamycin in suppressing hypertrophy, senescent morphology, Oil Red O staining and in increasing so-called “chronological life span (CLS)”. We suggest that, at doses lower than anti-cancer concentrations, pan-mTOR inhibitors can be developed as anti-aging drugs.

MTOR Is Dispensable in Hematopoietic Stem Cells and GMP Progenitors, but Plays an Essential Role in the CMP Population, for Autophagy Regulation

Macroautophagy, or autophagy, is an intracellular self-degrading and recycling process that maintains cellular homeostasis. In blood system, autophagy is essential for life-long support and maintenance of hematopoietic stem cells (HSCs). Defect in autophagy process may lead to HSC aging, abnormal development in both myeloid and lymphoid lineages, and failure of blood reconstitution after transplantation. Current biochemical model of autophagy views the mTOR kinase activity, a therapeutic target for a variety of hematologic diseases including leukemia, as a critical inhibitory mechanism of autophagy initiation through phosphorylation of ULK1 and Atg13. Previously we showed that mTOR gene deletion in the blood system lead to hematopoietic failure, with a transient expansion of the HSC pool and differential blockage of various progenitor populations without detectable effects on survival. Since cellular responses to mTOR deletion vary in different blood cell populations, we hypothesize that autophagy dependence on mTOR may also differ among different HSC and progenitor populations. To examine the basal state and mTOR regulated autophagy flux in hematopoiesis, we used a reporter mouse carrying an autophagy reporter GFP-LC3 that allows a readout of autophagy flux in live or fixed cells. Crossing it to an Mx-Cre;mTORflox/flox or an Mx-Cre;mTORflox/knockin genetic background produced the mTOR null or the mTOR kinase-activity deficient D2338A mutant knockin genotype upon poly I:C induced Cre recombination in all blood lineages. Subpopulations of mTOR wild-type (WT), mTOR knockout (KO) and mTOR kinase-dead knockin (KI) cells from freshly harvested mouse bone marrow, including HSC (Lin-Sca-1+c-Kit+CD135-), CMP (Lin-Sca-1-c-Kit+CD34+CD16/32lo), and GMP (Lin-Sca-1-c-Kit+CD34+CD16/32hi, were isolated by flow cytometry. The sorted cells were treated with autophagy inhibitor Bafilomycin A1 (BA), mTOR kinase inhibitor AZD8055 (AZD), or a nonspecific AMPK inhibitor Compound C, and the autophagy flux activities of the various populations of HSC and progenitors were quantified by immunofluorescent microscopy of the GFP-LC3 puncta or FACS analysis. In wild-type mice, HSC and CMP populations had significantly higher basal autophagy flux than the GMP cells, with ~55% LC3 puncta positive cells in HSC and CMP versus ~15% in GMP. Surprisingly, the HSC and GMP populations of cells did not respond to either mTOR KO or KI in their autophagy activities while the CMP cells were highly responsive to the mTOR KO or KI showing a significant increase of autophagy flux in the KO and KI cells, compared with wild-type CMP cells. In the GMP enriched Lin-c-Kit+ population, p-Akt308 was significantly upregulated upon mTOR loss, possibly due to a compensatory mechanism, which may in turn suppress autophagy through Beclin 1. Upon treatment by the mTOR inhibitor AZD, autophagy flux was induced in wild-type HSC, CMP and GMP populations but not in the corresponding mTOR KO or KI populations, whereas Compound C, a non-specific kinase inhibitor, induced LC3 puncta accumulation in HSC, CMP and GMP populations of all three mTOR WT, KO, and KI genotypes. Our data shows that in HSPCs, basal autophagy flux varies in different populations, with higher autophagy activity found in the most primitive HSCs and lower autophagy activity in the GMP population. The dependence on mTOR in autophagy regulation also differs amongst different HSC and progenitor populations, with HSC and GMP populations showing a surprising independence whereas CMP population a dependence on mTOR in autophagy suppression. The autophagy flux of the mTOR KO genotype are similar to that of the mTOR KI genotype in the stem/progenitor populations, suggesting that the mTOR kinase activity, not a kinase-independent mTOR function, regulates autophagy. Lastly, the mTOR WT, KO and KI CMP cells respond to a non-specific kinase inhibitor Compound C in autophagy induction, suggesting that mTOR-independent kinases are involved in autophagy suppression. Thus, despite that the mTOR KO and KI HSPCs show similar deficiencies in the canonical mTOR signaling including dampened p-S6 and p-4EBP1, the molecular mechanisms underlying autophagy regulation is diverse amongst distinct blood stem/progenitor cell types. DisclosuresNo relevant conflicts of interest to declare.

