Unknown,Transcriptomics,Genomics,Proteomics

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Rapamycin effect on Wild Type and TSC deficient Mouse Embryonic Fibroblasts : Time Course

ABSTRACT: Aberrant activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a common molecular event in a large variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell intrinsic consequences of mTORC1 activation remain poorly defined. Here, we identify global trancriptional changes in TSC1 and TSC2 null MEFs, which exhibit constitutive activation of mTORC1, compared to wild-type littermate control lines. A rapamycin time course is included to determine those changes that are dependent on mTORC1 signaling, revealing mTORC1 induced and repressed transcripts. In order to identify mTORC1-dependent transcriptional changes, we compared wild-type MEFs to both Tsc1-/- and Tsc2-/- MEFs following serum starvation, where mTORC1 signaling is off in wild-type cells and fully active in TSC-deficient cells. All cell lines were serum-starved for 24 h, and the Tsc1-/- and Tsc2-/- cells were treated with a time course of rapamycin prior to the isolation of mRNA for microarray analysis. Immortalized wild-type (Tsc2+/+ p53-/-), Tsc1-/- (p53+/+, 3T3-immortalized), and Tsc2-/- (p53-/-, derived from a littermate of the wild-type cell line) MEFs are the three cell lines used in this study and were derived in the laboratory of David J. Kwiatkowski (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). Wild-type and null MEFs were grown to 70% confluence in 10 cm plates and were serum starved for 24 h in the presence of vehicle (DMSO) for 24 h or rapamycin (20 nM) for 2, 6, 12, or 24 h. All vehicle-treated samples (0 h time points) were plated in triplicate and all rapamycin time course samples were plated in duplicate. For each replicate, expression analysis was performed by hybridization to an Affymetrix Mouse 430_2 oligonucleotide microarray chip.

ORGANISM(S): Mus musculus

SUBMITTER: Pichai Raman

PROVIDER: E-GEOD-21755 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Activation of a metabolic gene regulatory network downstream of mTOR complex 1.

Düvel Katrin K Yecies Jessica L JL Menon Suchithra S Raman Pichai P Lipovsky Alex I AI Souza Amanda L AL Triantafellow Ellen E Ma Qicheng Q Gorski Regina R Cleaver Stephen S Vander Heiden Matthew G MG MacKeigan Jeffrey P JP Finan Peter M PM Clish Clary B CB Murphy Leon O LO Manning Brendan D BD

Molecular cell 20100701 2

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative ...[more]

PMID: 20670887

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Project description:Tuberous sclerosis complex (TSC) is an inherited multi-system disorder caused by mutations in the TSC1 or TSC2 gene. TSC patients are often diagnosed with a range of neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD), intellectual disability (ID), anxiety and mood disorders. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) kinase signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using neural progenitor cells (NPCs) from patient-derived induced pluripotent stem cells (iPSCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in CRISPR-modified TSC1-null NPCs, which were unaffected by mTORC1 inhibition by rapamycin,the only approved therapy for TSC1. Here, to assess TSC1-dependent gene expression programs in NPCs, we used polysome-profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level. In addition to changes in mRNA abundance, this revealed numerous TSC1-dependent alterations in translational efficiencies. To assess the relevance of these gene expression alterations we performed polysome-profiling in post-mortem brains originating from ASD donors and matched controls. Strikingly, TSC1-dependent alterations in mRNA translation observed in NPCs were largely recapitulated in human brains. Furthermore, although polysome-profiling revealed a partial reversal of TSC1-associated gene expression alterations following rapamycin treatment, most genes related to neural activity/synaptic regulation or ASD that showed TSC1-dependent translation were rapamycin-insensitive. Therefore, we also examined whether early ND rapamycin-insensitive phenotypes in TSC1-null NPCs could be rescued by a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272 (Revolution Medicines, Inc.). Unlike rapamycin, RMC-6272 strongly inhibited translation and reversed TSC1-associated proliferation and neurite outgrowth phenotypes. In summary, we reveal ample translational alterations in TSC1 patient-derived NPCs recapitulating human brain expression profiles and potential implications for treatment of TAND.

Dataset Information

Rapamycin effect on Wild Type and TSC deficient Mouse Embryonic Fibroblasts : Time Course

Publications

Activation of a metabolic gene regulatory network downstream of mTOR complex 1.

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