The GCN2 kinase is required for activating autophagy in response to indispensable amino acid deficiencies

ORGANIZING COMMITTEEChairs: Didier Attaix - Lydie Combaret - Daniel TaillandierDaniel Béchet - Agnès Claustre - Cécile Coudy-Gandilhon - Christiane Deval - Gérard Donadille - Cécile PolgeSCIENTIFIC COMMITTEEDidier Attaix - Lydie Combaret - Alfred L. Goldberg - Ron Hay - Germana Meroni - Marco Sandri - Daniel Taillandier - Keiji Tanaka - Simon S. WingPoster Session 3 - AutophagyImbalances in dietary amino acid (AA) supply, including deficits in one or more indispensable amino acids (IAA), are stressful conditions for the organism that needs to modulate a number of physiological functions to adapt to this situation. In particular, since there is no system dedicated for storing AA in the body, the release of free AA occurs by proteolysis at the expense of functional proteins, notably in the liver by up-regulating autophagy. This process can be rapidly mobilized within the cell in response to a number of stresses, by post-translational regulations of autophagy-related proteins already present in the cytosol. The protein kinase GCN2 is activated upon IAA scarcity in order to promote cell adaptation to a nutritional stress condition. In response to IAA limitation, GCN2 couples the accumulation of uncharged transfer RNAs to the phosphorylation of eIF2a on serine 51. By this mean, GCN2 diminishes the overall protein synthesis rate, while simultaneously activating a gene expression program mediated by the translational upregulation of the transcription factor ATF4. Our recent work has shown that the GCN2/p-eIF2a/ATF4 signaling pathway plays an essential role in the induction of transcription of a number of autophagy-related genes involved in the maintenance of the autophagic process in response to an IAA deficiency (B’chir et al., 2013). In the present study we sought to determine whether GCN2 could play a role in regulating the early stages of autophagy. The most upstream complex for triggering the autophagic process (initiation complex) is notably composed of the ULK kinase and the ATG13 bridging protein, and is classically viewed to be controlled by mTORC1. Indeed, the activity of the autophagy initiation complex has been shown to be modulated according to AA availability by the activity of mTORC1, which phosphorylates different sites in ULK. Here, by using a GCN2 knock-out mouse model we investigated the role of GCN2 in the upregulation of autophagy in the first hour of an IAA deficiency. Our results show that 1) GCN2 is required for upregulating liver autophagy in response to an IAA-deficient diet, which is confirmed in cell culture model; 2) this early activation of the autophagic process does not require the transcription factor ATF4; 3) moreover, while this effect can occur without concomitant inhibition of mTORC1 activity, our results suggest that ULK/ATG13 couple is involved in the GCN2-dependent activation of autophagy. Our results demonstrate that in the particular model of an IAA deficiency GCN2 plays a preponderant role in triggering the adaptive autophagy upregulation, a mechanism which can operate without concomitant inhibition of mTORC1 activit

Locus Coeruleus Activation Patterns Differentially Modulate Odor Discrimination Learning and Odor Valence in Rats

The locus coeruleus (LC) produces phasic and tonic firing patterns that are theorized to have distinct functional consequences. However, how different firing modes affect learning and valence encoding of sensory information are unknown. Here, we show bilateral optogenetic activation of rat LC neurons using 10-Hz phasic trains of either 300 ms or 10 s accelerated acquisition of a similar odor discrimination. Similar odor discrimination learning was impaired by noradrenergic blockade in the piriform cortex (PC). However, 10-Hz phasic light-mediated learning facilitation was prevented by a dopaminergic antagonist in the PC, or by ventral tegmental area (VTA) silencing with lidocaine, suggesting a LC–VTA–PC dopamine circuitry involvement. Ten-hertz tonic stimulation did not alter odor discrimination acquisition, and was ineffective in activating VTA DA neurons. For valence encoding, tonic stimulation at 25 Hz induced conditioned odor aversion, whereas 10-Hz phasic stimulations produced an odor preference. Both conditionings were prevented by noradrenergic blockade in the basolateral amygdala (BLA). Cholera Toxin B retro-labeling showed larger engagement of nucleus accumbens-projecting neurons in the BLA with 10-Hz phasic activation, and larger engagement of central amygdala projecting cells with 25-Hz tonic light. These outcomes argue that the LC activation patterns differentially influence both target networks and behavior

