Molecular,pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain

G protein‐coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin‐like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²⁺‐([Ca²⁺]_i) or 3′,5′‐cyclic adenosine monophosphate ([cAMP]_i) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long‐lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²⁺]_i or [cAMP]_i. To date, only one honeybee octopamine receptor that induces Ca²⁺ signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC_50s in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin >> cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees.

     

GM1 ganglioside enhances Ret signaling in striatum

It has been proposed that GM1 ganglioside promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. Our studies tested the hypothesis that GM1 exerts its neurotrophic action on dopaminergic neurons, in part, by interacting with the GDNF (glia cell‐derived neurotrophic factor) receptor complex, Ret tyrosine kinase and GFRα1 co‐receptor. GM1 addition to striatal slices in situ increased Ret activity in a concentration‐ and time‐dependent manner. GM1‐induced Ret activation required the whole GM1 molecule and was inhibited by the kinase inhibitors PP2 and PP1. Ret activation was followed by Tyr1062 phosphorylation and PI3 kinase/Akt recruitment. The Src kinase was associated with Ret and GM1 enhanced its phosphorylation. GM1 responses required the presence of GFRα1, and there was a GM1 concentration‐dependent increase in the binding of endogenous GDNF which paralleled that of Ret activation. Neutralization of the released GDNF did not influence the Ret response to GM1, and GM1 had no effect on GDNF release. Our in situ studies suggest that GM1 via GFRα1 modulates Ret activation and phosphorylation in the striatum and provide a putative mechanism for its effects on dopaminergic neurons. Indeed, chronic GM1 treatment enhanced Ret activity and phosphorylation in the striatum of the MPTP‐mouse and kinase activation was associated with recovery of dopamine and DOPAC deficits.

     

Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release

The nucleus accumbens is highly heterogeneous, integrating regionally distinct afferent projections and accumbal interneurons, resulting in diverse local microenvironments. Dopamine (DA) neuron terminals similarly express a heterogeneous collection of terminal receptors that modulate DA signaling. Cyclic voltammetry is often used to probe DA terminal dynamics in brain slice preparations; however, this method traditionally requires electrical stimulation to induce DA release. Electrical stimulation excites all of the neuronal processes in the stimulation field, potentially introducing simultaneous, multi‐synaptic modulation of DA terminal release. We used optogenetics to selectively stimulate DA terminals and used voltammetry to compare DA responses from electrical and optical stimulation of the same area of tissue around a recording electrode. We found that with multiple pulse stimulation trains, optically stimulated DA release increasingly exceeded that of electrical stimulation. Furthermore, electrical stimulation produced inhibition of DA release across longer duration stimulations. The GABA_B antagonist, CGP 55845, increased electrically stimulated DA release significantly more than light stimulated release. The nicotinic acetylcholine receptor antagonist, dihydro‐β‐erythroidine hydrobromide, inhibited single pulse electrically stimulated DA release while having no effect on optically stimulated DA release. Our results demonstrate that electrical stimulation introduces local multi‐synaptic modulation of DA release that is absent with optogenetically targeted stimulation.

     

Larvae of the small white butterfly,Pieris rapae,express a novel serotonin receptor

The biogenic amine serotonin ( 5‐hydroxytryptamine, 5‐HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G‐protein‐coupled receptors. Five 5‐HT receptor subtypes have been reported in Drosophila that share high similarity with mammalian 5‐HT_1A, 5‐HT_1B, 5‐HT_2A, 5‐HT_2B, and 5‐HT₇ receptors. We isolated a cDNA (Pr5‐HT₈) from larval Pieris rapae, which shares relatively low similarity to the known 5‐HT receptor classes. After heterologous expression in HEK293 cells, Pr5‐HT₈ mediated increased [Ca²⁺]_i in response to low concentrations (< 10 nM) of 5‐HT. The receptor did not affect [cAMP]_i even at high concentrations (> 10 μM) of 5‐HT. Dopamine, octopamine, and tyramine did not influence receptor signaling. Pr5‐HT₈ was also activated by various 5‐HT receptor agonists including 5‐methoxytryptamine, (±)‐8‐Hydroxy‐2‐(dipropylamino) tetralin, and 5‐carboxamidotryptamine. Methiothepin, a non‐selective 5‐HT receptor antagonist, activated Pr5‐HT₈. WAY 10635, a 5‐HT_1A antagonist, but not SB‐269970, SB‐216641, or RS‐127445, inhibited 5‐HT‐induced [Ca²⁺]_i increases. We infer that Pr5‐HT₈ represents the first recognized member of a novel 5‐HT receptor class with a unique pharmacological profile. We found orthologs of Pr5‐HT₈ in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee or parasitoid wasps. This is likely to be an invertebrate‐specific receptor because there were no similar receptors in mammals.

