Developmental neurotoxicity of reserpine exposure in zebrafish larvae (Danio rerio).

Reserpine is widely used for treatment of hypertension and schizophrenia. As a specific inhibitor of monoamine transporters, reserpine is known to deplete monoamine neurotransmitters and cause decreased movement symptoms. However, how zebrafish larvae respond to reserpine treatment is not well studied. Here we show that swimming distance and average velocity are significantly reduced after reserpine exposure under various stimulatory conditions. Using liquid chromatograph-mass spectrometer analysis, decreased levels of monoamines (e.g. dopamine, noradrenaline, and serotonin) were detected in reserpine-treated larvae. Moreover, reserpine treatment significantly reduced the number of dopaminergic neurons, which was identified with th (Tyrosine Hydroxylase) in situ hybridization in the preoptic area. Interestingly, dopaminergic neuron development-associated genes, such as otpa, otpb, wnt1, wnt3, wnt5 and manf, were downregulated in reserpine treated larvae. Our data indicates that 2 mg/L reserpine exposure induces dopaminergic neuron damage in the brain, demonstrating a chemical induced depression-like model in zebrafish larvae for future drug development.

Sphingosine 1-phosphate receptor 1 regulates cell-surface localization of membrane proteins in endothelial cells.

The endothelial cell (EC) barrier disruption has been implicated in vascular leakage and pulmonary edema. Many reports have shown that the EC barrier dysfunction is regulated by the sphingosine-1-phophate (S1P)/S1P receptor-1 (S1PR1) axis. Identifying downstream effectors for the S1P/S1PR1 axis in pulmonary vasculature has been limited by mixed populations in vitro cultures that do not retain physiological EC phenotype and complex of tedious proteomics. In this study, we used a combination of in vivo biotinylation and liquid chromatograph tandem mass spectrometry on three mouse models of S1pr1 expression, namely normal, knockout (KO) and high, to identify EC membrane proteins whose cell-surface expression is S1pr1-dependent. EC-specific KO of S1pr1 caused severe pulmonary vascular disruption and reduction of many membrane proteins on ECs. Using the MaxQuant software we were able to identify novel membrane targets of S1pr1, for instance, Cd105 and Plvap, by comparison with their membrane expressions among the three EC model systems. Moreover, regulation of Cd105 and Plvap by S1pr1 were validated with Western blot and immunostaining in vivo and in vitro. Our data suggest that S1pr1 dictates cell-surface localization of several apical membrane proteins in ECs. Our results are insightful for development of novel therapeutics to specifically target EC barrier function.

Phosphorylation of ELAVL1 (Ser219/Ser316) mediated by PKC is required for erythropoiesis.

Elavl1 (also known as HuR), an RNA binding protein highly conserved between zebrafish and human, regulates gene expression by stabilizing target mRNA. Our previous studies have uncovered that the predominant isoform elavl1a is required for zebrafish embryonic erythropoiesis. However, the exact mechanism of how elav11 spatiotemporally stabilizes target mRNAs to regulate specific erythropoiesis is not yet understood. Here we show that phosphorylation of elavl1a at Ser219 and Ser316 by PKC is necessarily required for cytosolic shuttling from the nucleus to stabilize gata1 mRNA and thus promotes erythropoiesis. Knockdown of elavl1a resulted in the hindrance of erythropoiesis and Hemin-induced erythroid differentiation of human myeloid leukemia K562 cells. Interestingly, inhibition of PKC reproduced the phenotype seen during zebrafish embryogenesis and erythroid differentiation of myeloid leukemia. Mechanistically, Hemin induced elavl1a export from nuclear to cytoplasmic space in K562 cells in a manner dependent on phosphorylation on Ser219 and Ser316, as overexpression of elavl1a with mutations on Ser219 and Ser316 resulted in erythropoiesis failure. Additionally, co-administration of low doses of elavl1a morpholino (MO) and three PKC inhibitors showed a combined effect in zebrafish embryonic erythropoiesis dysplasia. In conclusion, our study reveals that PKC-mediated phosphorylation of elavl1a at Ser219 and Ser316 sites controls its nucleo-cytoplasmic translocation in zebrafish, thereby regulating embryonic erythropoiesis.

Fasudil exerts a cardio-protective effect on mice with coxsackievirus B3-induced acute viral myocarditis.

AIMS: To investigate whether there exists a cardio-protective effect of Fasudil, a selective Rho kinase (ROCK) inhibitor, in an experimental murine model of acute viral myocarditis. METHODS: Male BALB/c mice were randomly assigned to three groups: control, myocarditis treated with placebo and myocarditis treated with Fasudil (n = 40 animals per group). Myocarditis was established by intraperitoneal injection with coxsackievirus B3 (CVB3). Twenty-four hours after infection, Fasudil was intraperitoneally administered for 14 consecutive days. Twenty mice were randomly selected from each group to monitor a 14-day survival rate. On day 7 and day 14, eight surviving mice from each group were sacrificed and their hearts and blood were obtained to perform serological and histological examinations. Expression of ROCKs, IL-17, IL-1b, TNFα, RORgt, and Foxp3 were quantified with RT-PCR. Plasma levels of TNF alpha, IL-1 beta, and IL-17 were measured by ELISA. In addition, protein levels of IL-17 and ROCK2 in cardiac tissues were analyzed with Western blot. RESULTS: Fasudil treatment significantly increased survival, attenuated myocardial necrotic lesions, reduced CVB3 replication and expression of ROCK2 and IL-17 in the infected hearts. This treatment also imposed a T-cell subpopulation shift, from Th17 to Treg, in cardiac tissues. CONCLUSIONS: ROCK pathway inhibition was cardio-protective in viral myocarditis with increased survival, decreased viral replication, and inflammatory response. These findings suggest that Fasudil might be a potential therapeutic agent for patients with viral myocarditis.