Spatially controlled cell adhesion on three-dimensional substrates

The microenvironment of cells in vivo is defined by spatiotemporal patterns of chemical and biophysical cues. Therefore, one important goal of tissue engineering is the generation of scaffolds with defined biofunctionalization in order to control processes like cell adhesion and differentiation. Mimicking extrinsic factors like integrin ligands presented by the extracellular matrix is one of the key elements to study cellular adhesion on biocompatible scaffolds. By using special thermoformable polymer films with anchored biomolecules micro structured scaffolds, e.g. curved and µ-patterned substrates, can be fabricated. Here, we present a novel strategy for the fabrication of µ-patterned scaffolds based on the “Substrate Modification and Replication by Thermoforming” (SMART) technology: The surface of a poly lactic acid membrane, having a low forming temperature of 60°C and being initially very cell attractive, was coated with a photopatterned layer of poly(L-lysine) (PLL) and hyaluronic acid (VAHyal) to gain spatial control over cell adhesion. Subsequently, this modified polymer membrane was thermoformed to create an array of spherical microcavities with diameters of 300 µm for 3D cell culture. Human hepatoma cells (HepG2) and mouse fibroblasts (L929) were used to demonstrate guided cell adhesion. HepG2 cells adhered and aggregated exclusively within these cavities without attaching to the passivated surfaces between the cavities. Also L929 cells adhering very strongly on the pristine substrate polymer were effectively patterned by the cell repellent properties of the hyaluronic acid based hydrogel. This is the first time cell adhesion was controlled by patterned functionalization of a polymeric substrate with UV curable PLL-VAHyal in thermoformed 3D microstructures

Influence of endogenous and exogenous butyrate on protein kinase B (Akt) phosphorylation in broilers

Butyrate is a short chain fatty acid that is produced by microbial fermentation in the gut as well as applied as a feed additive in poultry nutrition. It was suspected based on previous studies that butyrate acts as an insulin sensitizer in liver and muscle of chicken and that it would enhance the phosphorylation of Akt, a key member of the insulin signalling pathway. In this study the influence of endogenous and exogenous butyrate on Akt phosphorylation and its association with the insulin signalling cascade was investigated. Broiler chickens (Ross-308 strain) were fed with two different basal diets (maize or wheat), supplemented with sodium (n-)butyrate (1.5 g/kg diet) or without butyrate addition. Wheat-based diet, rich in soluble non-starch polysaccharides, was supposed to increase caecal production of short chain fatty acids, primarily that of butyrate. The animals were slaughtered at 21 days of age by decapitation. Liver and muscle (m. gastrocnemius) samples were taken from each individual and thereafter homogenized and diluted to appropriate equal total protein concentrations. Proteins were separated by gel electrophoresis and were blotted to nitrocellulose membranes by turbo-blotting. For protein detection, phospho-Akt was examined in both liver and muscle samples by heterologous antibodies. As housekeeping protein, β-actin was assayed in liver and γ-actin in skeletal muscle

Mit Wölfen leben: Über die Rückkehr des Wolfes nach Sachsen

Aufgrund der langen Abwesenheit des Wolfes gibt es in der Bevölkerung viele Fragen bezüglich des Tieres und den möglichen Änderungen im alltäglichen Leben die sich aus der Anwesenheit von Wölfen ergeben könnten. Die vorliegende Broschüre soll helfen, die eindrucksvolle Tierart besser kennenzulernen, Sorgen und Ängste abzubauen und Konflikte möglichst zu vermeiden. Informationen für Jäger, Förster und Tierhalter in Sachsen und Brandenburg zum Wolf. Redaktionsschluss: 31.10.201

Mit Wölfen leben: Über die Rückkehr des Wolfes nach Sachsen

Aufgrund der langen Abwesenheit des Wolfes gibt es in der Bevölkerung viele Fragen bezüglich des Tieres und den möglichen Änderungen im alltäglichen Leben die sich aus der Anwesenheit von Wölfen ergeben könnten. Die vorliegende Broschüre soll helfen, die eindrucksvolle Tierart besser kennenzulernen, Sorgen und Ängste abzubauen und Konflikte möglichst zu vermeiden. Informationen für Jäger, Förster und Tierhalter in Sachsen und Brandenburg zum Wolf. Redaktionsschluss: 31.10.201

Impact of Endothelial Progenitor Cells in the Vascularization of Osteogenic Scaffolds

The microvascular endothelial network plays an important role in osteogenesis, bone regeneration and bone tissue engineering. Endothelial progenitor cells (EPCs) display a high angiogenic and vasculogenic potential. The endothelialization of scaffolds with endothelial progenitor cells supports vascularization and tissue formation. In addition, EPCs enhance the osteogenic differentiation and bone formation of mesenchymal stem cells (MSCs). This study aimed to investigate the impact of EPCs on vascularization and bone formation of a hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP)–fibrin scaffold. Three groups were designed: a scaffold-only group (A), a scaffold and EPC group (B), and a scaffold and EPC/MSC group (C). The HA/ß–TCP–fibrin scaffolds were placed in a porous titanium chamber permitting extrinsic vascularization from the surrounding tissue. Additionally, intrinsic vascularization was achieved by means of an arteriovenous loop (AV loop). After 12 weeks, the specimens were explanted and investigated by histology and CT. We were able to prove a strong scaffold vascularization in all groups. No differences regarding the vessel number and density were detected between the groups. Moreover, we were able to prove bone formation in the coimplantation group. Taken together, the AV loop is a powerful tool for vascularization which is independent from scaffold cellularization with endothelial progenitor cells’ prior implantation

