GABAA receptor transmembrane amino acids are critical for alcohol action: disulfide cross‐linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites

Alcohols and inhaled anesthetics modulate GABA_A receptor (GABA_AR) function via putative binding sites within the transmembrane regions. The relative position of the amino acids lining these sites could be either inter‐ or intra‐subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA‐mediated currents in wild‐type and mutant α1β2γ2 GABA_ARs expressed in Xenopus laevis oocytes. We tested for: (i) inter‐subunit cross‐linking: a cysteine located in α1TM1 [either α1(Q229C) or α1(L232C)] was paired with a cysteine in different positions of β2TM2 and TM3; (ii) intra‐subunit cross‐linking: a cysteine located either in β2TM1 [β2(T225C)] or in TM2 [β2(N265C)] was paired with a cysteine in different locations along β2TM3. Three inter‐subunit cysteine pairs and four intra‐subunits cross‐linked. In three intra‐subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra‐subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter‐ or intra‐subunit sites is determined by the specific receptor and ligand.

     

Assembly,trafficking and function of α1β2γ2 GABAA receptors are regulated by N‐terminal regions,in a subunit‐specific manner

GABA_A receptors are pentameric ligand‐gated ion channels that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pentameric ligand‐gated ion channels structures, sequence analysis predicts an α‐helix near the N‐terminus of each GABA_A receptor subunit. Preceding each α‐helix are 8–36 additional residues, which we term the N‐terminal extension. In homomeric GABA_C receptors and nicotinic acetylcholine receptors, the N‐terminal α‐helix is functionally essential. Here, we determined the role of the N‐terminal extension and putative α‐helix in heteromeric α1β2γ2 GABA_A receptors. This role was most prominent in the α1 subunit, with deletion of the N‐terminal extension or further deletion of the putative α‐helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the β2 or γ2 N‐terminal extension had little effect on the number of functional cell surface receptors. Additional deletion of the putative α‐helix in the β2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the β2 subunit only, α‐helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N‐terminal extensions and α‐helices make key subunit‐specific contributions to assembly, consistent with both regions being involved in inter‐subunit interactions.

     

Effects of the selective sigma‐1 receptor antagonist S1RA on formalin‐induced pain behavior and neurotransmitter release in the spinal cord in rats

We have previously shown that the selective sigma‐1 receptor (σ₁R) antagonist S1RA (E‐52862) inhibits neuropathic pain and activity‐induced spinal sensitization in various pre‐clinical pain models. In this study we characterized both the behavioral and the spinal neurochemical effects of S1RA in the rat formalin test. Systemic administration of S1RA produced a dose‐related attenuation of flinching and lifting/licking behaviors in the formalin test. Neurochemical studies using concentric microdialysis in the ipsilateral dorsal horn of awake, freely moving rats revealed that the systemic S1RA‐induced antinociceptive effect occurs concomitantly with an enhancement of noradrenaline levels and an attenuation of formalin‐evoked glutamate release in the spinal dorsal horn. Intrathecal pre‐treatment with idazoxan prevented the systemic S1RA antinociceptive effect, suggesting that the S1RA antinociception depends on the activation of spinal α₂‐adrenoceptors which, in turn, could induce an inhibition of formalin‐evoked glutamate release. When administered locally, intrathecal S1RA inhibited only the flinching behavior, whereas intracerebroventricularly or intraplantarly injected also attenuated the lifting/licking behavior. These results suggest that S1RA supraspinally activates the descending noradrenergic pain inhibitory system, which may explain part of its antinociceptive properties in the formalin test; however, effects at other central and peripheral sites also account for the overall effect.

     

γ‐glutamyl transpeptidase 1 specifically suppresses green‐light avoidance via GABAA receptors in Drosophila

Drosophila larvae innately show light avoidance behavior. Compared with robust blue‐light avoidance, larvae exhibit relatively weaker green‐light responses. In our previous screening for genes involved in larval light avoidance, compared with control w¹¹¹⁸ larvae, larvae with γ‐glutamyl transpeptidase 1 (Ggt‐1) knockdown or Ggt‐1 mutation were found to exhibit higher percentage of green‐light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt‐1 in different tissues, we found that Ggt‐1 in malpighian tubules was both necessary and sufficient for green‐light avoidance. Our results showed that glutamate levels were lower in Ggt‐1 null mutants compared with controls. Feeding Ggt‐1 null mutants glutamate can normalize green‐light avoidance, indicating that high glutamate concentrations suppressed larval green‐light avoidance. However, rather than directly, glutamate affected green‐light avoidance indirectly through GABA, the level of which was also lower in Ggt‐1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABA_A receptor, both exhibit elevated levels of green‐light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green‐light avoidance, which was inhibited in wild‐type larvae.

