Neuroendocrine transcriptional programs adapt dynamically to the supply and demand for neuropeptides as revealed in NSF mutant zebrafish

<p>Abstract</p> <p>Background</p> <p>Regulated secretion of specialized neuropeptides in the vertebrate neuroendocrine system is critical for ensuring physiological homeostasis. Expression of these cell-specific peptide markers in the differentiating hypothalamus commences prior to birth, often predating the physiological demand for secreted neuropeptides. The conserved function and spatial expression of hypothalamic peptides in vertebrates prompted us to search for critical neuroendocrine genes in newly hatched zebrafish larvae.</p> <p>Results</p> <p>We screened mutant 5 days post-fertilization zebrafish larvae that fail to undergo visually mediated background adaptation for disruption in hypothalamic <it>pomc </it>expression. To our surprise, the ATPase <it>N-ethylmaleimide sensitive factor </it>(<it>nsf</it>) was identified as an essential gene for maintenance of neuroendocrine transcriptional programs during the embryo-to-larva transition. Despite normal hypothalamic development in <it>nsf</it>^{<it>st</it>53}mutants, neuropeptidergic cells exhibited a dramatic loss of cell-specific markers by 5 days post-fertilization that is accompanied by elevated intracellular neuropeptide protein. Consistent with the role of NSF in vesicle-membrane fusion events and intracellular trafficking, cytoplasmic endoplasmic reticulum-like membranes accumulate in <it>nsf</it>^-/-hypothalamic neurons similar to that observed for <it>SEC18 </it>(<it>nsf ortholog</it>) yeast mutants. Our data support a model in which unspent neuropeptide cargo feedbacks to extinguish transcription in neuropeptidergic cells just as they become functionally required. In support of this model we found that <it>gnrh3 </it>transcripts remained unchanged in pre-migratory, non-functional gonadotropin-releasing hormone (GnRH) neurons in <it>nsf</it>^-/-zebrafish. Furthermore, <it>oxytocin-like </it>(<it>oxtl</it>, <it>intp</it>) transcripts, which are found in osmoreceptive neurons and persist in mutant zebrafish, drop precipitously after mutant zebrafish are acutely challenged with high salt.</p> <p>Conclusion</p> <p>Our analyses of <it>nsf </it>mutant zebrafish reveal an unexpected role for NSF in hypothalamic development, with mutant 5 days post-fertilization larvae exhibiting a stage-dependent loss of neuroendocrine transcripts and a corresponding accumulation of neuropeptides in the soma. Based on our collective findings, we speculate that neuroendocrine transcriptional programs adapt dynamically to both the supply and demand for neuropeptides to ensure adequate homeostatic responses.</p

Rooftop Photovoltaics Market Penetration Scenarios

The goal of this study was to model the market penetration of rooftop photovoltaics (PV) in the United States under a variety of scenarios, on a state-by-state basis, from 2007 to 2015

The effect of 24-week belimumab treatment withdrawal followed by treatment restart in patients with SLE: an open-label, non-randomised 52-week study

Abstract Background Treatment goals for patients with systemic lupus erythematosus (SLE) include minimising disease activity and reducing the risk of flares. Although belimumab is effective at reducing disease activity and risk of severe flares, it was previously unknown what the clinical effects were upon treatment discontinuation. The objective of this study was to assess the impact of temporary withdrawal of intravenous (IV) belimumab in patients with SLE. Methods This multicentre, open-label, non-randomised, 52-week study (GSK Study BEL116027; NCT02119156) recruited patients with SLE and stable low disease activity, of whom those on belimumab 10 mg/kg IV plus standard therapy either discontinued belimumab for 24 weeks and then restarted belimumab 10 mg/kg IV every 4 weeks (q4w) for 28 weeks (treatment holiday [TH] group), or continued on belimumab 10 mg/kg IV plus standard therapy q4w for 52 weeks (treatment continuation [TC] group). The primary endpoint was median time to first Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index (SELENA-SLEDAI) Flare Index flare. Secondary and other endpoints included rate of any flare, time to severe flare, time to renal flare and rebound (SELENA-SLEDAI score exceeding parent study baseline). Data on rebound phenomenon in patients with any disease level of SLE who had permanently withdrawn from further belimumab treatment (long-term discontinuation group [LTD]) were also assessed. Safety was assessed. Results The primary endpoint was not evaluable in the TH (n = 12) and TC (n = 29) groups as fewer than half of patients flared. Unadjusted flare rates per patient-year were 1.0 during treatment discontinuation and 0.3 during treatment restart (0.6 overall) in the TH group and 0.6 in the TC group; there were no severe or renal flares. No TH patients rebounded; 2 (6.9%) TC patients rebounded; 2 (5.1%) patients in the LTD group rebounded. There were no new safety signals. Conclusions Twenty-four-week belimumab discontinuation did not appear to increase the risk of flares or rebound in patients with low SLE disease activity; flare rates were low in both groups. Further studies may help to fully determine the effect of belimumab discontinuation. Trial registration ClinicalTrials.gov, NCT02119156 . Registered on April 21, 2014

