Modulation de la phosphorylation endogène 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 épileptogène de patients souffrants d épilepsies pharamacorésistantes. Nous avons proposé deux cibles potentielles qui sont impliquées dans un mécanisme intrinsèque du système GABAergique : d une part la GAPDH, kinase du GABAAR et d autre part la phosphatase membranaire qui contrecarre la phosphorylation endogène et la fonction du GABAAR. La première partie de mon travail de thèse a concerné l étude du rôle de la spermine dans la phosphorylation endogène du GABAAR ainsi que son rôle dans le rundown du récepteur. Les résultats obtenus ont montré que la spermine activait la phosphorylation endogène 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 deuxième partie de ma thèse 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 endogène identifiés de la boucle I2a1 du GABAAR comme substrats. Par cette méthode nous avons caractérisé les profils pharmacologiques des activités phosphatasiques détectées, qui s avèrent atypiques. Nous avons purifié et identifié ces activités par nano-LC-MALDI-TOF/TOF. En parallèle, 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

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_A Receptors

<div><p>Background</p><p>GABA_A receptor (GABA_AR) 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 I₂ 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_AR 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_AR. 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_AR 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_AR 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_AR.</p><p>Conclusion/Significance</p><p>Our results provide new insights on the regulation of GABA_AR 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_A current rundown.

<p>GABA_A 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 µM 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 µM 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 (n = 17) or in absence (Control, n = 11) 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: ‘Very Slow’ (n = 3), ‘Mid Slow’ (n = 5), ‘Mid Rapid’ (n = 4) and ‘Very Rapid’ (n = 5). For each group the plot (Left) is the average of normalized currents. Error bars indicate the SEM. One-way ANOVA with Dunnett’s 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_AR α1-subunit phosphatase activities.

<p>(A) Dose-dependency at various protein concentrations; (B) time course of phosphatase activities using 50 µg/ml of total proteins of washed cortical membranes from bovine brain and (C) from human epileptic tissue. The N-terminal phosphopeptide (pI₂α1N-P) used as substrate at 10 µM was incubated during 10 min at 30°C in 10 mM Hepes (pH 7.4) in the presence of 1 mM Mg²⁺. 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 (pI₂α1N) and of the remaining phosphorylated substrate (pI₂α1N-P). Error bars represent SEM of two experiments. Kinetic analysis of GABA_AR α1-subunit phosphatase activities was investigated using N-terminal (pI₂α1N-P) and C-terminal (pI₂α1C-P) phosphopeptides. All experiments were carried out with 50 µg/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_i versus [pI₂α1N-P] and Lineweaver-Burk plots of 1/V versus 1/[pI₂α1N-P] (inset) showing two significantly different slopes. (E) Saturation plot of V_i versus [pI₂α1C-P] and Lineweaver-Burk plot of 1/V versus 1/[pI₂α1C-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 (I₂α1) of type A gamma-amino butyric acid receptor (GABA_AR). The sequences indicate the synthetic N- and C-terminal peptides (AA334–346 and AA407–420 respectively) used in this study, with the two identified phosphorylation sites (PO₄) 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 “b” (N-flanking) or “y” (C-flanking) when the ion is broken at peptide bounds (break position in the amino acid sequence) or by losses (−) 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