     

Retracted: Superoxide‐dependent uptake of vitamin C in human glioma cells

Glioblastomas are lethal brain tumors that resist current cytostatic therapies. Vitamin C may antagonize the effects of reactive oxygen species (ROS) generating therapies; however, it is often used to reduce therapy‐related side effects despite its effects on therapy or tumor growth. Because the mechanisms of vitamin C uptake in gliomas are currently unknown, we evaluated the expression of the sodium‐vitamin C cotransporter (SVCT) and facilitative hexose transporter (GLUT) families in human glioma cells. In addition, as microglial cells can greatly infiltrate high‐grade gliomas (constituting up to 45% of cells in glioblastomas), the effect of TC620 glioma cell interactions with microglial‐like HL60 cells on vitamin C uptake (Bystander effect) was determined. Although glioma cells expressed high levels of the SVCT isoform‐2 (SVCT2), low functional activity, intracellular localization and the expression of the dominant‐negative isoform (dnSVCT2) were observed. The increased glucose metabolic activity of glioma cells was evident by the high 2‐Deoxy‐d ‐glucose and dehydroascorbic acid (DHA) uptake rates through the GLUT isoform‐1 (GLUT1), the main DHA transporter in glioblastoma. Co‐culture of glioma cells and activated microglial‐like HL60 cells resulted in extracellular ascorbic acid oxidation and high DHA uptake by glioma cells. This Bystander effect may explain the high antioxidative potential observed in high‐grade gliomas.

     

Positive feedback of NR2B‐containing NMDA receptor activity is the initial step toward visual imprinting: a model for juvenile learning

Imprinting in chicks is a good model for elucidating the processes underlying neural plasticity changes during juvenile learning. We recently reported that neural activation of a telencephalic region, the core region of the hyperpallium densocellulare (HDCo), was critical for success of visual imprinting, and that N‐Methyl‐D‐aspartic (NMDA) receptors containing the NR2B subunit (NR2B/NR1) in this region were essential for imprinting. Using electrophysiological and multiple‐site optical imaging techniques with acute brain slices, we found that long‐term potentiation (LTP) and enhancement of NR2B/NR1 currents in HDCo neurons were induced in imprinted chicks. Enhancement of NR2B/NR1 currents as well as an increase in surface NR2B expression occurred even following a brief training that was too weak to induce LTP or imprinting behavior. This means that NR2B/NR1 activation is the initial step of learning, well before the activation of alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors which induces LTP. We also showed that knockdown of NR2B/NR1 inhibited imprinting, and inversely, increasing the surface NR2B expression by treatment with a casein kinase 2 inhibitor successfully reduced training time required for imprinting. These results suggest that imprinting stimuli activate post‐synaptic NR2B/NR1 in HDCo cells, increase NR2B/NR1 signaling through up‐regulation of its expression, and induce LTP and memory acquisition.

     

Dopamine denervation of the prefrontal cortex increases expression of the astrocytic glutamate transporter GLT‐1

Both dopamine and glutamate are critically involved in cognitive processes such as working memory. Astrocytes, which express dopamine receptors, are essential elements in the termination of glutamatergic signaling: the astrocytic glutamate transporter GLT‐1 is responsible for > 90% of cortical glutamate uptake. The effect of dopamine depletion on glutamate transporters in the prefrontal cortex (PFC) remains unknown. In an effort to determine if astrocytes are a locus of cortical dopamine–glutamate interactions, we examined the effects of chronic dopamine denervation on PFC protein and mRNA levels of glutamate transporters. PFC dopamine denervation elicited a marked increase in GLT‐1 protein levels, but had no effect on levels of other glutamate transporters; high‐affinity glutamate transport was positively correlated with the extent of dopamine depletion. GLT‐1 gene expression was not altered. Our data suggest that dopamine depletion may lead to post‐translational modifications that result in increased expression and activity of GLT‐1 in PFC astrocytes.

