Differential associations of leptin with adiposity across early childhood

Objective: We examined associations of perinatal and 3-year leptin with weight gain and adiposity through 7 years. Design and Methods In Project Viva, we assessed plasma leptin from mothers at 26–28 weeks’ gestation (n=893), umbilical cord vein at delivery (n=540), and children at 3 years (n=510) in relation to body mass index (BMI) z-score, waist circumference, skinfold thicknesses, and dual X-ray absorptiometry body fat. Results: 50.1% of children were male and 29.5% non-white. Mean(SD) maternal, cord, and age 3 leptin concentrations were 22.9(14.2), 8.8(6.4), and 1.8(1.7) ng/mL, respectively, and 3- and 7-year BMI z-scores were 0.46(1.00) and 0.35(0.97), respectively. After adjusting for parental and child characteristics, higher maternal and cord leptin was associated with less 3- year adiposity. For example, mean 3-year BMI z-score was 0.5 lower (95%CI:−0.7,−0.2; p-trend=0.003) among children whose mothers’ leptin concentrations were in the top vs. bottom quintile. In contrast, higher age 3 leptin was associated with greater weight gain and adiposity through age 7 [e.g., change in BMI z-score from 3 to 7 years was 0.2 units (95%CI:−0.0,0.4; p-trend=0.05)]. Conclusions: Higher perinatal leptin was associated with lower 3-year adiposity, whereas higher age 3 leptin was associated with greater weight gain and adiposity by 7 years

The REST remodeling complex protects genomic integrity during embryonic neurogenesis

The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation

Reductions in hypothalamic Gfap expression, glial cells and α-tanycytes in lean and hypermetabolic Gnasxl-deficient mice

BACKGROUND: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Gαs, termed XLαs, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. RESULTS: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive α-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3(rd) ventricle, while β-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. CONCLUSION: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since α-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections

Methylation Defect in Imprinted Genes Detected in Patients with an Albright's Hereditary Osteodystrophy Like Phenotype and Platelet Gs Hypofunction

Pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of hormones that activate Gsalpha, encoded by the imprinted GNAS gene. PHP-Ib patients have isolated Parathormone (PTH) resistance and GNAS epigenetic defects while PHP-Ia cases present with hormone resistance and characteristic features jointly termed as Albright's Hereditary Osteodystrophy (AHO) due to maternally inherited GNAS mutations or similar epigenetic defects as found for PHP-Ib. Pseudopseudohypoparathyroidism (PPHP) patients with an AHO phenotype and no hormone resistance and progressive osseous heteroplasia (POH) cases have inactivating paternally inherited GNAS mutations.We here describe 17 subjects with an AHO-like phenotype that could be compatible with having PPHP but none of them carried Gsalpha mutations. Functional platelet studies however showed an obvious Gs hypofunction in the 13 patients that were available for testing. Methylation for the three differentially methylated GNAS regions was quantified via the Sequenom EpiTYPER. Patients showed significant hypermethylation of the XL amplicon compared to controls (36 ± 3 vs. 29 ± 3%; p<0.001); a pattern that is reversed to XL hypomethylation found in PHPIb. Interestingly, XL hypermethylation was associated with reduced XLalphaS protein levels in the patients' platelets. Methylation for NESP and ExonA/B was significantly different for some but not all patients, though most patients have site-specific CpG methylation abnormalities in these amplicons. Since some AHO features are present in other imprinting disorders, the methylation of IGF2, H19, SNURF and GRB10 was quantified. Surprisingly, significant IGF2 hypermethylation (20 ± 10 vs. 14 ± 7%; p<0.05) and SNURF hypomethylation (23 ± 6 vs. 32 6%; p<0.001) was found in patients vs. controls, while H19 and GRB10 methylation was normal.In conclusion, this is the first report of methylation defects including GNAS in patients with an AHO-like phenotype without endocrinological abnormalities. Additional studies are still needed to correlate the methylation defect with the clinical phenotype

Postnatal Changes in the Expression Pattern of the Imprinted Signalling Protein XLαs Underlie the Changing Phenotype of Deficient Mice