The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway.

The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway.

MTOR signaling regulates myotube hypertrophy by modulating protein synthesis, rDNA transcription, and chromatin remodeling.

Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy; however, the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than rapamycin indicating that mTOR function during hypertrophy is not on general, but rather on specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and, unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcriptional level. Third, Pol I activity is essential for rDNA transcription and, surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis, and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to rapamycin. Inhibition of mTOR signaling by rapamycin disrupted mTOR-rDNA promoter interaction and resulted in altered histone marks indicative of repressed transcription and formation of higher-order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression, and chromatin remodeling.

RPL23 Links Oncogenic RAS Signaling to p53-Mediated Tumor Suppression.

The ribosomal protein (RP)-MDM2 interaction is a p53 response pathway critical for preventing oncogenic c-MYC-induced tumorigenesis. To investigate whether the RP-MDM2-p53 pathway is a broad antioncogenic mechanism, we crossed mice bearing an MDM2(C305F) mutation, which disrupts RPL11 binding to MDM2, with mice expressing an oncogenic Hras(G12V) transgene. Interestingly, the MDM2(C305F)-mutant mice, which are hypersensitive to c-MYC-induced tumorigenesis, are not hypersensitive to oncogenic Hras(G12V)-induced tumorigenesis. Unlike c-MYC, which induces expression of RPL11, RAS overexpression leads to an increase in RPL23 mRNA and protein whereas RPL11 expression remains unchanged. The induction of RPL23 involves both MEK and PI3K signaling pathways and requires mTOR function. Increased expression of RPL23, which maintains binding to MDM2(C305F) mutant, correlates with increased p53 expression in MDM2(C305F) cells. Furthermore, RAS overexpression can induce p53 in the absence of p19ARF, and the induction can be abolished by downregulation of RPL23. Thus, although the RPL11-MDM2-p53 pathway coordinates with the p19ARF-MDM2-p53 pathway against oncogenic c-MYC-induced tumorigenesis, the RPL23-MDM2-p53 pathway coordinates with the p19ARF-MDM2-p53 pathway against oncogenic RAS-induced tumorigenesis. Cancer Res; 76(17); 5030-9. ©2016 AACR.

The underlying mechanism of proinflammatory NF-κB activation by the mTORC2/Akt/IKKα pathway during skin aging.

Mammalian target of rapamycin complex 2 (mTORC2), one of two different enzymatic complexes of mTOR, regulates a diverse set of substrates including Akt. mTOR pathway is one of well-known mediators of aging process, however, its role in skin aging has not been determined. Skin aging can be induced by physical age and ultraviolet (UV) irradiation which are intrinsic and extrinsic factors, respectively. Here, we report increased mTORC2 pathway in intrinsic and photo-induced skin aging, which is implicated in the activation of nuclear factor-κB (NF-κB). UVB-irradiated or aged mice skin revealed that mTORC2 activity and its component, rictor were significantly upregulated which in turn increased Akt activation and Akt-dependent IκB kinase α (IKKα) phosphorylation at Thr23 in vivo. We also confirmed that UVB induced the mTORC2/Akt/IKKα signaling pathway with HaCaT human normal keratinocytes. The increased mTORC2 signaling pathway during skin aging were associated to NF-κB activation. Suppression of mTORC2 activity by the treatment of a mTOR small inhibitor or knockdown of RICTOR partially rescued UVB-induced NF-κB activation through the downregulation of Akt/IKKα activity. Our data demonstrated the upregulation of mTORC2 pathway in intrinsic and photo-induced skin aging and its role in IKKα/NF-κB activation. These data not only expanded the functions of mTOR to skin aging but also revealed the therapeutic potential of inhibiting mTORC2 in ameliorating both intrinsic skin aging and photoaging.

