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1.
J Neurosci ; 33(18): 7799-810, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23637172

ABSTRACT

The mammalian target of rapamycin (mTOR) regulates cell growth in response to various intracellular and extracellular signals. It assembles into two multiprotein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-insensitive mTORC2. In this study, we inactivated mTORC1 in mice by deleting the gene encoding raptor in the progenitors of the developing CNS. Mice are born but never feed and die within a few hours. The brains deficient for raptor show a microcephaly starting at E17.5 that is the consequence of a reduced cell number and cell size. Changes in cell cycle length during late cortical development and increased cell death both contribute to the reduction in cell number. Neurospheres derived from raptor-deficient brains are smaller, and differentiation of neural progenitors into glia but not into neurons is inhibited. The differentiation defect is paralleled by decreased Stat3 signaling, which is a target of mTORC1 and has been implicated in gliogenesis. Together, our results show that postnatal survival, overall brain growth, and specific aspects of brain development critically depend on mTORC1 function.


Subject(s)
Brain , Cell Differentiation/genetics , Gene Expression Regulation, Developmental/genetics , Microcephaly/genetics , Microcephaly/pathology , Neuroglia/pathology , Proteins/metabolism , Animals , Animals, Newborn , Apoptosis/genetics , Brain/embryology , Brain/growth & development , Brain/pathology , Bromodeoxyuridine/metabolism , Caspase 3/metabolism , Cell Cycle/genetics , Cell Proliferation , Disease Models, Animal , Embryo, Mammalian , Female , Glial Fibrillary Acidic Protein/metabolism , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Male , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Microcephaly/mortality , Multiprotein Complexes , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin , Proteins/genetics , STAT3 Transcription Factor/metabolism , TOR Serine-Threonine Kinases , Transcription Factors/genetics , Transcription Factors/metabolism , Tubulin/metabolism
2.
J Cell Biol ; 201(2): 293-308, 2013 Apr 15.
Article in English | MEDLINE | ID: mdl-23569215

ABSTRACT

The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons.


Subject(s)
Brain/metabolism , Carrier Proteins/metabolism , Cell Shape , Cell Size , Multiprotein Complexes/metabolism , Purkinje Cells/metabolism , Purkinje Cells/pathology , TOR Serine-Threonine Kinases/metabolism , Animals , Blotting, Western , Brain/enzymology , Brain/pathology , Cell Count , Cerebellum/enzymology , Cerebellum/pathology , Enzyme Activation , Gene Deletion , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Knockout , Microcephaly/enzymology , Microcephaly/pathology , Phenotype , Purkinje Cells/enzymology , Rapamycin-Insensitive Companion of mTOR Protein , Synapses/metabolism
3.
PLoS One ; 7(1): e30011, 2012.
Article in English | MEDLINE | ID: mdl-22253858

ABSTRACT

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.


Subject(s)
Genetic Loci/genetics , Integrases/metabolism , Mutagenesis, Insertional/methods , Promoter Regions, Genetic/genetics , Proteins/genetics , Transgenes/genetics , Animals , Attachment Sites, Microbiological/genetics , Embryonic Stem Cells/metabolism , Genes, Reporter/genetics , Green Fluorescent Proteins/metabolism , Luciferases/metabolism , Mice , Mice, Inbred BALB C , Mice, Transgenic , Organ Specificity/genetics , RNA, Untranslated
4.
Cell Metab ; 8(5): 411-24, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19046572

ABSTRACT

Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival. In contrast, muscles lacking the mTORC2 component rictor are indistinguishable from wild-type controls. Raptor-deficient muscles become progressively dystrophic, are impaired in their oxidative capacity, and contain increased glycogen stores, but they express structural components indicative of oxidative muscle fibers. Biochemical analysis indicates that these changes are probably due to loss of activation of direct downstream targets of mTORC1, downregulation of genes involved in mitochondrial biogenesis, including PGC1alpha, and hyperactivation of PKB/Akt. Finally, we show that activation of PKB/Akt does not require mTORC2. Together, these results demonstrate that muscle mTORC1 has an unexpected role in the regulation of the metabolic properties and that its function is essential for life.


Subject(s)
Carrier Proteins/physiology , Mitochondria/physiology , Muscle, Skeletal/metabolism , Muscular Dystrophies/metabolism , Transcription Factors/physiology , Adaptor Proteins, Signal Transducing , Animals , Carrier Proteins/genetics , Enzyme Activation , Gene Expression Regulation , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Multiprotein Complexes , Muscle, Skeletal/pathology , Muscular Dystrophies/pathology , Oncogene Protein v-akt/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphorylation , Proteins , Proto-Oncogene Proteins c-akt/metabolism , Rapamycin-Insensitive Companion of mTOR Protein , Regulatory-Associated Protein of mTOR , TOR Serine-Threonine Kinases , Trans-Activators/metabolism , Transcription Factors/genetics
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