Hypothalamic eIF2α signaling regulates food intake.

The reversible phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) is a highly conserved signal implicated in the cellular adaptation to numerous stresses such as the one caused by amino acid limitation. In response to dietary amino acid deficiency, the brain-specific activation of the eIF2α kinase GCN2 leads to food intake inhibition. We report here that GCN2 is rapidly activated in the mediobasal hypothalamus (MBH) after consumption of a leucine-deficient diet. Furthermore, knockdown of GCN2 in this particular area shows that MBH GCN2 activity controls the onset of the aversive response. Importantly, pharmacological experiments demonstrate that the sole phosphorylation of eIF2α in the MBH is sufficient to regulate food intake. eIF2α signaling being at the crossroad of stress pathways activated in several pathological states, our study indicates that hypothalamic eIF2α phosphorylation could play a critical role in the onset of anorexia associated with certain diseases.This work was supported by grants from “Fondation pour la Recherche Médicale,” “Société Française de Nutrition,” Ajinomoto Amino Acid Research Program (3ARP), and “Agence Nationale pour la Recherche.”This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.celrep.2014.01.00

Genetic changes in human pluripotent stem cells: implications for basic biology and regenerative medicine

Chronic tissue and organ failure caused by an injury, disease, ageing or congenital defects represents some of the most complex therapeutic challenges and poses a significant financial healthcare burden. Regenerative medicine strategies aim to fulfil the unmet clinical need by restoring the normal tissue function either through stimulating the endogenous tissue repair or by using transplantation strategies to replace the missing or defective cells. Stem cells represent an essential pillar of regenerative medicine efforts as they provide a source of progenitors or differentiated cells for use in cell replacement therapies. Whilst significant leaps have been made in controlling the stem cell fates and differentiating them to cell types of interest, transitioning bespoke cellular products from an academic environment to off-the-shelf clinical treatments brings about a whole new set of challenges which encompass manufacturing, regulatory and funding issues. Notwithstanding the need to resolve such issues before cell replacement therapies can benefit global healthcare, mounting progress in the field has highlighted regenerative medicine as a realistic prospect for treating some of the previously incurable conditions

Elevated Intraocular Pressure, Optic Nerve Atrophy, and Impaired Retinal Development in ODAG Transgenic Mice

PURPOSE. In an earlier study, a cDNA was cloned that showed abundant expression in the eye at postnatal day (P)2 but was downregulated at P10; it was named ODAG (ocular development-associated gene). Its biological function was examined by generating and analyzing transgenic mice overexpressing ODAG (ODAG Tg) in the eye and by identifying ODAG-binding proteins. METHODS. Transgenic mice were generated by using the mouse Crx promoter. EGFP was designed to be coexpressed with transgenic ODAG, to identify transgene-expressing cells. Overexpression of ODAG was confirmed by Northern and Western blot analysis. IOP was measured with a microneedle technique. The eyes were macroscopically examined and histologically analyzed. EGFP expression was detected by confocal microscope. Proteins associated with ODAG were isolated by pulldown assay in conjugation with mass spectrometry. RESULTS. Macroscopically, ODAG Tg exhibited gradual protrusion of the eyeballs. The mean IOP of ODAG Tg was significantly higher than that of wild-type (WT) littermates. Histologic analysis exhibited optic nerve atrophy and impaired retinal development in the ODAG Tg eye. EGFP was expressed highly in the presumptive outer nuclear layer and weakly in the presumptive inner nuclear layer in the ODAG Tg retina. Rab6-GTPase-activating protein (Rab6-GAP) and its substrate, Rab6, were identified as ODAG-binding proteins. CONCLUSIONS. Deregulated expression of ODAG in the eye induces elevated intraocular pressure and optic nerve atrophy and impairs retinal development, possibly by interfering with the Rab6/Rab6-GAP-mediated signaling pathway. These results provide new insights into the mechanisms regulating ocular development, and ODAG Tg would be a novel animal model for human diseases caused by ocular hypertension. (Invest Ophthalmol Vis Sci. 2009;50:242-248) DOI:10.1167/iovs.08-2206 O cular development is a complex process, involving several genes with expression that is strictly controlled in a spatial and temporal manner. Although several genes, including Pax6, Rx, and Crx, are essential for normal ocular formation, 1-3 the molecular mechanism(s) governing eye development has not been fully elucidated. To identify genes that are preferentially expressed in the developing eye, we performed a differential display using mRNAs extracted from postnatal day (P)2 and P10 mouse eyes. 4 At P2, ODAG was highly expressed in all the retinal layers (presumptive outer nuclear layer [ONL], presumptive inner nuclear layer [INL], and ganglion cell layer [GCL]), but at P7, its expression decreases, especially in the GCL, and at P14, no apparent expression is detected. To investigate, we generated transgenic mice overexpressing ODAG (ODAG Tg). The mouse Crx promoter, which directs transgene expression in photoreceptors, From th

