Image_1_Oxytocin receptors in the Magel2 mouse model of autism: Specific region, age, sex and oxytocin treatment effects.tif

The neurohormone oxytocin (OXT) has been implicated in the regulation of social behavior and is intensively investigated as a potential therapeutic treatment in neurodevelopmental disorders characterized by social deficits. In the Magel2-knockout (KO) mouse, a model of Schaaf-Yang Syndrome, an early postnatal administration of OXT rescued autistic-like behavior and cognition at adulthood, making this model relevant for understanding the actions of OXT in (re)programming postnatal brain development. The oxytocin receptor (OXTR), the main brain target of OXT, was dysregulated in the hippocampus of Magel2-KO adult males, and normalized upon OXT treatment at birth. Here we have analyzed male and female Magel2-KO brains at postnatal day 8 (P8) and at postnatal day 90 (P90), investigating age, genotype and OXT treatment effects on OXTR levels in several regions of the brain. We found that, at P8, male and female Magel2-KOs displayed a widespread, substantial, down-regulation of OXTR levels compared to wild type (WT) animals. Most intriguingly, the postnatal OXT treatment did not affect Magel2-KO OXTR levels at P8 and, consistently, did not rescue the ultrasonic vocalization deficits observed at this age. On the contrary, the postnatal OXT treatment reduced OXTR levels at P90 in male Magel2-KO in a region-specific way, restoring normal OXTR levels in regions where the Magel2-KO OXTR was upregulated (central amygdala, hippocampus and piriform cortex). Interestingly, Magel2-KO females, previously shown to lack the social deficits observed in Magel2-KO males, were characterized by a different trend in receptor expression compared to males; as a result, the dimorphic expression of OXTR observed in WT animals, with higher OXTR expression observed in females, was abolished in Magel2-KO mice. In conclusion, our data indicate that in Magel2-KO mice, OXTRs undergo region-specific modifications related to age, sex and postnatal OXT treatment. These results are instrumental to design precisely-timed OXT-based therapeutic strategies that, by acting at specific brain regions, could modify the outcome of social deficits in Schaaf-Yang Syndrome patients.</p

Table_4_Oxytocin receptors in the Magel2 mouse model of autism: Specific region, age, sex and oxytocin treatment effects.XLSX

The neurohormone oxytocin (OXT) has been implicated in the regulation of social behavior and is intensively investigated as a potential therapeutic treatment in neurodevelopmental disorders characterized by social deficits. In the Magel2-knockout (KO) mouse, a model of Schaaf-Yang Syndrome, an early postnatal administration of OXT rescued autistic-like behavior and cognition at adulthood, making this model relevant for understanding the actions of OXT in (re)programming postnatal brain development. The oxytocin receptor (OXTR), the main brain target of OXT, was dysregulated in the hippocampus of Magel2-KO adult males, and normalized upon OXT treatment at birth. Here we have analyzed male and female Magel2-KO brains at postnatal day 8 (P8) and at postnatal day 90 (P90), investigating age, genotype and OXT treatment effects on OXTR levels in several regions of the brain. We found that, at P8, male and female Magel2-KOs displayed a widespread, substantial, down-regulation of OXTR levels compared to wild type (WT) animals. Most intriguingly, the postnatal OXT treatment did not affect Magel2-KO OXTR levels at P8 and, consistently, did not rescue the ultrasonic vocalization deficits observed at this age. On the contrary, the postnatal OXT treatment reduced OXTR levels at P90 in male Magel2-KO in a region-specific way, restoring normal OXTR levels in regions where the Magel2-KO OXTR was upregulated (central amygdala, hippocampus and piriform cortex). Interestingly, Magel2-KO females, previously shown to lack the social deficits observed in Magel2-KO males, were characterized by a different trend in receptor expression compared to males; as a result, the dimorphic expression of OXTR observed in WT animals, with higher OXTR expression observed in females, was abolished in Magel2-KO mice. In conclusion, our data indicate that in Magel2-KO mice, OXTRs undergo region-specific modifications related to age, sex and postnatal OXT treatment. These results are instrumental to design precisely-timed OXT-based therapeutic strategies that, by acting at specific brain regions, could modify the outcome of social deficits in Schaaf-Yang Syndrome patients.</p

Functional analysis of basal and agonist induced total IP accumulation in HEK293 cells transiently expressing the double mutant E129V/E240V of human TP receptor without and following rescue with 1

<p> µ<b>M SQ29,548 for 18 </b><b>h.</b> A. Total IP accumulation in basal condition and following 30 min stimulation with 1 µM U46619. B. Concentration-response curves of U46619. Error bars represent mean±SE of at least two independent experiments each performed in duplicates.</p

Total IP dose-response parameters for different agonists in HEK293 cells transiently expressing the WT or the E129V TP receptor.

<p>Values of EC₅₀ and E_max were obtained by simultaneous analysis with GraphPad Prism (see Data and statistical analysis) of at least three independent experiments each performed in duplicates.</p>a<p>Activity ratio was calculated as the ratio of the EC₅₀ for the WT TP receptor over the EC₅₀ of the E129V mutant.</p>**<p>p<0.01 vs. WT.</p

Agonist-induced total IP accumulation in HEK293 cells transiently expressing equal amounts of the WT (A) or E129V mutant (B) of human TP receptor.