The alternatively spliced trimeric G-protein subunit XLαs, which is involved in cAMP signalling, is encoded by the Gnasxl transcript of the imprinted Gnas locus. XLαs deficient mice show neonatal feeding problems, leanness, inertia and a high mortality rate. Mutants that survive to weaning age develop into healthy and fertile adults, which remain lean despite elevated food intake. The adult metabolic phenotype can be attributed to increased energy expenditure, which appears to be caused by elevated sympathetic nervous system activity. To better understand the changing phenotype of Gnasxl deficient mice, we compared XLαs expression in neonatal versus adult tissues, analysed its co-localisation with neural markers and characterised changes in the nutrient-sensing mTOR1-S6K pathway in the hypothalamus. Using a newly generated conditional Gnasxl lacZ gene trap line and immunohistochemistry we identified various types of muscle, including smooth muscle cells of blood vessels, as the major peripheral sites of expression in neonates. Expression in all muscle tissues was silenced in adults. While Gnasxl expression in the central nervous system was also developmentally silenced in some midbrain nuclei, it was upregulated in the preoptic area, the medial amygdala, several hypothalamic nuclei (e.g. arcuate, dorsomedial, lateral and paraventricular nuclei) and the nucleus of the solitary tract. Furthermore, expression was detected in the ventral medulla as well as in motoneurons and a subset of sympathetic preganglionic neurons of the spinal cord. In the arcuate nucleus of Gnasxl-deficient mice we found reduced activity of the nutrient sensing mTOR1-S6K signalling pathway, which concurs with their metabolic status. The expression in these brain regions and the hypermetabolic phenotype of adult Gnasxl-deficient mice imply an inhibitory function of XLαs in energy expenditure and sympathetic outflow. By contrast, the neonatal phenotype of mutant mice appears to be due to a transient role of XLαs in muscle tissues

Spinal cord expression of <i>Gnasxl</i>.

<p>(<b>A–C</b>) Immunohistochemistry of neonatal (P1) thoracic spinal cords from <i>Nestin-Cre/+</i>; +/<i>XLlacZGT</i> mice using an anti-βGalactosidase antibody (red, A and C), or from wild-type mice using an anti-XLαs antibody (purple DAB/Ni staining, B). Transverse (A, B) and sagittal (C) sections are shown. <i>Gnasxl</i> expression is detected in scattered neurons of the intermediolateral region as well as in the ventrolateral, motoneuron containing area. (<b>D–G</b>) Co-staining for XL-βGal fusion protein (red) and Choline acetyltransferase (ChAT; green) on sagittal sections from neonatal (D, E) and adult (F, G) spinal cords. ChAT marks cholinergic sympathetic preganglionic neurons of the intermediolateral layer as well as ventrolateral motoneurons. While XLαs and ChAT are co-expressed in the majority of motoneurons, they are only occasionally co-localised in neurons of the intermediolateral layer (white arrows in (E–G)). Epifluorescent (D, F, G) and confocal (E) images are shown. (G) Shows a magnification of the area indicated in (F). Note the plasma membrane association of XL-βGal fusion protein, due to palmitoylation of the XL domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#pone.0029753-Ugur1" target="_blank">[97]</a>. Neonatal tissues in (D, E) were obtained from <i>Nestin-Cre</i>/+; +/<i>XLlacZGT</i> offspring, while adult samples in (F, G) were derived from <i>CMV-Cre</i>/+; +/<i>XLlacZGT</i> mice.</p

Simplified scheme of the imprinted murine <i>Gnas</i> locus.

<p>The differential, parental allele-specific expression of the various transcripts of the locus is indicated by arrows. Open and filled boxes represent non-coding and coding exons, respectively. Spliced transcripts and encoded proteins are only shown for <i>Gnas</i> and <i>Gnasxl</i> above and below the parental alleles. On the paternal allele <i>Gnas</i> expression is silenced in specific tissues (shaded <i>Gnas</i> exon 1 box). <i>Gnasxl</i> splices in frame onto exon 2 and encodes an ‘extra large’ α-subunit. Splicing onto exon N1 is limited to neural tissue, results in premature termination of the open reading frame and expression of a truncated XLN1 protein. The alternatively spliced exon A20 is found in a minority of <i>Gnasxl</i> transcripts, mainly in combination with exon N1, and introduces a frameshift and early termination codon in exon 2. <i>Nespas</i> and <i>exon 1A</i> promoters generate non-coding regulatory RNAs. The <i>Nesp</i> transcript splices onto exon 2 and has a regulatory as well as protein coding function. Regions of differential DNA methylation, including the imprinting control region (ICR) at <i>Nespas</i>, are symbolized by M.</p

Expression of <i>Gnasxl</i> in neonatal brain (P1).

