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1.  Genetic Removal of p70 S6 Kinase 1 Corrects Molecular, Synaptic, and Behavioral Phenotypes in Fragile X Syndrome Mice 
Neuron  2012;76(2):325-337.
Fragile X syndrome (FXS) is the leading inherited cause of autism and intellectual disability. Aberrant synaptic translation has been implicated in the etiology of FXS, but most lines of research on therapeutic strategies have targeted protein synthesis indirectly, far upstream of the translation machinery. We sought to perturb p70 ribosomal S6 kinase 1 (S6K1), a key translation initiation and elongation regulator, in FXS model mice. We found that genetic reduction of S6K1 prevented elevated phosphorylation of translational control molecules, exaggerated protein synthesis, enhanced mGluR-dependent long-term depression (LTD), weight gain, and macro-orchidism in FXS model mice. In addition, S6K1 deletion prevented immature dendritic spine morphology and multiple behavioral phenotypes, including social interaction deficits, impaired novel object recognition, and behavioral inflexibility. Our results support the model that dysregulated protein synthesis is the key causal factor in FXS, and that restoration of normal translation can stabilize peripheral and neurological function in FXS.
PMCID: PMC3479445  PMID: 23083736
3.  Interspecific prediction of photosynthetic light response curves using specific leaf mass and leaf nitrogen content: effects of differences in soil fertility and growth irradiance 
Annals of Botany  2012;109(6):1149-1157.
Background and Aims
Previous work has shown that the entire photosynthetic light response curve, based on both Mitscherlich and Michaelis–Menten functions, could be predicted in an interspecific context through allometric relations linking the parameters of these functions to two static leaf traits: leaf nitrogen (N) content and leaf mass per area (LMA). This paper describes to what extent these allometric relations are robust to changes in soil fertility and the growth irradiance of the plants.
Plants of 25 herbaceous species were grown under controlled conditions in factorial combinations of low/high soil fertility and low/high growth irradiance. Net photosynthetic rates per unit dry mass were measured at light intensities ranging from 0 to 700 µmol m−2 s−1 photosynthetically active radiation (PAR).
Key Results
The differing growth environments induced large changes in N, LMA and in each of the parameter estimates of the Mitscherlich and Michaelis–Menten functions. However, the differing growth environments induced only small (although significant) changes in the allometric relationships linking N and LMA to the parameters of the two functions. As a result, 88 % (Mitcherlich) and 89 % (Michaelis–Menten) of the observed net photosynthetic rates over the full range of light intensities (0–700 µmol m−2 s−1 PAR) and across all four growth environments could be predicted using only N and LMA using the same allometric relations.
These results suggest the possibility of predicting net photosynthetic rates in nature across species over the full range of light intensities using readily available data.
PMCID: PMC3336948  PMID: 22442344
Quantum yield; Mitscherlich curve; Michaelis–Menten curve; leaf respiration rate; maximum photosynthetic rate; SLA; LMA
4.  BDNF activation of CaM-kinase kinase via TRPC channels induces the translation and synaptic incorporation of GluA1 containing calcium-permeable AMPARs 
The Journal of Neuroscience  2012;32(24):8127-8137.
Glutamatergic synapses in early postnatal development transiently express calcium-permeable AMPA receptors (CP-AMPARs). Although these GluA2-lacking receptors are essential and are elevated in response to brain-derived neurotrophic factor (BDNF), little is known regarding molecular mechanisms that govern their expression and synaptic insertion. Here we show that BDNF-induced GluA1 translation in rat primary hippocampal neurons requires the activation of mTOR via calcium calmodulin-dependent protein kinase kinase (CaMKK). Specifically, BDNF-mediated phosphorylation of T308 in AKT, a known substrate of CaMKK and an upstream activator of mTOR-dependent translation, was prevented by 1) pharmacological inhibition of CaMKK with STO-609, 2) overexpression of a dominant-negative CaMKK, or 3) short hairpin-mediated knockdown of CaMKK. GluA1 surface expression induced by BDNF, as assessed by immunocytochemistry using an extracellular N-terminal GluA1 antibody or by surface biotinylation, was impaired following knockdown of CaMKK or treatment with STO-609. Activation of CaMKK by BDNF requires TRPC channels as SKF-96365, but not the NMDA receptor antagonist D-APV, prevented BDNF-induced GluA1 surface expression as well as phosphorylation of CaMKI, AKTT308 and mTOR. Using siRNA we confirmed the involvement of TRPC5 and -6 subunits in BDNF-induced AKTT308 phosphorylation. The BDNF-induced increase in mEPSC was blocked by IEM-1460, a selected antagonist of CP-AMPARs, as well as by the specific repression of acute GluA1 translation via siRNA to GluA1 but not GluA2. Taken together these data support the conclusion that newly synthesized GluA1 subunits, induced by BDNF, are readily incorporated into synapses where they enhance the expression of CP-AMPARs and synaptic strength.