     

PERK,mTORC1 and eEF2 interplay during long term potentiation

This Editorial highlights a study by Zimmermann and coworkers in the current issue of Journal of Neurochemistry. The authors' link suppression of PKR‐like endoplasmatic reticulum kinase (PERK) activity to eukaryotic elongation factor 2 (eEF2) dephosphorylation and mTORC1‐independent high‐frequency stimulation (HFS)‐induced long‐term potentiation (LTP) in acute hippocampal slices from PERK forebrain conditional knockout mice. The results suggest that functional interaction between the signaling pathways controlling different phases of the mRNA translation process is necessary for long‐term plasticity in the hippocampus.

     

Endothelin‐1 increases the expression of VEGF‐R1/Flt‐1 receptors in rat cultured astrocytes through ETB receptors

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin‐1 (ET‐1) were examined in rat cultured astrocytes. Expressions of VEGF‐R1 and ‐R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF‐R3 and Tie‐2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF‐R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF‐E, a selective VEGF‐R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF₁₆₅ were reduced by selective antagonists for VEGF‐R1 and ‐R2. Treatment with ET‐1 increased VEGF‐R1 mRNA and protein levels. The effects of ET‐1 on VEGF‐R1 mRNA were mimicked by Ala^1,3,11,15‐ET‐1, a selective agonist for ET_B receptors, and inhibited by BQ788, an ET_B antagonist. ET‐1 did not affect the mRNA levels of VEGF‐R2, ‐R3, and Tie‐2. Pre‐treatment with ET‐1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET‐1 induces up‐regulation of VEGF‐R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.

     

Opposite control of frontocortical 2‐arachidonoylglycerol turnover rate by cannabinoid type‐1 receptors located on glutamatergic neurons and on astrocytes

This study examined the respective influences of cannabinoid type‐1 (CB₁) receptors expressed either in forebrain GABAergic neurons, in cortical glutamatergic neurons, or in astrocytes on the turnover rates of the endocannabinoids N‐arachidonoylethanolamide (AEA) and 2‐arachidonoylglycerol (2‐AG), and the non‐cannabinoid N‐acylethanolamides, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), in mouse forebrain regions. To this end, conditional mutant mice lacking CB₁ receptors from either of these cell types were pre‐treated systemically with JZL195, a dual inhibitor of fatty acid amide hydrolase, the enzyme degrading AEA, PEA, and OEA, and of monoacylglycerol lipase, the main 2‐AG‐degrading enzyme. The analyses of frontocortical, hippocampal, and striatal AEA, 2‐AG, PEA, and OEA concentrations revealed that their respective baseline concentrations were not influenced by the mouse genotype. On the other hand, the accumulation of frontocortical and/or hippocampal 2‐AG levels in JZL195‐pre‐treated mice was dependent on the mouse genotype. Thus, JZL195‐induced 2‐AG accumulation rates were diminished in the frontal cortex of mice lacking CB₁ receptors in glutamatergic neurons while their respective values were increased in the frontal cortex and hippocampus of mice lacking these receptors in astrocytes. These genotypic differences occurred with parallel and proportionate changes in the fractional rate constants for degradation of 2‐AG, thus providing a mechanism whereby the baseline levels of 2‐AG remained constant between genotypes. Besides suggesting a cell‐type‐specific control of frontocortical and/or hippocampal 2‐AG synthesis and degradation rates by CB₁ receptors, this study highlights the interest of assessing endocannabinoid turnover rates when questioning the status of the endocannabinoid system.

     

Adenosine transiently modulates stimulated dopamine release in the caudate–putamen via A1 receptors

Adenosine modulates dopamine in the brain via A₁ and A_2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast‐scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate–putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 μM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2 s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7%, similar to the 54 ± 6% decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 min. The A₁ receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A_2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A₁ receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain.