Analysis of Transcriptional Regulatory Pathways of Photoreceptor Genes by Expression Profiling of the Otx2-Deficient Retina

In the vertebrate retina, the Otx2 transcription factor plays a crucial role in the cell fate determination of both rod and cone photoreceptors. We previously reported that Otx2 conditional knockout (CKO) mice exhibited a total absence of rods and cones in the retina due to their cell fate conversion to amacrine-like cells. In order to investigate the entire transcriptome of the Otx2 CKO retina, we compared expression profile of Otx2 CKO and wild-type retinas at P1 and P12 using microarray. We observed that expression of 101- and 1049-probe sets significantly decreased in the Otx2 CKO retina at P1 and P12, respectively, whereas, expression of 3- and 4149-probe sets increased at P1 and P12, respectively. We found that expression of genes encoding transcription factors involved in photoreceptor development, including Crx, Nrl, Nr2e3, Esrrb, and NeuroD, was markedly down-regulated in the Otx2 CKO at both P1 and P12. Furthermore, we identified three human retinal disease loci mapped in close proximity to certain down-regulated genes in the Otx2 CKO retina including Ccdc126, Tnfsf13 and Pitpnm1, suggesting that these genes are possibly responsible for these diseases. These transcriptome data sets of the Otx2 CKO retina provide a resource on developing rods and cones to further understand the molecular mechanisms underlying photoreceptor development, function and disease

BAC-Crx-EGFPトランスジェニックマウスを用いた胎児期網膜視細胞に発現する遺伝子の解析について

京都大学0048新制・課程博士博士(医学)甲第15271号医博第3471号新制||医||981(附属図書館)27749京都大学大学院医学研究科医学専攻(主査)教授 影山 龍一郎, 教授 吉村 長久, 教授 河野 憲二学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

BAC-Dkk3-EGFP Transgenic Mouse: An In Vivo Analytical Tool for Dkk3 Expression

Dickkopf (DKK) family proteins are secreted modulators of the Wnt signaling pathway and are capable of regulating the development of many organs and tissues. We previously identified Dkk3 to be a molecule predominantly expressed in the mouse embryonic retina. However, which cell expresses Dkk3 in the developing and mature mouse retina remains to be elucidated. To examine the precise expression of the Dkk3 protein, we generated BAC-Dkk3-EGFP transgenic mice that express EGFP integrated into the Dkk3 gene in a BAC plasmid. Expression analysis using the BAC-Dkk3-EGFP transgenic mice revealed that Dkk3 is expressed in retinal progenitor cells (RPCs) at embryonic stages and in Müller glial cells in the adult retina. Since Müller glial cells may play a potential role in retinal regeneration, BAC-Dkk3-EGFP mice could be useful for retinal regeneration studies

LPS Administration during Fertilization Affects Epigenetic Inheritance during Embryonic Development

Intrauterine inflammation can cause infertility by disrupting reproductive function. The pathogenesis underlying this process may primarily involve endotoxins from lipopolysaccharides (LPS), which are produced by Gram-negative bacteria. However, the long-term effects of endotoxins in mammalian pregnancy following LPS exposure during fertilization have not been clarified. In this study, we performed experiments to analyze the influence of LPS on early embryonic development and fetal development in mice. Mice uteruses were examined for the expression of genes related to the inflammatory response. The expression of Il-1β and Il-6 increased following the administration of 200 and 1000 µg/kg LPS. Exposure to LPS using in vitro fertilization (IVF) significantly decreased the embryonic developmental rate. A concentration of 100 µg/kg LPS significantly increased the placental weight and fetal crown –rump length (CRL), whereas a concentration of 200 µg/kg LPS significantly decreased the placenta weight and fetal weight in vivo. These findings indicate that maternal LPS during fertilization affects fetal development until the late stage of pregnancy. Thus, maternal endotoxins may affect epigenetic inheritance during embryonic development from the early to late stages of pregnancy

The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression

International audienc