PMCID: PMC3390208  PMID: 22699894
BDNF; AMPA receptors; translation; CaM-kinase; TRPC
5.  Large G3BP-induced granules trigger eIF2α phosphorylation 
Molecular Biology of the Cell  2012;23(18):3499-3510.
Increasing size of G3BP-induced stress granules is associated with a threshold or switch that must be triggered for eIF2α phosphorylation and subsequent translational repression to occur. Stress granules are active in signaling to the translational machinery and may be important regulators of the innate immune response.
Stress granules are large messenger ribonucleoprotein (mRNP) aggregates composed of translation initiation factors and mRNAs that appear when the cell encounters various stressors. Current dogma indicates that stress granules function as inert storage depots for translationally silenced mRNPs until the cell signals for renewed translation and stress granule disassembly. We used RasGAP SH3-binding protein (G3BP) overexpression to induce stress granules and study their assembly process and signaling to the translation apparatus. We found that assembly of large G3BP-induced stress granules, but not small granules, precedes phosphorylation of eIF2α. Using mouse embryonic fibroblasts depleted for individual eukaryotic initiation factor 2α (eIF2α) kinases, we identified protein kinase R as the principal kinase that mediates eIF2α phosphorylation by large G3BP-induced granules. These data indicate that increasing stress granule size is associated with a threshold or switch that must be triggered in order for eIF2α phosphorylation and subsequent translational repression to occur. Furthermore, these data suggest that stress granules are active in signaling to the translational machinery and may be important regulators of the innate immune response.
PMCID: PMC3442399  PMID: 22833567
6.  Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae) 
PLoS ONE  2012;7(10):e48253.
Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.
PMCID: PMC3484996  PMID: 23133579
7.  Nuclear translation visualized by ribosome-bound nascent chain puromycylation 
The Journal of Cell Biology  2012;197(1):45-57.
A new method for visualizing translation in cells via standard immunofluorescence microscopy provides evidence for translation in the nucleoplasm and nucleolus.
Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.
PMCID: PMC3317795  PMID: 22472439
8.  Brain-specific Disruption of the eIF2α Kinase PERK Decreases ATF4 Expression and Impairs Behavioral Flexibility 
Cell Reports  2012;1(6):676-688.
Translational control depends on phosphorylation of eIF2α by PKR-like ER kinase (PERK). To examine the role of PERK in cognitive function, we selectively disrupted PERK expression in the adult mouse forebrain. In the prefrontal cortex (PFC) of PERK-deficient mice, eIF2α phosphorylation and ATF4 expression were diminished and associated with enhanced behavioral perseveration, decreased prepulse inhibition, reduced fear extinction, and impaired behavioral flexibility. Treatment with the glycine transporter inhibitor SSR504734 normalized eIF2α phosphorylation, ATF4 expression, and behavioral flexibility in PERK-deficient mice. Moreover, PERK and ATF4 expression were reduced in the frontal cortex of human schizophrenic patients. Together, our findings reveal that PERK plays a critical role in information processing and cognitive function, and that modulation of eIF2α phosphorylation and ATF4 expression may represent an effective strategy for treating behavioral inflexibility associated with several neurological disorders including schizophrenia.
PMCID: PMC3401382  PMID: 22813743
PERK; translational control; eIF2α; ATF4; prefrontal cortex; cognitive control; glycine transporter-1 inhibitor; behavioral flexibility; schizophrenia
9.  Induction of GADD34 Is Necessary for dsRNA-Dependent Interferon-β Production and Participates in the Control of Chikungunya Virus Infection 
PLoS Pathogens  2012;8(5):e1002708.
Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection.
Author Summary
Nucleic acids detection by multiple molecular sensors results in type-I interferon production, which protects cells and tissues from viral infections. At the intracellular level, the detection of double-stranded RNA by one of these sensors, the dsRNA-dependent protein kinase also leads to the profound inhibition of protein synthesis. We describe here that the inducible phosphatase 1 co-factor Ppp1r15a/GADD34, a well known player in the endoplasmic reticulum unfolded protein response (UPR), is activated during double-stranded RNA detection and is absolutely necessary to allow cytokine production in cells exposed to poly I:C or Chikungunya virus. Our data shows that the cellular response to nucleic acids can reveal unanticipated connections between innate immunity and fundamental stress pathways, such as the ATF4 branch of the UPR.
PMCID: PMC3355096  PMID: 22615568

Results 1-9 (9)