     

The GLT‐1 (EAAT2; slc1a2) glutamate transporter is essential for glutamate homeostasis in the neocortex of the mouse

Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT‐1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT‐1 and GLAST, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we injected GLT‐1 knockout (KO) mice and wild‐type littermates with [1‐¹³C]glucose and [1,2‐¹³C]acetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and ¹³C labeling in neocortex. Whereas the cerebellum in GLT‐1‐deficient mice had normal levels of glutamate, glutamine, and ¹³C labeling of metabolites, glutamate level was decreased but labeling from [1‐¹³C] glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from [1,2‐¹³C] acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the cortex.

     

Kisspeptin1 modulates odorant‐evoked fear response via two serotonin receptor subtypes (5‐HT1A and 5‐HT2) in zebrafish

Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5‐HT) system to decrease odorant cue [alarm substance (AS)]‐evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5‐HT system as well as to determine the involvement of the 5‐HT receptor subtypes in AS‐evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5‐HT_1A receptor antagonist, to observe the effects of Kiss1 administration on AS‐evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p < 0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5‐HT₁ and 5‐HT₂ receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p < 0.001). From this, we conclude that Kiss1 modulates AS‐evoked fear responses mediated by the 5‐HT_1A and 5‐HT₂ receptors.

     

Impaired clustered protocadherin‐α leads to aggregated retinogeniculate terminals and impaired visual acuity in mice

Clustered protocadherins (cPcdhs) comprising cPcdh‐α, ‐β, and ‐γ, encode a large family of cadherin‐like cell‐adhesion molecules specific to neurons. Impairment of cPcdh‐α results in abnormal neuronal projection patterns in specific brain areas. To elucidate the role of cPcdh‐α in retinogeniculate projections, we investigated the morphological patterns of retinogeniculate terminals in the lateral geniculate (LG) nucleus of mice with impaired cPcdh‐α. We found huge aggregated retinogeniculate terminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in the olivary pretectal nucleus and the ventral LG nucleus. These aggregated terminals appeared between P10 and P14, just before eye opening and at the beginning of the refinement stage of the retinogeniculate projections. Reduced visual acuity was observed in adult mice with impaired cPcdh‐α, whereas the orientation selectivity and direction selectivity of neurons in the primary visual cortex were apparently normal. These findings suggest that cPcdh‐α is required for adequate spacing of retinogeniculate projections, which may be essential for normal development of visual acuity.

     

Profiling the genes affected by pathogenic TDP‐43 in astrocytes

Mutation in TAR DNA binding protein 43 (TDP‐43) is a causative factor of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Neurodegeneration may not require the presence of pathogenic TDP‐43 in all types of relevant cells. Rather, expression of pathogenic TDP‐43 in neurons or astrocytes alone is sufficient to cause cell‐autonomous or non‐cell‐autonomous neuron death in transgenic rats. How pathogenic TDP‐43 in astrocytes causes non‐cell‐autonomous neuron death, however, is not clear. Here, we examined the effect of pathogenic TDP‐43 on gene expression in astrocytes. Microarray assay revealed that pathogenic TDP‐43 in astrocytes preferentially altered expression of the genes encoding secretory proteins. Whereas neurotrophic genes were down‐regulated, neurotoxic genes were up‐regulated. Representative genes Lcn2 and chitinase‐3‐like protein 1 were markedly up‐regulated in astrocytes from primary culture and intact transgenic rats. Furthermore, synthetic chitinase‐3‐like protein 1 induced neuron death in a dose‐dependent manner. Our results suggest that TDP‐43 pathogenesis is associated with the simultaneous induction of multiple neurotoxic genes in astrocytes, which may synergistically produce adverse effects on neuronal survival and contribute to non‐cell‐autonomous neuron death.

     

Sulforaphane enhances temozolomide‐induced apoptosis because of down‐regulation of miR‐21 via Wnt/β‐catenin signaling in glioblastoma

Temozolomide (TMZ) has been widely used in the treatment of glioblastoma (GBM), although inherent or acquired resistance restricts the application. This study was aimed to evaluate the efficacy of sulforaphane (SFN) to TMZ‐induced apoptosis in GBM cells and the potential mechanism. Biochemical assays and subcutaneous tumor establishment were used to characterize the function of SFN in TMZ‐induced apoptosis. Our results revealed that β‐catenin and miR‐21 were concordantly expressed in GBM cell lines, and SFN significantly reduced miR‐21 expression through inhibiting the Wnt/β‐catenin/TCF4 pathway. Furthermore, down‐regulation of miR‐21 enhanced the pro‐apoptotic efficacy of TMZ in GBM cells. Finally, we observed that SFN strengthened TMZ‐mediated apoptosis in a miR‐21‐dependent manner. In conclusion, SFN effectively enhances TMZ‐induced apoptosis by inhibiting miR‐21 via Wnt/β‐catenin signaling in GBM cells. These findings support the use of SFN for potential therapeutic approach to overcome TMZ resistance in GBM treatment.