<p>Total IP accumulation was measured after incubation in the absence (basal) or presence of increasing concentrations of the indicated agonists for 30 min. Data are expressed as dpm/well. Error bars represent mean±SE of at least three independent experiments each performed in duplicates or triplicates (For the sake of clarity, in panel B, error bar direction of U46619 and I-BOP data is above and below, respectively). Curves are computer generated from the simultaneous analysis of at least three independent experiments. Values for EC₅₀ and significant differences from WT are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060475#pone-0060475-t002" target="_blank">Table 2</a>.</p

BRET² measurement of Gα_qβ₁γ₂ complex activation in HEK293 living cells expressing equal amounts of the WT of human TP receptor or its E129V mutant.

<p>A. BRET² was measured between the donor Rluc8 and the acceptor GFP¹⁰ introduced at the residue 97 of the Gα_q subunit and the N-terminal domain of the Gγ₂ subunit, respectively. Agonist-induced coupling of TP receptor and Gq protein distances Gα_q-Rluc8 and GFP¹⁰-Gγ₂ giving rise to a decrease in the BRET signal. B. Protein expression levels of the constructs used for BRET experiments were set to be constant and able to assure the same level of basal BRET ratio in the presence of WT and E129V mutant of the human TP receptors. Total Gα_q-Rluc8 luminescence was evaluated in HEK293 cells co-expressing Gα_q-Rluc8 together with GFP¹⁰-Gγ₂ and Gβ₁ in the presence of WT or E129V mutant of the human TP receptor measuring the light emission in aliquots of the transfected cells incubated with 5 µM coelenterazine for 8 min. In the same cells stimulated with PBS, basal BRET ratio was calculated as the ratio of the light emitted by GFP¹⁰ (510–540 nm) over the light emitted by Rluc8 (370–450 nm). C. BRET was measured in HEK293 cells co-expressing Gα_q-Rluc8 together with GFP¹⁰-Gγ₂ and Gβ₁ in the presence of WT (left) or E129V (right) mutant of the human TP receptor and stimulated with increasing concentrations of the indicated full and partial agonists. Results are the differences in the BRET signal measured in the presence and the absence of agonists, and are expressed as the mean value±SE of at least two independent determinations.</p

Zwitterion-Coated Iron Oxide Nanoparticles: Surface Chemistry and Intracellular Uptake by Hepatocarcinoma (HepG2) Cells

Nanoparticles (NPs) have received much attention in recent years for their diverse potential biomedical applications. However, the synthesis of NPs with desired biodistribution and pharmacokinetics is still a major challenge, with NP size and surface chemistry being the main factors determining the behavior of NPs in vivo. Here we report on the surface chemistry and in vitro cellular uptake of magnetic iron oxide NPs coated with zwitterionic dopamine sulfonate (ZDS). ZDS-coated NPs were compared to similar iron oxide NPs coated with PEG-like 2-[2-(2-methoxyethoxy)­ethoxy]­acetic acid (MEEA) to investigate how surface chemistry affects their in vitro behavior. ZDS-coated NPs had a very dense coating, guaranteeing high colloidal stability in several aqueous media and negligible interaction with proteins. Treatment of HepG2 cells with increasing doses (2.5–100 μg Fe/mL) of ZDS-coated iron oxide NPs had no effect on cell viability and resulted in a low, dose-dependent NP uptake, inferior than most reported data for the internalization of iron oxide NPs by HepG2 cells. MEEA-coated NPs were scarcely stable and formed micrometer-sized aggregates in aqueous media. They decreased cell viability for dose ≥50 μg Fe/mL, and were more efficiently internalized than ZDS-coated NPs. In conclusion, our data indicate that the ZDS layer prevented both aggregation and sedimentation of iron oxide NPs and formed a biocompatible coating that did not display any biocorona effect. The very low cellular uptake of ZDS-coated iron NPs can be useful to achieve highly selective targeting upon specific functionalization

Affinities of the agonists for the binding site of the receptor labelled by [³H]SQ29,548 in HEK293 cells transiently expressing the WT or the mutant human TP receptors.

<p>Ki values were obtained by simultaneous analysis of at least 3 independent competition experiments analyzed with GraphPad Prism implemented with the LIGAND model (see Data and statistical analysis).</p>a<p>Affinity ratio was calculated as the ratio of the K_i for the WT TP receptor over the K_i of the E129V mutant.</p>**<p>p<0.01 vs. WT.</p

Pharmacodynamic analysis of antagonists SQ29,548 and PTA₂ in functional assay.

<p>Total IP accumulation in HEK293 cells transiently expressing the WT or E129V mutant of human TP receptor was assayed in the absence and presence of Gαq overexpression at increasing concentrations of the antagonists for 30 min. TP and Gαq plasmids were added in a 1∶5 (5x) ratio. Data are expressed as dpm/well. Error bars represent mean±SE of at least three independent experiments each performed in duplicates or triplicates. Curves are computer generated from the simultaneous analysis of several independent experiments.</p

Analysis of basal and agonist induced total IP accumulation in HEK293 cells transiently expressing the WT or SAM mutants of human TP receptor in the absence and presence of Gαq overexpression.

<p>TP and Gαq plasmids were added in a 1∶3 (3x) and 1∶5 ratio (5x), respectively. A. Total IP accumulation in basal conditions. B. Total IP accumulation induced by 30 min stimulation with 1 µM U46619. C. Fold increase over basal of total IP accumulation. D. Basal activity for WT and SAMs with increasing receptor expression in pmol/mg protein. Data are expressed as dpm/well. Error bars represent mean±SE of at least three independent experiments each performed in duplicates or triplicates.</p