16 research outputs found
A Key Glycolytic Enzyme Plays a Dual Role in GABAergic Neurotransmission and in Human Epilepsy
Lâenzyme de la glycolyse GAPDH joue un double rĂŽle dans la neurotransmission GABAergique et lâĂ©pilepsie humaine
Modulation de la phosphorylation endogÚne du récepteur GABA A (nouvelles approches thérapeutiques pour le traitement des épilepsies pharmacorésistantes)
Le but de notre travail est de corriger le déficit fonctionnel du récepteur GABAA (GABAAR) mis en évidence dans le tissu épileptogÚne de patients souffrants d épilepsies pharamacorésistantes. Nous avons proposé deux cibles potentielles qui sont impliquées dans un mécanisme intrinsÚque du systÚme GABAergique : d une part la GAPDH, kinase du GABAAR et d autre part la phosphatase membranaire qui contrecarre la phosphorylation endogÚne et la fonction du GABAAR. La premiÚre partie de mon travail de thÚse a concerné l étude du rÎle de la spermine dans la phosphorylation endogÚne du GABAAR ainsi que son rÎle dans le rundown du récepteur. Les résultats obtenus ont montré que la spermine activait la phosphorylation endogÚne et diminuait le rundown du récepteur. De plus, les études enzymologiques ont montré un effet activateur de la spermine sur l activité déshydrogénase de la GAPDH pure. La deuxiÚme partie de ma thÚse est relative à la caractérisation et l identification des phosphatases membranaires qui déphosphorylent la sous-unité a1 du GABAAR. Pour cela, nous avons développé un nouvel outil analytique par LC-MS/MS et des phosphopeptides incluant les deux sites de phosphorylation endogÚne identifiés de la boucle I2a1 du GABAAR comme substrats. Par cette méthode nous avons caractérisé les profils pharmacologiques des activités phosphatasiques détectées, qui s avÚrent atypiques. Nous avons purifié et identifié ces activités par nano-LC-MALDI-TOF/TOF. En parallÚle, nous avons développé un test ELISA pour isoler des molécules inhibitrices des phosphatases membranaires dans le but de développer un nouvel antiépileptique.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF
Usefulness of preschool and school versions of the Behavioral Rating Inventory of Executive Functions in the evaluation of the daily life executive function in myoclonicâatonic epilepsy
Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors
When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells
Mass Spectrometric Detection and Characterization of Atypical Membrane-Bound Zinc-Sensitive Phosphatases Modulating GABA<sub>A</sub> Receptors
<div><p>Background</p><p>GABA<sub>A</sub> receptor (GABA<sub>A</sub>R) function is maintained by an endogenous phosphorylation mechanism for which the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase. This phosphorylation is specific to the long intracellular loop I<sub>2</sub> of the α1 subunit at two identified serine and threonine residues. The phosphorylation state is opposed by an unknown membrane-bound phosphatase, which inhibition favors the phosphorylated state of the receptor and contributes to the maintenance of its function. In cortical nervous tissue from epileptogenic areas in patients with drug-resistant epilepsies, both the endogenous phosphorylation and the functional state of the GABA<sub>A</sub>R are deficient.</p><p>Methodology/Principal Findings</p><p>The aim of this study is to characterize the membrane-bound phosphatases counteracting the endogenous phosphorylation of GABA<sub>A</sub>R. We have developed a new analytical tool for <i>in vitro</i> detection of the phosphatase activities in cortical washed membranes by liquid chromatography coupled to mass spectrometry. The substrates are two synthetic phosphopeptides, each including one of the identified endogenous phosphorylation sites of the I2 loop of GABA<sub>A</sub>R α1 subunit. We have shown the presence of multiple and atypical phosphatases sensitive to zinc ions. Patch-clamp studies of the rundown of the GABA<sub>A</sub>R currents on acutely isolated rat pyramidal cells using the phosphatase inhibitor okadaic acid revealed a clear heterogeneity of the phosphatases counteracting the function of the GABA<sub>A</sub>R.</p><p>Conclusion/Significance</p><p>Our results provide new insights on the regulation of GABA<sub>A</sub>R endogenous phosphorylation and function by several and atypical membrane-bound phosphatases specific to the α1 subunit of the receptor. By identifying specific inhibitors of these enzymes, novel development of antiepileptic drugs in patients with drug-resistant epilepsies may be proposed.</p></div
Effects of okadaic acid on the GABA<sub>A</sub> current rundown.
