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There are 39 results for Rapamycin (displaying 1 to 10).

Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition

In addition to key roles in embryonic neurogenesis and myelinogenesis, γ‐aminobutyric acid ( GABA ) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6 K 1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm …

Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls. After sacrifice, homogenates were used to measure S6 phosphorylation. Quantification of S6 phosphorylation of liver lysates from WT (n = 5) and Aldh5a1−/− mice treated with vehicle (n = 4) or rapamycin (n = 5) after normalization (WT set to 1). S6 phosphorylation of liver lysates …

WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin). WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD). To monitor autophagy, WT cells expressing GFP‐Atg8 along with the uga2∆ strain over‐expressing the GAD1 gene and expressing GFP‐Atg8 were grown in SD medium and transferred to SD‐N.

Peroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig . Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm. Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .

… can be overridden by the Tor1 inhibitor, rapamycin, via signaling pathways we have identified in this study. Furthermore, we show that rapamycin can reduce elevated mitochondrial numbers and normalize aberrant antioxidant levels found in the murine model of the disease. These results demonstrate a proof of concept for using autophagy‐inducing or mTOR‐inhibiting drugs as treatment for disorders characterized by elevated levels of GABA. Patients with SSADH deficiency have up to a threefold …

Ronak Lakhani et al. EMBO molecular medicine April 2014

Binding of the Atg1/ULK1 kinase to the ubiquitin‐like protein Atg8 regulates autophagy

… by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)‐inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3‐interaction region (LIR)‐dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers …

… to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 and the GFP‐tagged proteins was analysed by immunoblotting. Extract inputs are shown in Supplementary Figure S1B. (D) atg1Δ and atg1Δatg13Δ cells expressing HA‐tagged Atg1 and wild‐type (wt) cells containing an empty plasmid were grown to mid log phase (rich), and then treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1 was immunoprecipitated and its association with Atg13 analysed …

… composed of several proteins including Atg13 ( ). Atg1 activity is regulated by the Target of rapamycin (TOR) kinase, which regulates cell growth and autophagy as part of the TOR complex 1 (TORC1) in response to the nutrient availability ( ; ; ). Both Atg1 and Atg13 are highly phosphorylated under nutrient‐rich conditions, and Atg13 phosphorylation is partly mediated by TORC1 ( ). Atg13 is rapidly dephosphorylated upon starvation in both yeast and mammalian cells ( ; ; ), which might be part …

Claudine Kraft et al. The EMBO Journal September 2012

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. Here, we have characterized the fly mutants of Autophagy‐specific gene 1 ( ATG1 ), an autophagy‐regulating kinase, and found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. Our Drosophila studies have shown that ATG1 inhibits TOR/S6K‐dependent cell growth and development by interfering …

Involvement of Drosophila Autophagy‐specific gene 1 in Drosophila Target of rapamycin‐dependent cell growth and development. (A) Genomic structure of CG10967. The P‐element insertion site of EP3348 (DmATG11) is denoted. (B) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT–PCR. Ribosomal protein 49 (rp49) was used as an internal control; n=3. Bars indicate mean±s.d. (C) Images of the larvae of denoted genotypes at 3 days (top) and 6 days (middle) after egg …

… The nutritional environment is a crucial determinant of important cellular decisions, such as growth, proliferation and development. Recently, a series of outstanding studies have demonstrated that nutrient availability tightly regulates cell growth through an evolutionarily highly conserved signalling pathway, the target of rapamycin (TOR)/p70 S6 kinase (S6K) pathway (reviewed in ). S6K was originally discovered as a kinase that phosphorylates 40S ribosomal protein S6 at many sites and its …

Sung Bae Lee et al. EMBO Reports April 2007

Inhibition of insulin/IGF‐1 receptor signaling protects from mitochondria‐mediated kidney failure

… progressive proteinuria, kidney failure, and death of the animals and resulted in hyperphosphorylation of S6 ribosomal protein (S6RP), a known mediator of the mTOR signaling pathway. Inhibition of the insulin/IGF‐1 signaling system through genetic deletion of the insulin receptor alone or in combination with the IGF‐1 receptor or treatment with rapamycin prevented hyperphosphorylation of S6RP without affecting the mitochondrial structural defect, alleviated renal disease, and delayed the onset of kidney failure in PHB2‐deficient animals. Evidently, perturbation of insulin/IGF‐1 receptor signaling contributes to tissue damage in mitochondrial disease, which may allow therapeutic intervention against a wide spectrum of diseases.