What will my child's future hold? phenotypes of intellectual development in 2-8-year-olds with autism spectrum disorder.

We examined phenotypes of autism spectrum disorder (ASD) based on trajectories of intellectual development from early (ages 2-3 ½) to middle (ages 5-8) childhood in a recent clinically ascertained cohort. Participants included 102 children (82 males) initially diagnosed with ASD from the Autism Phenome Project longitudinal sample. Latent class growth analysis was used to identify distinct IQ trajectories. Baseline and developmental course differences among groups were assessed using univariate techniques and repeated measures regression models, respectively. A four class model best represented the data. Using the highest posterior probability, participants were assigned to High Challenges (25.5%), Stable Low (17.6%), Changers (35.3%), and Lesser Challenges (21.6%) groups. The High Challenges and Stable Low groups exhibited persistently low IQ, although, the High Challenges group experienced declines while the Stable Low group's scores remained more constant. Changers showed IQ improvement of > 2 standard deviations. The Lesser Challenges group had IQs in the average range at both times that were about 1 standard deviation higher at T2. In summation, 75% of the participants experienced some relative improvements in intellectual and/or other areas of functioning between ages 2 and 8 years. The Changers group demonstrated the most significant IQ change that was accompanied by adaptive communication improvement and declining externalizing symptoms. Only the Lesser Challenges group showed a significant reduction in ASD symptom severity, such that by age 8, 14% of them no longer met ADOS-2 criteria for ASD. All groups showed reductions in internalizing symptoms. Intervention history was not associated with group status. Autism Res 2018, 11: 121-132. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.Lay summaryWe examined how the IQs of children with autism spectrum disorder change between ages 2 and 8, and identified four patterns. Two groups exhibited persistently lower IQs. One group showed IQ increases of greater than 30 points with improved communicate abilities and declining disruptive behaviors. The final group had IQs in the average or better range at both time points, and 14% of them lost their diagnoses. Over half of the children experienced improved intellectual functioning between ages 2 and 8, whereas about 25% showed declines. Findings were not associated with intervention history

What will my child's future hold? phenotypes of intellectual development in 2-8-year-olds with autism spectrum disorder.

We examined phenotypes of autism spectrum disorder (ASD) based on trajectories of intellectual development from early (ages 2-3 ½) to middle (ages 5-8) childhood in a recent clinically ascertained cohort. Participants included 102 children (82 males) initially diagnosed with ASD from the Autism Phenome Project longitudinal sample. Latent class growth analysis was used to identify distinct IQ trajectories. Baseline and developmental course differences among groups were assessed using univariate techniques and repeated measures regression models, respectively. A four class model best represented the data. Using the highest posterior probability, participants were assigned to High Challenges (25.5%), Stable Low (17.6%), Changers (35.3%), and Lesser Challenges (21.6%) groups. The High Challenges and Stable Low groups exhibited persistently low IQ, although, the High Challenges group experienced declines while the Stable Low group's scores remained more constant. Changers showed IQ improvement of > 2 standard deviations. The Lesser Challenges group had IQs in the average range at both times that were about 1 standard deviation higher at T2. In summation, 75% of the participants experienced some relative improvements in intellectual and/or other areas of functioning between ages 2 and 8 years. The Changers group demonstrated the most significant IQ change that was accompanied by adaptive communication improvement and declining externalizing symptoms. Only the Lesser Challenges group showed a significant reduction in ASD symptom severity, such that by age 8, 14% of them no longer met ADOS-2 criteria for ASD. All groups showed reductions in internalizing symptoms. Intervention history was not associated with group status. Autism Res 2018, 11: 121-132. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.Lay summaryWe examined how the IQs of children with autism spectrum disorder change between ages 2 and 8, and identified four patterns. Two groups exhibited persistently lower IQs. One group showed IQ increases of greater than 30 points with improved communicate abilities and declining disruptive behaviors. The final group had IQs in the average or better range at both time points, and 14% of them lost their diagnoses. Over half of the children experienced improved intellectual functioning between ages 2 and 8, whereas about 25% showed declines. Findings were not associated with intervention history