     

Effects of the beta‐adrenergic receptor antagonist Propranolol on dyskinesia and L‐DOPA‐induced striatal DA efflux in the hemi‐parkinsonian rat

Dopamine (DA) replacement therapy with L‐DOPA continues to be the primary treatment of Parkinson's disease; however, long‐term therapy is accompanied by L‐DOPA‐induced dyskinesias (LID). Several experimental and clinical studies have established that Propranolol, a β‐adrenergic receptor antagonist, reduces LID without affecting L‐DOPA's efficacy. However, the exact mechanisms underlying these effects remain to be elucidated. The aim of this study was to evaluate the anti‐dyskinetic profile of Propranolol against a panel of DA replacement strategies, as well as elucidate the underlying neurochemical mechanisms. Results indicated that Propranolol, in a dose‐dependent manner, reduced LID, without affecting motor performance. Propranolol failed to alter dyskinesia produced by the D₁ receptor agonist, SKF81297 (0.08 mg/kg, sc), or the D₂ receptor agonist, Quinpirole (0.05 mg/kg, sc). These findings suggested a pre‐synaptic mechanism for Propranolol's anti‐dyskinetic effects, possibly through modulating L‐DOPA‐mediated DA efflux. To evaluate this possibility, microdialysis studies were carried out in the DA‐lesioned striatum of dyskinetic rats and results indicated that co‐administration of Propranolol (20 mg/kg, ip) was able to attenuate L‐DOPA‐ (6 mg/kg, sc) induced DA efflux. Therefore, Propranolol's anti‐dyskinetic properties appear to be mediated via attenuation of L‐DOPA‐induced extraphysiological efflux of DA.

     

Glutathione reductase mediates drug resistance in glioblastoma cells by regulating redox homeostasis

Glutathione (GSH) and GSH‐related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)‐resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ‐resistant cells than in sensitive cells. Furthermore, silencing GSR in drug‐resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over‐expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro‐oxidant L‐buthionine ‐sulfoximine. The modulation of redox state by GSH or L‐buthionine –sulfoximine regulated GSR‐mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress‐free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment.

     

Expression of the synaptic exocytosis‐regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.

     

Ca2+‐dependent down‐regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein‐coupled human histamine H₁ receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin‐dependent endocytosis followed by proteasome/lysosome‐mediated down‐regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca²⁺ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca²⁺ ionophore, on the endocytosis and down‐regulation of H₁ receptors in Chinese hamster ovary cells. All cellular and cell‐surface H₁ receptors were detected by the binding of [³H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [³H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca²⁺ and partially by a ubiquitin‐activating enzyme inhibitor (UBEI‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH₄Cl), proteasomes (lactacystin or MG‐132), and a Ca²⁺‐dependent non‐lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down‐regulated by a sustained increase in intracellular Ca²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

     

Rac1‐mediated indentation of resting neurons promotes the chain migration of new neurons in the rostral migratory stream of post‐natal mouse brain

New neurons generated in the ventricular‐subventricular zone in the post‐natal brain travel toward the olfactory bulb by using a collective cell migration process called ‘chain migration.’ These new neurons show a saltatory movement of their soma, suggesting that each neuron cycles through periods of ‘rest’ during migration. Here, we investigated the role of the resting neurons in chain migration using post‐natal mouse brain, and found that they undergo a dynamic morphological change, in which a deep indentation forms in the cell body. Inhibition of Rac1 activity resulted in less indentation of the new neurons in vivo. Live cell imaging using a Förster resonance energy transfer biosensor revealed that Rac1 was activated at the sites of contact between actively migrating and resting new neurons. On the cell surface of resting neurons, Rac1 activation coincided with the formation of the indentation. Furthermore, Rac1 knockdown prevented the indentation from forming and impaired migration along the resting neurons. These results suggest that Rac1 regulates a morphological change in the resting neurons, which allows them to serve as a migratory scaffold, and thereby non‐cell‐autonomously promotes chain migration.