Lipopolysaccharide and Tumor Necrosis Factor Regulate Parkin Expression via Nuclear Factor-Kappa B

Inflammation and oxidative stress have been implicated in the pathophysiology of Parkinson's disease (PD) and inhibition of microglial activation attenuates degeneration of dopaminergic (DA) neurons in animal models of PD. Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, cause autosomal recessive parkinsonism. While most studies on Parkin have focused on its function in neurons, here we demonstrate that Parkin mRNA and protein is detectable in brain-resident microglia and peripheral macrophages. Using pharmacologic and genetic approaches, we found that Parkin levels are regulated by inflammatory signaling. Specifically, exposure to LPS or Tumor Necrosis Factor (TNF) induced a transient and dose-dependent decrease in Parkin mRNA and protein in microglia, macrophages and neuronal cells blockable by inhibitors of Nuclear Factor-Kappa B (NF-κB) signaling and not observed in MyD88-null cells. Moreover, using luciferase reporter assays, we identified an NF-κB response element in the mouse parkin promoter responsible for mediating the transcriptional repression, which was abrogated when the consensus sequence was mutated. Functionally, activated macrophages from Parkin-null mice displayed increased levels of TNF, IL-1β, and iNOS mRNA compared to wild type macrophages but no difference in levels of Nrf2, HO-1, or NQO1. One implication of our findings is that chronic inflammatory conditions may reduce Parkin levels and phenocopy parkin loss-of-function mutations, thereby increasing the vulnerability for degeneration of the nigrostriatal pathway and development of PD

The Nutritional Induction of COUP-TFII Gene Expression in Ventromedial Hypothalamic Neurons Is Mediated by the Melanocortin Pathway

BACKGROUND: The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues. METHODOLOGY/PRINCIPAL FINDINGS: Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression. CONCLUSIONS/SIGNIFICANCE: These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation

The Neuro-Glial Properties of Adipose-Derived Adult Stromal (ADAS) Cells Are Not Regulated by Notch 1 and Are Not Derived from Neural Crest Lineage

We investigated whether adipose-derived adult stromal (ADAS) are of neural crest origin and the extent to which Notch 1 regulates their growth and differentiation. Mouse ADAS cells cultured in media formulated for neural stem cells (NSC) displayed limited capacity for self-renewal, clonogenicity, and neurosphere formation compared to NSC from the subventricular zone in the hippocampus. Although ADAS cells expressed Nestin, GFAP, NSE and Tuj1 in vitro, exposure to NSC differentiation supplements did not induce mature neuronal marker expression. In contrast, in mesenchymal stem cell (MSC) media, ADAS cells retained their ability to proliferate and differentiate beyond 20 passages and expressed high levels of Nestin. In neuritizing cocktails, ADAS cells extended processes, downregulated Nestin expression, and displayed depolarization-induced Ca2+ transients but no spontaneous or evoked neural network activity on Multi-Electrode Arrays. Deletion of Notch 1 in ADAS cell cultures grown in NSC proliferation medium did not significantly alter their proliferative potential in vitro or the differentiation-induced downregulation of Nestin. Co-culture of ADAS cells with fibroblasts that stably expressed the Notch ligand Jagged 1 or overexpression of the Notch intracellular domain (NICD) did not alter ADAS cell growth, morphology, or cellular marker expression. ADAS cells did not display robust expression of neural crest transcription factors or genes (Sox, CRABP2, and TH); and lineage tracing analyses using Wnt1–Cre;Rosa26R-lacZ or -EYFP reporter mice confirmed that fewer than 2% of the ADAS cell population derived from a Wnt1-positive population during development. In summary, although media formulations optimized for MSCs or NSCs enable expansion of mouse ADAS cells in vitro, we find no evidence that these cells are of neural crest origin, that they can undergo robust terminal differentiation into functionally mature neurons, and that Notch 1 is likely to be a key regulator of their cellular and molecular characteristics