Rapamycin increases RSV RNA levels and survival of RSV-infected dendritic cell depending on T cell contact

The macrolide rapamycin inhibits mTOR (mechanist target of rapamycin) function and has been broadly used to unveil the role of mTOR in immune responses. Inhibition of mTOR on dendritic cells (DC) can influence cellular immune response and the survival of DC. RSV is the most common cause of hospitalization in infants and is a high priority candidate to vaccine development. In this study we showed that rapamycin treatment on RSV-infected murine bone marrow-derived DC (BMDC) decreases the frequency of CD8+CD44high T cells. However, inhibition of mTOR on RSV-infected BMDC did not modify the activation phenotype of these cells. RSV-RNA levels increase when infected BMDC were treated with rapamycin. Moreover, we observed that rapamycin diminishes apoptosis cell death of RSV-infected BMDC co-culture with T cells and this effect was abolished when the cells were co-cultured in a transwell system that prevents cell-to-cell contact or migration. Taken together, these data indicate that rapamycin treatment present a toxic effect on RSV-infected BMDC increasing RSV-RNA levels, affecting partially CD8 T cell differentiation and also increasing BMDC survival in a mechanism dependent on T cell contact.

Abstract 393A: Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620

Abstract Introduction: The mammalian target of rapamycin (mTOR) signaling pathway is an integrating factor in cell physiology that influences many processes like growth, metabolism and proliferation. mTOR signaling is constitutively activated in many cancers. Rapamycin is an allosteric inhibitor of mTOR that targets a subset of mTOR functions via inhibition of the mTORC1 complex. An ATP site-directed mTORC1/2 inhibitor that fully blocks all mTOR functions is desirable as cancer therapeutic. PQR620 is a novel, ATP site directed inhibitor of mTOR that is currently in pre-clinical development. PQR620 potently binds to its target (Kd = 6 nM) and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR620 inhibits mTOR signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been observed in mice (MTD = 150 mg/kg). A 14 day GLP toxicological study in rats showed very good tolerability (MTD = 30 mg/kg). Only minor toxicities such as dose-related changes in body weight and blood count were observed. PQR620 was administered to male C57BL/6J mice for a pharmacokinetic (PK) and pharmacodynamics (PD) evaluation. After oral application PQR620 exhibited dose-proportional PK, a maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (4.8 μg/ml and 7.7 μg/ml, respectively). In muscle, Cmax (7.6 μg/ml) was reached after 2 hours. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC0-tz (area under the curve)) was 20.5 μg*h/ml in plasma, while it was approximately 30% higher in both, brain and thigh muscle (30.6 and 32.3 μg*h/ml, respectively). PQR620 potently inhibited mTOR signaling in vivo after administration of a single oral dose of 50 mg/kg. Importantly, no effect on plasma insulin levels was observed. In an OVCAR-3, ovarian carcinoma mouse xenograft, PQR620 effectively attenuated tumor growth using daily, oral dosing. Conclusion: PQR620 potently inhibits mTORC1/2 in vitro and in vivo. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Beaufils F, Rageot D, et al., Structure-Activity Relationship Studies, Synthesis and Biological Evaluation of PQR620, a Highly Potent and Selective mTORC1/2 Inhibitor, AACR annual meeting 2016 Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Alexander Sele, Marc Lang, Juergen Mestan, Robert A. Ettlin, Petra Hillmann, Vladimir Cmiljanovic, Carolin Walter, Elisabeth Singer, Hoa HP Nguyen, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393A.