Phb2‐deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t‐test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein. Immunohistochemistry for phosphorylated S6 ribosomal protein (pS6RP) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl/fl mice (arrows point …

… of the insulin and IGF‐1 receptor or treatment with the mTORC1 inhibitor rapamycin partially rescued the phenotype of Phb2 knockout mice and prolonged survival. To understand the contribution of mitochondrial dysfunction to kidney disease, we deleted the Phb2 gene specifically in podocytes ( Phb2 pko ) by mating a conditional Phb2 fl/fl mouse line (Merkwirth et al , ) to podocyte‐specific Cre mice ( NPHS2.cre ) (Moeller et al , ). Mice of all genotypes were born following Mendelian rules ( ). At birth …

Christina Ising et al. EMBO molecular medicine March 2015

Mitochondria regulate autophagy by conserved signalling pathways

… respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1–Atg13 kinase complex to the pre‐autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors—mitochondrial dysfunction and autophagy inhibition—for ageing, cancerogenesis, and neurodegeneration.

Role of TORC1 in autophagy regulation under amino‐acid starvation. (A) Wild‐type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8‐GFP‐ATG8 (upper panel) or prATG8‐GFP (lower panel) were exposed to amino‐acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated. (B) Wild‐type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1‐NPR1‐HA were …

… signalling pathways regulate autophagy including the conserved serine/threonine protein kinase target of rapamycin kinase complex I (TORC1) and cAMP‐dependent protein kinase A (PKA) pathways, which also sense nutrient availability and control cellular growth ( ; ; , ; ; ). In yeast, TORC1 and PKA negatively regulate autophagy at least in part by directly and independently phosphorylating the conserved kinase Atg1 (ULK1/2 in mammals) and Atg13 (mammalian Atg13), and by blocking the assembly …

Martin Graef et al. The EMBO Journal June 2011

TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells

… epithelial cells treated with TRAIL. TRAIL‐induced autophagy is mediated by the AMP‐activated protein kinase (AMPK) that inhibits mammalian target of rapamycin complex 1, a potent inhibitor of autophagy. Interestingly, the TRAIL‐induced AMPK activation is refractory to the depletion of the two known AMPK‐activating kinases, LKB1 and Ca(2+)/calmodulin‐dependent kinase kinase‐β, but depends on transforming growth factor‐β‐activating kinase 1 (TAK1) and TAK1‐binding subunit 2. As TAK1 and AMPK …

TRAIL induces cytoprotective autophagy in breast epithelial cells. (A) MCF10A–eGFP–LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown. (B) MCF10A–eGFP–LC3 cells were transfected with the indicated siRNAs for 48 h …

TRAIL activates AMPK and AMPK‐dependent cytoprotective autophagy. (A) Protein lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa, positive control) for 24 h were analysed by immunoblotting for phosphorylated p70S6K (P‐p70S6K), total p70S6K and GAPDH (loading control). (B) Immunoblot analysis of the levels of P‐ACC and tubulin (loading control) in lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL for 24 h, 1 mM AICAR for 4 h …

… Macroautophagy (hereafter referred to as autophagy) is a lysosomal pathway involved in the turnover of long‐lived proteins, cytoplasm and whole organelles ( ; ; ; ). In unstressed cells, autophagy is inhibited by the mammalian target of rapamycin complex 1 (mTORC1). The inhibition of mTORC1 activity (e.g., by starvation or rapamycin) leads to the activation of a set of evolutionarily conserved autophagy‐regulating proteins (Atg proteins) and formation of autophagosomes, which then fuse …

Griselda Herrero‐Martín et al. The EMBO Journal March 2009

mTORC2 sustains thermogenesis via Akt‐induced glucose uptake and glycolysis in brown adipose tissue

Activation of non‐shivering thermogenesis ( NST ) in brown adipose tissue ( BAT ) has been proposed as an anti‐obesity treatment. Moreover, cold‐induced glucose uptake could normalize blood glucose levels in insulin‐resistant patients. It is therefore important to identify novel regulators of NST and cold‐induced glucose uptake. Mammalian target of rapamycin complex 2 ( mTORC 2) mediates insulin‐stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC 2 …

Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins. Immunoblot analysis of BAT cells stimulated with NE for 5 min in the presence of rapamycin (Rapa), Torin, or wortmannin (Wrtm) for the indicated proteins. Immunoblot analysis of BAT cells stimulated with 8‐Br‐cAMP for 5 min in the presence of Rapa, Torin, or Wrtm for the indicated proteins. Immunoblot analysis of BAT cells stimulated with NE for 5 min in the presence of Wrtm, H89, or ESI‐09 …

… , identifying novel regulators of NST could provide new drug targets for anti‐obesity and diabetes treatments. The mammalian target of rapamycin (mTOR) signaling network is a central regulator of cell growth and metabolism (Laplante & Sabatini, ; Dibble & Manning, ; Albert & Hall, ; Shimobayashi & Hall, ). mTOR is a highly conserved protein kinase found in two structurally and functionally distinct complexes named mTOR complex 1 (mTORC1) and mTORC2. mTORC1 is sensitive to the macrolide rapamycin

Verena Albert et al. EMBO molecular medicine March 2016

Comprehensive establishment and characterization of orthoxenograft mouse models of malignant peripheral nerve sheath tumors for personalized medicine

… . Sorafenib (a BRAF inhibitor), in combination with doxorubicin or rapamycin, was found to be the most effective treatment for reducing MPNST growth. The development of genomically well‐characterized preclinical models for MPNST allowed the evaluation of novel therapeutic strategies for personalized medicine.