<p>GABA<sub>A</sub> currents were measured by whole-cell patch clamp on acutely dissociated cortical pyramidal neurons from Sprague Dawley rats. (A) Micrographs of a patched cell at two different magnifications, showing the rapid application device (left). (B) Rundown in cortical neuron: GABA was applied at a concentration of 100 ”M during 1 second every 3 min. The maximal amplitude of GABAergic currents gradually decreased with time in control conditions. In presence of okadaic acid (10 ”M in the pipette) variable effects were observed. (C) In some cells the rundown was even totally abolished. (D Left) Rundown profiles of normalized currents in presence (nâ=â17) or in absence (Control, nâ=â11) of okadaic acid; (D Right) color-coded hierarchical clustering tree for the recorded okadaic acid-treated cells in which a maximum of 4 groups are significantly distinguished: âVery Slowâ (nâ=â3), âMid Slowâ (nâ=â5), âMid Rapidâ (nâ=â4) and âVery Rapidâ (nâ=â5). For each group the plot (Left) is the average of normalized currents. Error bars indicate the SEM. One-way ANOVA with Dunnettâs test of mean currents and Student <i>t</i>-test were used (see in Results) indicating very likely that more than one phosphatase are involved.</p
LC-MS/MS detection and kinetics analysis of GABA<sub>A</sub>R α1-subunit phosphatase activities.
<p>(A) Dose-dependency at various protein concentrations; (B) time course of phosphatase activities using 50 ”g/ml of total proteins of washed cortical membranes from bovine brain and (C) from human epileptic tissue. The N-terminal phosphopeptide (pI<sub>2</sub>α1N-P) used as substrate at 10 ”M was incubated during 10 min at 30°C in 10 mM Hepes (pH 7.4) in the presence of 1 mM Mg<sup>2+</sup>. The enzymatic reactions were stopped with 10% acetic acid and the samples were analyzed by LC-MS/MS as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100612#pone-0100612-g001" target="_blank">figure 1E, F</a>. The dephosphorylation rate was quantified by measuring simultaneously the quantity of the produced native peptide (pI<sub>2</sub>α1N) and of the remaining phosphorylated substrate (pI<sub>2</sub>α1N-P). Error bars represent SEM of two experiments. Kinetic analysis of GABA<sub>A</sub>R α1-subunit phosphatase activities was investigated using N-terminal (pI<sub>2</sub>α1N-P) and C-terminal (pI<sub>2</sub>α1C-P) phosphopeptides. All experiments were carried out with 50 ”g/ml membrane proteins concentration, incubated at 30°C during 10 min, in presence of different concentrations of substrates. (D) Saturation plot of the initial velocity V<sub>i</sub> versus [pI<sub>2</sub>α1N-P] and Lineweaver-Burk plots of 1/V versus 1/[pI<sub>2</sub>α1N-P] (inset) showing two significantly different slopes. (E) Saturation plot of V<sub>i</sub> versus [pI<sub>2</sub>α1C-P] and Lineweaver-Burk plot of 1/V versus 1/[pI<sub>2</sub>α1C-P] (inset). The data points are means ± SEM of 3 experiments.</p
Mass spectrometry (MS) and liquid chromatography (LC)-MS/MS analysis of N- and C-terminal peptides.
<p>The insert (A) shows the representation of the α1 subunit TM3-TM4 intracellular loop (I<sub>2</sub>α1) of type A gamma-amino butyric acid receptor (GABA<sub>A</sub>R). The sequences indicate the synthetic N- and C-terminal peptides (AA334â346 and AA407â420 respectively) used in this study, with the two identified phosphorylation sites (PO<sub>4</sub>) on threonine (337Thr) and serine (416Ser) residues involved in the endogenous phosphorylation. EXT, extracellular; INT, intracellular. These peptides are detected in positive mode electrospray ionization (ESI) from m/z 200 to 2000 (mass in Da to charge ratio). The most abundant peptides (in blue) are selected for the subsequent ESI-MS/MS studies (parent peptides) and the other labeled ions have two, three or four charges. MS of native (A), and phosphorylated (B) N-terminal peptides. MS of native (C) and phosphorylated (D) C-terminal peptides. The major fragment ions produced by collision induced dissociation of parent peptide ions are labeled and the nature of fragmentation is indicated by âbâ (N-flanking) or âyâ (C-flanking) when the ion is broken at peptide bounds (break position in the amino acid sequence) or by losses (â) or gains (+) of small neutral molecules (water, ammonia). The highest intensity peaks used for identification and quantification of the different peptides are indicated in blue. LC-MS/MS analysis was performed simultaneously for the native and phosphorylated peptides. (E) Spectrum of native (F) phosphorylated N-terminal peptides. (G) Spectrum of native (H) phosphorylated C-terminal peptides. The inserts show the chromatograms of the chosen fragmentation products.</p