LSD but not lisuride disrupts prepulse inhibition in rats by activating the 5-HT2A receptor

Compounds that activate the 5-HT2A receptor, such as lysergic acid diethylamide (LSD), act as hallucinogens in humans. One notable exception is the LSD congener lisuride, which does not have hallucinogenic effects in humans even though it is a potent 5-HT2A agonist. LSD and other hallucinogens have been shown to disrupt prepulse inhibition (PPI), an operational measure of sensorimotor gating, by activating 5-HT2A receptors in rats. We tested whether lisuride disrupts PPI in male Sprague–Dawley rats. Experiments were also conducted to identify the mechanism(s) responsible for the effect of lisuride on PPI and to compare the effects of lisuride to those of LSD. Confirming a previous report, LSD (0.05, 0.1, and 0.2 mg/kg, s.c.) reduced PPI, and the effect of LSD was blocked by pretreatment with the selective 5-HT2A antagonist MDL 11,939. Administration of lisuride (0.0375, 0.075, and 0.15 mg/kg, s.c.) also reduced PPI. However, the PPI disruption induced by lisuride (0.075 mg/kg) was not blocked by pretreatment with MDL 11,939 or the selective 5-HT1A antagonist WAY-100635 but was prevented by pretreatment with the selective dopamine D2/D3 receptor antagonist raclopride (0.1 mg/kg, s.c). The effect of LSD on PPI is mediated by the 5-HT2A receptor, whereas activation of the 5-HT2A receptor does not appear to contribute to the effect of lisuride on PPI. These findings demonstrate that lisuride and LSD disrupt PPI via distinct receptor mechanisms and provide additional support for the classification of lisuride as a non-hallucinogenic 5-HT2A agonist

GABAergic and Cortical and Subcortical Glutamatergic Axon Terminals Contain CB1 Cannabinoid Receptors in the Ventromedial Nucleus of the Hypothalamus

Background: Type-1 cannabinoid receptors (CB1R) are enriched in the hypothalamus, particularly in the ventromedial hypothalamic nucleus (VMH) that participates in homeostatic and behavioral functions including food intake. Although CB1R activation modulates excitatory and inhibitory synaptic transmission in the brain, CB1R contribution to the molecular architecture of the excitatory and inhibitory synaptic terminals in the VMH is not known. Therefore, the aim of this study was to investigate the precise subcellular distribution of CB1R in the VMH to better understand the modulation exerted by the endocannabinoid system on the complex brain circuitries converging into this nucleus. Methodology/Principal Findings: Light and electron microscopy techniques were used to analyze CB1R distribution in the VMH of CB1R-WT, CB1R-KO and conditional mutant mice bearing a selective deletion of CB1R in cortical glutamatergic (Glu-CB1R-KO) or GABAergic neurons (GABA-CB1R-KO). At light microscopy, CB1R immunolabeling was observed in the VMH of CB1R-WT and Glu-CB1R-KO animals, being remarkably reduced in GABA-CB1R-KO mice. In the electron microscope, CB1R appeared in membranes of both glutamatergic and GABAergic terminals/preterminals. There was no significant difference in the percentage of CB1R immunopositive profiles and CB1R density in terminals making asymmetric or symmetric synapses in CB1R-WT mice. Furthermore, the proportion of CB1R immunopositive terminals/preterminals in CB1R-WT and Glu-CB1R-KO mice was reduced in GABA-CB1R-KO mutants. CB1R density was similar in all animal conditions. Finally, the percentage of CB1R labeled boutons making asymmetric synapses slightly decreased in Glu-CB1R-KO mutants relative to CB1R-WT mice, indicating that CB1R was distributed in cortical and subcortical excitatory synaptic terminals. Conclusions/Significance: Our anatomical results support the idea that the VMH is a relevant hub candidate in the endocannabinoid-mediated modulation of the excitatory and inhibitory neurotransmission of cortical and subcortical pathways regulating essential hypothalamic functions for the individual's survival such as the feeding behavior.L. Reguero is in receipt of a Predoctoral Fellowship from the Basque Country Government (BFI 07.286); I. Buceta is in receipt of a Predoctoral Fellowship from the Basque Country University. Dr. Pedro Grandes' laboratory is supported by The Basque Country Government grant GIC07/70-IT-432-07, by Ministerio de Ciencia e Innovacion (SAF2009-07065) and by Red de Trastornos Adictivos, RETICS, Instituto de Salud Carlos III, MICINN, grant RD07/0001/2001. Dr. Giovanni Marsicano's laboratory is supported by AVENIR/INSERM (with the Fondation Bettencourt-Schueller), by ANR (ANR-06-NEURO-043-01), by European Foundation for the Study of Diabetes (EFSD), by the EU-FP7 (REPROBESITY, contract number HEALTH-F2-2008-223713) and European Commission Coordination Action ENINET (contract number LSHM-CT-2005-19063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Integrated Expression Profiling and Genome-Wide Analysis of ChREBP Targets Reveals the Dual Role for ChREBP in Glucose-Regulated Gene Expression

The carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper transcription factor, plays a critical role in the control of lipogenesis in the liver. To identify the direct targets of ChREBP on a genome-wide scale and provide more insight into the mechanism by which ChREBP regulates glucose-responsive gene expression, we performed chromatin immunoprecipitation-sequencing and gene expression analysis. We identified 1153 ChREBP binding sites and 783 target genes using the chromatin from HepG2, a human hepatocellular carcinoma cell line. A motif search revealed a refined consensus sequence (CABGTG-nnCnG-nGnSTG) to better represent critical elements of a functional ChREBP binding sequence. Gene ontology analysis shows that ChREBP target genes are particularly associated with lipid, fatty acid and steroid metabolism. In addition, other functional gene clusters related to transport, development and cell motility are significantly enriched. Gene set enrichment analysis reveals that ChREBP target genes are highly correlated with genes regulated by high glucose, providing a functional relevance to the genome-wide binding study. Furthermore, we have demonstrated that ChREBP may function as a transcriptional repressor as well as an activator

Genetic labeling of steroidogenic factor‐1 (SF‐1) neurons in mice reveals ventromedial nucleus of the hypothalamus (VMH) circuitry beginning at neurogenesis and development of a separate non‐SF‐1 neuronal cluster in the ventrolateral VMH

The ventromedial nucleus of the hypothalamus (VMH) influences a wide variety of physiological responses. Here, using two distinct but complementary genetic tracing approaches in mice, we describe the development of VMH efferent projections, as marked by steroidogenic factor-1 (SF-1; NR5A1). SF-1 neurons were visualized by Tau-green fluorescent protein (GFP) expressed from the endogenous Sf-1 locus (Sf-1(TauGFP)) or by crossing the transgenic Sf1:Cre driver to a GFP reporter strain (Z/EG(Sf1:Cre)). Strikingly, VMH projections were visible early, at embryonic (E) 10.5, when few postmitotic SF1 neurons have been born, suggesting that formation of VMH circuitry begins at the onset of neurogenesis. At E14.5, comparison of these two reporter lines revealed that SF1-positive neurons in the ventrolateral VMH (VMH(vl)) persist in Z/EG(Sf1:Cre) embryos but are virtually absent in Sf-1(TauGFP). Therefore, although the entire VMH including the VMH(vl) shares a common lineage, the VMH(vl) further differentiates into a neuronal cluster devoid of SF-1. At birth, extensive VMH projections to broad regions of the brain were observed in both mouse reporter lines, matching well with those previously discovered by injection of axonal anterograde tracers in adult rats. In summary, our genetic tracing studies show that VMH efferent projections are highly conserved in rodents and are established far earlier than previously appreciated. Moreover, our results imply that neurons in the VMH(vl) adopt a distinct fate early in development, which might underlie the unique physiological functions associated with this VMH subregion