Regulation of PERK-eIF2α signalling by tuberous sclerosis complex-1 controls homoeostasis and survival of myelinating oligodendrocytes.

Tuberous sclerosis complex-1 or 2 (TSC1/2) mutations cause white matter abnormalities, including myelin deficits in the CNS; however, underlying mechanisms are not fully understood. TSC1/2 negatively regulate the function of mTOR, which is required for oligodendrocyte differentiation. Here we report that, unexpectedly, constitutive activation of mTOR signalling by Tsc1 deletion in the oligodendrocyte lineage results in severe myelination defects and oligodendrocyte cell death in mice, despite an initial increase of oligodendrocyte precursors during early development. Expression profiling analysis reveals that Tsc1 ablation induces prominent endoplasmic reticulum (ER) stress responses by activating a PERK–eIF2α signalling axis and Fas–JNK apoptotic pathways. Enhancement of the phospho-eIF2α adaptation pathway by inhibition of Gadd34-PP1 phosphatase with guanabenz protects oligodendrocytes and partially rescues myelination defects in Tsc1 mutants. Thus, TSC1-mTOR signalling acts as an important checkpoint for maintaining oligodendrocyte homoeostasis, pointing to a previously uncharacterized ER stress mechanism that contributes to hypomyelination in tuberous sclerosis.

Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function.

The kinase mammalian target of rapamycin (mTOR) integrates signals triggered by energy, stress, oxygen levels, and growth factors. It regulates ribosome biogenesis, mRNA translation, nutrient metabolism, and autophagy. mTOR participates in various functions of the brain, such as synaptic plasticity, adult neurogenesis, memory, and learning. mTOR is present during early neural development and participates in axon and dendrite development, neuron differentiation, and gliogenesis, among other processes. Furthermore, mTOR has been shown to modulate lifespan in multiple organisms. This protein is an important energy sensor that is present throughout our lifetime its role must be precisely described in order to develop therapeutic strategies and prevent diseases of the central nervous system. The aim of this review is to present our current understanding of the functions of mTOR in neural development, the adult brain and aging.

MTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice.

The mammalian target of rapamycin (Mtor) gene encodes a serine/threonine kinase that acts as a master regulator of processes as diverse as cell growth, protein synthesis, cytoskeleton reorganization, and cell survival. In the testis, physiological roles for Mtor have been proposed in perinatal Sertoli cell proliferation and blood–testis barrier (BTB) remodeling during spermatogenesis, but no in vivo studies of Mtor function have been reported. Here, we used a conditional knockout approach to target Mtor in Sertoli cells. The resulting Mtorflox/flox; Amhr2cre/+ mice were characterized by progressive, adult-onset testicular atrophy associated with disorganization of the seminiferous epithelium, loss of Sertoli cell polarity, increased germ cell apoptosis, premature release of germ cells, decreased epididymal sperm counts, increased sperm abnormalities, and infertility. Histopathologic analysis and quantification of the expression of stage-specific markers showed a specific loss of pachytene spermatocytes and spermatids. Although the BTB and the ectoplasmic specializations did not appear to be altered in Mtorflox/flox;Amhr2cre/+ mice, a dramatic redistribution of gap junction alpha-1 (GJA1) was detected in their Sertoli cells. Phosphorylation of GJA1 at Ser373, which is associated with its internalization, was increased in the testes of Mtorflox/flox; Amhr2cre/+ mice, as was the expression and phosphorylation of AKT, which phosphorylates GJA1 at this site. Together, these results indicate that Mtor expression in Sertoli cells is required for the maintenance of spermatogenesis and the progression of germ cell development through the pachytene spermatocyte stage. One mechanism of mTOR action may be to regulate gap junction dynamics by inhibiting AKT, thereby decreasing GJA1 phosphorylation and internalization. mTOR regulates gap junction alpha-1 protein distribution in Sertoli cells and is necessary for progression through the pachytene spermatocyte stage.