Tumor growth effects of treatment with doxorubicin, sorafenib, rapamycin and combinations thereof in the five MPNST xenograft models. Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value. Statistically significant differences are shown as *P < 0.05 and **P < 0.001 versus control group by the Bonferroni test. For long‐term studies, a subgroup of treated mice (n = 3–5 mice/group) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses (usually 1,500–2,000 mm3). The graph illustrates differences in the time delay (in days) of relapsed tumor masses for the different treatments.

… signaling pathway and the mTOR pathway (Basu et al , ; DeClue et al , ; Guha et al , ; Downward, ; Watson et al , ), which is expected to be over‐activated upon NF1 mutation (Guha et al , ; Sherman et al , ). However, the results of these assays are inconclusive or limited to certain models. Two recent phase II clinical trials assessed the monotherapy activity of sorafenib or rapamycin analogs (temsirolimus) in patients with different types of sarcoma, including MPNSTs (Maki et al , ; Okuno et al …

Joan Castellsagué et al. EMBO molecular medicine May 2015

Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4

… /mechanistic target of rapamycin complex 1), an upstream regulator of autophagy. We also found that Rab12 promotes constitutive degradation of PAT4 (proton‐coupled amino‐acid transporter 4), whose accumulation in Rab12‐knockdown cells modulates mTORC1 activity and autophagy. Our findings reveal a new mechanism of regulation of mTORC1 signalling and autophagy, that is, quality control of PAT4 by Rab12.

… recycling endosomes) in autophagosome formation and maturation [ , ]. However, the functional relationships between individual members of the mammalian Rab family and autophagy remain largely unknown. In this study, we comprehensively screened for Rabs that are involved in autophagy by using RNA‐mediated interference technologies and found that Rab12 is a new autophagic regulator. We showed that Rab12 regulates activity of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), an upstream …

Takahide Matsui et al. EMBO Reports May 2013

Mitochondria are required for pro‐ageing features of the senescent phenotype

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro‐inflammatory and pro‐oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent‐associated changes are dependent on mitochondria, particularly the pro‐inflammatory phenotype. Mechanistically, we show that the ATM , Akt and mTORC 1 phosphorylation cascade integrates signals from the DNA damage response ( DDR ) towards PGC ‐1β‐dependent mitochondrial biogenesis, contributing to a ROS ‐mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo , by either mTORC 1 inhibition or PGC ‐1β deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.

(left) Representative images of γH2A.X (red foci) immunofluorescence in MRC5 fibroblasts at different time points after 20‐Gy X‐ray with or without 100 nM rapamycin supplementation (scale bar = 5 μm) and (right) representative images of 53BP1 (green foci) immunofluorescence in MEFs, 3 days after 10‐Gy X‐ray with or without 100 nM rapamycin supplementation (scale bar = 5 μm). Effect of genomic damage‐ or oxidative stress‐induced senescence on the mean number (N) of γH2A.X foci in MRC5 …

Representative Western blot of mTORC1 activity measured by phosphorylated p70S6K (T389) from 6 to 72 h after 20 Gy in MRC5 fibroblasts. Data are representative of three independent experiments. Representative Western blots of the mitochondrial proteins TOMM20, NDUFB8 (complex I), SDHA (complex II), UQCR2 (complex III) and MT‐CO1 (complex IV) following 20‐Gy irradiation with or without 100 nM rapamycin treatment in MRC5 fibroblasts. Data are representative of 4 independent experiments …

… protein expression in mouse liver tissue at 3 and 12 months of age. Data are from n = 3 mice per group. Effect of 4 months of rapamycin‐supplemented diet on PGC‐1β expression in the liver tissue of 16‐month‐old mice. Data are from n = 3 mice per group. (top) Quantification of mitochondrial number per cross‐section and mitochondrial volume fraction (%Vv) in hepatocytes from 16‐month‐old mice with or without 4 months of rapamycin diet. Data are mean ± SEM of n = 3 mice per group (at least 20 …

… that mitochondrial mass increases following a DDR and establishes a causal relationship between mitochondrial biogenesis and senescence. We then proceeded to explore the signalling pathways linking the DDR to mitochondrial biogenesis. The mammalian target of rapamycin (mTOR) pathway has been widely implicated in processes governing mitochondrial turnover. mTOR complex 1 (mTORC1) has been shown to integrate stress signals into the regulation of protein and lipid synthesis and autophagy, all of which …

Clara Correia‐Melo et al. The EMBO Journal April 2016
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