Ro 15-4513 Antagonizes Alcohol-Induced Sedation in Mice Through aß¿2-type GABA(A) Receptors

Peer reviewedPublisher PD

Multiple actions of fenamates and other nonsteroidal anti-inflammatory drugs on GABA(A) receptors

The nonsteroidal anti-inflammatory drug (NSAID) niflumic acid, a fenamate in structure, has many molecular targets, one of them being specific subtypes of the main inhibitory ligand-gated anion channel, the GABA(A) receptor. Here, we report on the effects of other fenamates and other classes of NSAIDs on brain picrotoxinin-sensitive GABA A receptors, using an autoradiographic assay with [S-35]TBPS as a ligand on mouse brain sections. We found that the other fenamates studied (flufenamic acid, meclofenamic acid, mefenamic acid and tolfenamic acid) affected the autoradiographic signal at low micromolar concentrations in a facilitatory-like allosteric fashion, i.e., without having affinity to the [S-35]TBPS binding site. Unlike niflumic acid that shows clear preference for inhibiting cerebellar granule cell layer GABA(A) receptors, the other fenamates showed little brain regional selectivity, indicating that their actions are not receptor-subtype selective. Of the non-fenamate NSAIDs studied at 100 mu M concentration, diclofenac induced the greatest inhibition of the binding, which is not surprising as it has close structural similarity with the potent fenamate meclofenamic acid. Using two-electrode voltage-clamp assays on Xenopus oocytes, the effect of niflumic acid was found to be dependent on the beta subunit variant and the presence of gamma 2 subunit in rat recombinant alpha 1 beta and alpha 1 beta gamma 2 GABA(A) receptors, with the beta 1 allowing the niflumic acid inhibition and beta 3 the stimulation of the receptor-mediated currents. In summary, the fenamate NSAID5 constitute an interesting class of compounds that could be used for development of potent GABA(A) receptor allosteric agonists with other targets to moderate inflammation, pain and associated anxiety/depression.Peer reviewe

A53T-alpha-synuclein overexpression impairs dopamine signaling and striatal synaptic plasticity in old mice

BACKGROUND: Parkinson's disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice. CONCLUSIONS/SIGNIFICANCE: Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor α4 subunit

AbstractA cDNA of rat brain encoding the GABAA receptor α4 subunit has been cloned. Recombinant receptors composed of α4, β2 and γ2 subunits bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine ‘alcohol antagonist’ [3H]Ro 15-4513, but fail to bind benzodiazepine agonists. The α4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The α4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum

Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update

The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field

Membrane proteins and proteomics: Love is possible, but so difficult

Despite decades of extensive research, the large-scale analysis of membrane proteins remains a difficult task. This is due to the fact that membrane proteins require a carefully balanced hydrophilic and lipophilic environment, which optimum varies with different proteins, while most protein chemistry methods work mainly, if not only, in water-based media. Taking this review [Santoni, Molloy and Rabilloud, Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21, 1054-1070] as a pivotal paper, the current paper analyzes how the field of membrane proteomics exacerbated the trend in proteomics, i.e. developing alternate methods to the historical two-dimensional electrophoresis, and thus putting more and more pressure on the mass spectrometry side. However, in the case of membrane proteins, the incentive in doing so is due to the poor solubility of membrane proteins. This review also shows that in some situations, where this solubility problem is less acute, two-dimensional electrophoresis remains a method of choice. Last but not least, this review also critically examines the alternate approaches that have been used for the proteomic analysis of membrane proteins

A53T-Alpha-Synuclein Overexpression Impairs Dopamine Signaling and Striatal Synaptic Plasticity in Old Mice

Parkinson's disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons.Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice.Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches

Does ethanol act preferentially via selected brain GABAA receptor subtypes? the current evidence is ambiguous

In rodent models, γ-aminobutyric acid A (GABAA) receptors with the α6 and δ subunits, expressed in the cerebellar and cochlear nucleus granule cells, have been linked to ethanol sensitivity and voluntary ethanol drinking. Here, we review the findings. When considering both in vivo contributions and data on cloned receptors, the evidence for direct participation of the α6-containing receptors to increased ethanol sensitivity is poor. The α6 subunit-knockout mouse lines do not have any changed sensitivity to ethanol, although these mice do display increased benzodiazepine sensitivity. However, in general the compensations occurring in knockout mice (regardless of which particular gene is knocked out) tend to fog interpretations of drug actions at the systems level. For example, the α6 knockout mice have increased TASK-1 channel expression in their cerebellar granule cells, which could influence sensitivity to ethanol in the opposite direction to that obtained with the α6 knockouts. Indeed, TASK-1 knockout mice are more impaired than wild types in motor skills when given ethanol; this might explain why GABAA receptor α6 knockout mice have unchanged ethanol sensitivities. As an alternative to studying knockout mice, we examined the claimed δ subunit-dependent/γ2 subunit-independent ethanol/[3H]Ro 15-4513 binding sites on GABAA receptors. We looked at [3H]Ro 15-4513 binding in HEK 293 cell membrane homogenates containing rat recombinant α6/4β3δ receptors and in mouse brain sections. Specific high-affinity [3H]Ro 15-4513 binding could not be detected under any conditions to the recombinant receptors or to the cerebellar sections of γ2(F77I) knockin mice, nor was this binding to brain sections of wild-type C57BL/6 inhibited by 1–100 mM ethanol. Since ethanol may act on many receptor and channel protein targets in neuronal membranes, we consider the α6 (and α4) subunit-containing GABAA receptors unlikely to be directly responsible for any major part of ethanol's actions. Therefore, we finish the review by discussing more generally alcohol and GABAA receptors and by suggesting potential future directions for this research.This study and review was supported by the Finnish Foundation for Alcohol Studies, the Academy of Finland and the Sigrid Juselius Foundation.Peer reviewe