Flumazenil decreases surface expression of α4ß2δ GABAA receptors by increasing the rate of receptor internalization.

Increases in expression of α4ßδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[ß]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4ßδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4ßδ expression in vivo. We first determined that flumazenil (100 nM-100 µM, IC50=∼1 µM) acted as a negative modulator, reducing GABA (10 µM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4ß2δ expressed in HEK-293 cells. Surface expression of recombinant α4ß2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 µM)+THP (100 nM). Flumazenil (10 µM) decreased surface expression of α4F by ∼60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4ß2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4ß2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4ß2δ expression using flumazenil.

Neurosteroid effects at α4ßδ GABAA receptors alter spatial learning and synaptic plasticity in CA1 hippocampus across the estrous cycle of the mouse.

Fluctuations in circulating levels of ovarian hormones have been shown to regulate cognition (Sherwin and Grigorova, 2011. Fertil. Steril. 96, 399-403; Shumaker et al., 2004. JAMA. 291, 2947-2958), but increases in estradiol on the day of proestrus yield diverse outcomes: In vivo induction of long-term potentiation (LTP), a model of learning, is reduced in the morning, but optimal in the afternoon (Warren et al., 1995. Brain Res. 703, 26-30). The mechanism underlying this discrepancy is not known. Here, we show that impairments in both CA1 hippocampal LTP and spatial learning observed on the morning of proestrus are due to increased dendritic expression of α4ßδ GABAA receptors (GABARs) on CA1 pyramidal cells, as assessed by electron microscopic (EM) techniques, compared with estrus and diestrus. LTP induction and spatial learning were robust, however, when assessed on the morning of proestrus in α4-/- mice, implicating these receptors in mediating impaired plasticity. Although α4ßδ expression remained elevated on the afternoon of proestrus, increases in 3α-OH-THP (3α-OH-5α-pregnan-20-one) decreased inhibition by reducing outward current through α4ßδ GABARs (Shen et al., 2007. Nat. Neurosci. 10, 469-477), in contrast to the usual effect of this steroid to enhance inhibition. Proestrous levels of 3α-OH-THP reversed the deficits in LTP and spatial learning, an effect prevented by the inactive metabolite 3ß-OH-THP (10 mg/kg, i.p.), which antagonizes actions of 3α-OH-THP. In contrast, administration of 3α-OH-THP (10 mg/kg, i.p.) on the morning of proestrus improved spatial learning scores 150-300%. These findings suggest that cyclic fluctuations in ovarian steroids can induce changes in cognition via α4ßδ GABARs that are dependent upon 3α-OH-THP. This article is part of a Special Issue entitled SI: Brain and Memory.

Regulation of the surface expression of α4ß2δ GABAA receptors by high efficacy states.

α4ßδ GABA(A) receptors (GABARs) have low CNS expression, but their expression is increased by 48h exposure to the neurosteroid THP (3α-OH-5α[ß]-pregnan-20-one). THP also increases the efficacy of δ-containing GABARs acutely, where GABA is a partial agonist. Thus, we examined effects of THP (100 nM) and full GABA agonists at α4ß2δ (gaboxadol, 10 µM, and ß-alanine, 10 µM-1mM), on surface expression of α4ß2δ. To this end, we used an α4 construct tagged with a 3XFLAG (F) epitope or measured expression of native α4 and δ. HEK-293 cells or cultured hippocampal neurons were transfected with α4Fß2δ and treated 24h later with GABA agonists, THP, GABA plus THP or vehicle (0.01% DMSO) for 0.5 h-48 h. Immunocytochemistry was performed under both non-permeabilized and permeabilized conditions to detect surface and intracellular labeling, respectively, using confocal microscopy. The high efficacy agonists and GABA (1 or 10 µM) plus THP increased α4ß2δ surface expression up to 3-fold after 48h, an effect first seen by 0.5h. This effect was not dependent upon the polarity of GABAergic current, although expression was increased by KCC2. Intracellular labeling was decreased while functional expression was confirmed by whole cell patch clamp recordings of responses to GABA agonists. GABA plus THP treatment did not alter the rate of receptor removal from the surface membrane, suggesting that THP-induced α4ß2δ expression is likely via receptor insertion. Surface expression of α4ß2δ was decreased by rottlerin (10 µM), suggesting a role for PKC-δ. These results suggest that trafficking of α4ß2δ GABARs is regulated by high efficacy states.