Interference with the Cannabinoid Receptor CB1R Results in Miswiring of GnRH3 and AgRP1 Axons in Zebrafish Embryos

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron’s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake

Pathogenesis of peroxisomal deficiency disorders (Zellweger syndrome) may be mediated by misregulation of the GABAergic system via the diazepam binding inhibitor

BACKGROUND: Zellweger syndrome (ZS) is a fatal inherited disease caused by peroxisome biogenesis deficiency. Patients are characterized by multiple disturbances of lipid metabolism, profound hypotonia and neonatal seizures, and distinct craniofacial malformations. Median live expectancy of ZS patients is less than one year. While the molecular basis of peroxisome biogenesis and metabolism is known in considerable detail, it is unclear how peroxisome deficiency leads to the most severe neurological symptoms. Recent analysis of ZS mouse models has all but invalidated previous hypotheses. HYPOTHESIS: We suggest that a regulatory rather than a metabolic defect is responsible for the drastic impairment of brain function in ZS patients. TESTING THE HYPOTHESIS: Using microarray analysis we identify diazepam binding inhibitor/acyl-CoA binding protein (DBI) as a candidate protein that might be involved in the pathogenic mechanism of ZS. DBI has a dual role as a neuropeptide antagonist of GABA(A) receptor signaling in the brain and as a regulator of lipid metabolism. Repression of DBI in ZS patients could result in an overactivation of GABAergic signaling, thus eventually leading to the characteristic hypotonia and seizures. The most important argument for a misregulation of GABA(A) in ZS is, however, provided by the striking similarity between ZS and "benzodiazepine embryofetopathy", a malformation syndrome observed after the abuse of GABA(A) agonists during pregnancy. IMPLICATIONS OF THE HYPOTHESIS: We present a tentative mechanistic model of the effect of DBI misregulation on neuronal function that could explain some of the aspects of the pathology of Zellweger syndrome

Antagonism of neurosteroid modulation of native gamma-aminobutyric acid receptors by (3alpha,5alpha)-17-phenylandrost-16-en-3-ol.

Endogenous pregnane neurosteroids are allosteric modulators at ?-aminobutyric acid type-A (GABAA) receptors at nanomolar concentrations. There is direct evidence for multiple distinct neurosteroid binding sites on GABAA receptors, dependent upon subunit composition and stoichiometry. This view is supported by the biphasic kinetics of various neuroactive steroids, enantioselectivity of some neurosteroids, selective mutation studies of recombinantly expressed receptors and the selectivity of the neurosteroid antagonist (3?,5?)-17-phenylandrost-16-en-3-ol (17PA) on 5?-pregnane steroid effects on recombinant GABAA receptors expressed in Xenopus oocytes and native receptors in dissociated neurons. However, it is unclear whether this antagonist action is present in a mature mammalian system. The present study evaluated the antagonist activity of 17PA on neurosteroid agonists both in vivo and in vitro by examining the effects of 17PA on 5?-pregnane-induced sedation in rats, native mature GABAA receptor ion channels utilizing the chloride flux assay and further studies in recombinant ?1?2?2 receptors. The data show that 17PA preferentially inhibits 3?,5?-THP vs. alphaxalone in vivo, preferentially inhibits 3?,5?-THDOC vs. alphaxalone potentiation of GABA-mediated Cl? uptake in adult cerebral cortical synaptoneurosomes, but shows no specificity for 3?,5?-THDOC vs. alphaxalone in recombinant ?1?2?2 receptors. These data provide further evidence of the specificity of 17PA and the heterogeneity of neurosteroid recognition sites on GABAA receptors in the CNS

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812)

Parma consensus statement on metabolic disruptors

A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16–18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as “metabolic disruptors”, in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome

Combined in silico and in vitro studies of phthalates and organophosphorus compounds: effects on peroxisome proliferator activated receptors (PPARs) signalling pathways

Increasing evidence suggests that some environmental contaminants, including known endocrine disrupting chemicals (EDCs), are able to interfere with metabolic pathways by interacting with nuclear receptors. Recently, it has been demonstrated that phthalates and organometallic compounds bind to peroxisome proliferator activated receptors (PPARs) resulting in modulation of lipid metabolism at both the systemic and peripheral level. However, little is known about the metabolic impact of these pollutants on fish. In this context, we performed an in silico docking screen of an EDCs database to identify a set of ligands with conveniently high affinity for the PPARs. Kd values in the nanomolar to micromolar range, generated by the in silico model, suggest that piscine PPARs may be activated by phthalates (e.g. di-isononylphpthalate -DiNP; Di-isodecyl phthalate-DiDP) and some organophosphorus compounds (e.g. tri-m-cresyl phosphate-TmCP) at concentrations similar to those activating the homologous mammalian receptors. Because natural endogenous ligands for PPARs are involved in lipid homeostasis, we assessed the effects of compounds identified using in silico screening on Sparus aurata hepatocytes primary cultures. Generally, exposure of hepatocytes to 0.1, 1 or 10 microM of DiNP, DiDP or TmCP consistently increased both PPAR and its heterodimeric partner Retinoid X Receptor (RXR) mRNA levels at 48 h. In addition, all compounds investigated produced significant increases in the expression of the PPAR target genes, carnitine palmitoyltransferase (CPT) isoforms. In general our data show that phthalates and TmCP modulated PPAR signaling in the seabream in vitro system. The results also suggest the potential involvement of these pollutants in the modulation of mitochondrial fatty acid oxidation

Endocrine disruptors and nuclear receptors in the control of neural progenitors proliferation

Endocrine-disrupting chemicals (EDCs), in particular plasticizers present in food wrapping materials, are very relevant to human health. Several studies have shown that EDCs may pose the greatest risk during prenatal and early postnatal development, especially when binding to members of the nuclear receptor (NR) superfamily. EDCs can mimic/suppress estrogen actions in the developing brain by binding to estrogen receptors (ERs), and also interfere with neural progenitors (NPs) homeostasis through activation of peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs). We used the ST14A immortalized neural progenitor cell line to simulate the effects of prenatal NPs exposure to EDCs. We found by RT-PCR that the following NRs were expressed: ERα, ERβ, PPARα, PPARβ, PPARγ, RXR. Plasticizers were chosen based on their computational affinity for these receptors. NPs were exposed for 24-48 hours to endogenous and synthetic estrogens (17-β-estradiol and ethinyl estradiol) and to the following plasticizers: Bisphenol A, Diisononyl phthalate, Diisodecyl phthalate, Diethileneglicol benzoate. Both estrogens were able to increase cell proliferation by about 30% at 24hrs. Bisphenol-A (10nM) had an estrogen-like behavior in enhancing cell number, but its action was still significant at 48hrs. At 200nM concentration, all plasticizers determined an early increase in proliferation, however only DiDP and DGB were able to sustain this positive modulation for 48hrs. Ongoing studies in our lab will determine the cellular pathways activated by these EDCs in NPs, in order to earn greater insight into how these molecules may influence and perturb the neurogenic process