     

Valproic acid induces neuronal cell death through a novel calpain‐dependent necroptosis pathway

A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase inhibitor used to treat epilepsy and mood disorders, has histone deacetylase‐related and ‐unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain‐dependent necroptosis pathway that initiates with downstream activation of c‐Jun N‐terminal kinase 1 (JNK1) and increased expression of receptor‐interacting protein 1 (RIP‐1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis‐inducing factor, mitochondrial release of Smac/DIABLO, and inhibition of the anti‐apoptotic protein X‐linked inhibitor of apoptosis (XIAP). Coinciding with apoptosis‐inducing factor nuclear translocation, VPA induces phosphorylation of the necroptosis‐associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3‐K/Akt survival pathways, allowing them to resist VPA‐induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain‐dependent necroptosis pathway that includes JNK1 activation and RIP‐1 expression.

     

Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression

Blood–brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier; however, they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein, we employed oxygen–glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3‐h before co‐culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8‐D1A cells). Incubation of OGD‐treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin‐5 proteins as well as increased vascular endothelial growth factor (VEGF) secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.

     

Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A‐dependent manner in non‐neuronal and neuronal cell lines

Trafficking of G protein‐coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein‐coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non‐neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand‐mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand‐mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A‐dependent manner.

     

HIV‐1 Tat C modulates NOX2 and NOX4 expressions through miR‐17 in a human microglial cell line

HIV‐1 invades CNS in the early course of infection, which can lead to the cascade of neuroinflammation. NADPH oxidases (NOXs) are the major producers of reactive oxygen species (ROS), which play important roles during pathogenic insults. The molecular mechanism of ROS generation via microRNA‐mediated pathway in human microglial cells in response to HIV‐1 Tat protein has been demonstrated in this study. Over‐expression and knockdown of microRNAs, luciferase reporter assay, and site‐directed mutagenesis are main molecular techniques used in this study. A significant reduction in miR‐17 levels and increased NOX2, NOX4 expression levels along with ROS production were observed in human microglial cells upon HIV‐1 Tat C exposure. The validation of NOX2 and NOX4 as direct targets of miR‐17 was done by luciferase reporter assay. The over‐expression and knockdown of miR‐17 in human microglial cells showed the direct role of miR‐17 in regulation of NOX2, NOX4 expression and intracellular ROS generation. We demonstrated the regulatory role of cellular miR‐17 in ROS generation through over‐expression and knockdown of miR‐17 in human microglial cells exposed to HIV‐1 Tat C protein.

     

Botulinum protease‐cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

Soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal‐associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA‐transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations.

     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains

Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine‐71 in EAAT2 and Serine‐45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate‐binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain.

     

Tnfaip8 l1/Oxi‐β binds to FBXW5, increasing autophagy through activation of TSC2 in a Parkinson's disease model

Abnormal autophagy may contribute to neurodegeneration in Parkinson's disease (PD). However, it is largely unknown how autophagy is dysregulated by oxidative stress (OS), one of major pathogenic causes of PD. We recently discovered the potential autophagy regulator gene family including Tnfaip8/Oxi‐α, which is a mammalian target of rapamycin (mTOR) activator down‐regulated by OS in dopaminergic neurons (J. Neurochem., 112, 2010 , 366). Here, we demonstrate that the OS‐induced Tnfaip8 l1/Oxi‐β could increase autophagy by a unique mechanism that increases the stability of tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTOR. Tnfaip8 l1/Oxi‐β and Tnfaip8/Oxi‐α are the novel regulators of mTOR acting in opposition in dopaminergic (DA) neurons. Specifically, 6‐hydroxydopamine (6‐OHDA) treatment up‐regulated Tnfaip8 l1/Oxi‐β in DA neurons, thus inducing autophagy, while knockdown of Tnfaip8 l1/Oxi‐β prevented significantly activation of autophagic markers by 6‐OHDA. FBXW5 was identified as a novel binding protein for Tnfaip8 l1/Oxi‐β. FBXW5 is a TSC2 binding receptor within CUL4 E3 ligase complex, and it promotes proteasomal degradation of TSC2. Thus, Tnfaip8 l1/Oxi‐β competes with TSC2 to bind FBXW5, increasing TSC2 stability by preventing its ubiquitination. Our data show that the OS‐induced Tnfaip8 l1/Oxi‐β stabilizes TSC2 protein, decreases mTOR phosphorylation, and enhances autophagy. Therefore, altered regulation of Tnfaip8 l1/Oxi‐β may contribute significantly to dysregulated autophagy observed in dopaminergic neurons under pathogenic OS condition.