<p>(<b>A–H</b>) XGal staining of neonatal brain sections (A–F coronal sections; G, H sagittal sections), indicating XL-βGal fusion protein activity in <i>Nestin-Cre</i>/+; +/<i>XLlacZGT</i> offspring. Images are shown in posterior to anterior order covering the medulla oblongata (A, B), pons (C, D) and hypothalamus (E, F). Sagittal sections are located peripherally (G) and close to the midline (H). Red arrows indicate expression in blood vessels (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#pone-0029753-g007" target="_blank">Figure 7A–G</a>). (<b>I–M</b>) Immunohistochemistry for XLαs on coronal sections of the medulla oblongata (I, J), pons (K) and hypothalamus (L, M) of wild-type brain. 5N – motor trigeminal nucleus, 7N – facial nucleus, 12N – hypoglossal nucleus, A7 – A7 noradrenaline cells, Amb – ambiguus nucleus, Amy – medial amygdaloid nucleus, Arc – arcuate hypothalamic nucleus, DMH – dorsomedial hypothalamic nucleus, Gi – gigantocellular reticular nucleus, LC – locus coeruleus, LDTg – laterodorsal tegmental nucleus, LH – lateral hypothalamic area, PreOp – preoptic area, PTg – pedunculopontine reticular nucleus, PVH – paraventricular hypothalamic nucleus, ROb – raphe obscurus nucleus, SCh – suprachiasmatic nucleus, SubC – subcoeruleus nucleus. Scale bars = 500 µm or as indicated.</p

Generation of a conditional gene trap mouse line for

<p> (<b>A</b>) The targeting construct comprises the <i>Gnas</i> exon 2 splice acceptor (Ex2SA) fused in frame to LacZ-pA, an frt-flanked <i>neo</i>^r cassette and pairs of loxP/lox2272 sites assembled in head-to-head orientation. The gene trap cassette was inserted in the non-functional (antisense) orientation at the rarely used A20 exon position (see D–F). Cre recombination results in inversion at compatible lox sites followed by excision between head-to-tail sites, which results in a stably integrated gene trap that attracts splicing from <i>Gnasxl</i> exon 1. The resulting XL domain – β-Galactosidase (XL-βGal) fusion protein lacks XLαs function, but retains β-Galactosidase activity. Relative positions of probes and restriction sites used in Southern-blots are shown, as are PCR primers (arrows) used in genotyping. (<b>B</b>) Southern blots showing a correctly targeted ES-cell clone (<i>Spe</i>I: WT = 16.1 kbp, targeted = 20.4 kbp; <i>Afl</i>II: targeted = 13.7 kbp; <i>Mfe</i>I: targeted = 8.8 kb). (<b>C</b>) Genotyping PCRs for deletion of the <i>neo</i>^r cassette via <i>Flpe</i> mice (left), and for Cre recombinase mediated inversion of the gene trap (right). The Flpe / Cre status of the samples is given above the lanes. For primer locations see (A). (<b>D</b>) Scheme indicating splicing of the rarely used A20 exon in full-length <i>Gnasxl</i> and neural-specific <i>XLN1</i> transcripts. Arrows indicate primers used in (E). (<b>E</b>) RT-PCR from wild-type brain using a common <i>Gnasxl</i> exon 1 primer combined with reverse primers in exon 5 (full-length <i>Gnasxl</i>) or exon N1 (<i>XLN1</i>). Exon A20-containing products (size increase: 95 bp) are indicated by asterisks above the respective bands and are hardly detectable in full-length transcripts (XL lanes), but are more prominent in <i>XLN1</i> transcripts (N1 lanes). (<b>F</b>) Inclusion of the A20 exon results in a frame shift and termination codon in exon 2. The translated <i>Gnasxl</i>-A20 sequence from (E) is shown: <i>Gnasxl</i> exon 1 sequence (not highlighted), exon A20 sequence (highlighted black), exon 2 sequence (highlighted grey).</p