A Human TREK-1/HEK Cell Line: A Highly Efficient Screening Tool for Drug Development in Neurological Diseases

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model

Behavioral study of spadin and its analogs : new antidepressant concept

La dépression est une maladie psychiatrique qui atteint environ 20% de la population. En 2006 notre équipe a montré l’implication du canal potassique TREK-1 dans cette pathologie, et, en 2010, elle a identifié un bloqueur de TREK-1, la spadine, ayant des propriétés antidépressives. La spadine a la même efficacité après quatre jours de traitement que les antidépresseurs classiques qui en nécessitent vingt-et-un. Lors de mon doctorat, nous avons démontré la spécificité de la spadine pour les canaux TREK-1 et l’absence d’effet secondaire notamment au niveau cardiaque et sur les fonctions où l’activation du canal TREK-1 a des effets bénéfiques (ischémie, épilepsie, douleur). Nous avons pour cela utilisé des techniques d’électrophysiologiques et différents tests comportementaux adaptés à ces différentes pathologies. J’ai également démontré les effets antidépressifs de la spadine sur deux modèles animaux, un modèle génétique, les souris Rouen, et un modèle induit par un traitement à la coticostérone. La dernière partie de ma thèse a porté sur l’identification d’analogues de la spadine ayant une meilleure affinité et une meilleure efficacité in vivo, et ne présentant pas d’effet secondaire. Deux peptides synthétisés par la technique de retro-inverso ont présenté ces propriétés. Parallèlement, nous avons également recherché, en collaboration avec la société MedinCell, une formulation de polymère permettant la libération constante et prolongée de l’analogue sélectionné. Cette formulation a pour but de résoudre les problèmes engendrés par le non-respect de la prise de médicaments par les patients en administrant en une seule injection le traitement pour trois voire quatre semaines.Depression is a devastating psychiatric disorder which affects about 20% of the population. In 2006 our team demonstrated the involvement of TREK-1, a potassium channel, in this pathology, and that its inhibition has led to a depression’s resistant phenotype. The search of TREK-1 inhibitors, a potential antidepressant, has led to the discovery of spadin. Spadin has, after only a four day treatment, similar efficacy than classical antidepressants which require about three weeks of treatment to produce their therapeutic effects. My work was firstly focused on the potential side effects of spadin. Indeed, TREK- 1’s activation has beneficial effects in many pathophysiologies (ischemia, epilepsy, pain). Its inhibition by spadin could generate significant adverse effects. The use of animal models has allowed us to confirm that spadin has no side effects related to TREK-1 channel’s inhibition. The specificity of spadin has been demonstrated since it has no effect on other potassium channels belonging to the K2P family. My work was also to study the effects of spadin in two mice models of depression, a genetic model and an induced model. In both cases, spadin shows a specific antidepressant effect in different behavioral tests of depression. The last part of my work was focused on the development of spadin’s analogs in order to improve the affinity and in vivo efficiency. We also developed, in collaboration with the MedinCell society, a polymer formulation for the constant and prolonged release of the selected analog. This formulation will be administered as a single injection treatment for three to four weeks, solving the problems caused by non respected medication by patients

Étude comportementale de la spadine et de ses analogues : un nouveau concept d'antidépresseur

Depression is a devastating psychiatric disorder which affects about 20% of the population. In 2006 our team demonstrated the involvement of TREK-1, a potassium channel, in this pathology, and that its inhibition has led to a depression’s resistant phenotype. The search of TREK-1 inhibitors, a potential antidepressant, has led to the discovery of spadin. Spadin has, after only a four day treatment, similar efficacy than classical antidepressants which require about three weeks of treatment to produce their therapeutic effects. My work was firstly focused on the potential side effects of spadin. Indeed, TREK- 1’s activation has beneficial effects in many pathophysiologies (ischemia, epilepsy, pain). Its inhibition by spadin could generate significant adverse effects. The use of animal models has allowed us to confirm that spadin has no side effects related to TREK-1 channel’s inhibition. The specificity of spadin has been demonstrated since it has no effect on other potassium channels belonging to the K2P family. My work was also to study the effects of spadin in two mice models of depression, a genetic model and an induced model. In both cases, spadin shows a specific antidepressant effect in different behavioral tests of depression. The last part of my work was focused on the development of spadin’s analogs in order to improve the affinity and in vivo efficiency. We also developed, in collaboration with the MedinCell society, a polymer formulation for the constant and prolonged release of the selected analog. This formulation will be administered as a single injection treatment for three to four weeks, solving the problems caused by non respected medication by patients.La dépression est une maladie psychiatrique qui atteint environ 20% de la population. En 2006 notre équipe a montré l’implication du canal potassique TREK-1 dans cette pathologie, et, en 2010, elle a identifié un bloqueur de TREK-1, la spadine, ayant des propriétés antidépressives. La spadine a la même efficacité après quatre jours de traitement que les antidépresseurs classiques qui en nécessitent vingt-et-un. Lors de mon doctorat, nous avons démontré la spécificité de la spadine pour les canaux TREK-1 et l’absence d’effet secondaire notamment au niveau cardiaque et sur les fonctions où l’activation du canal TREK-1 a des effets bénéfiques (ischémie, épilepsie, douleur). Nous avons pour cela utilisé des techniques d’électrophysiologiques et différents tests comportementaux adaptés à ces différentes pathologies. J’ai également démontré les effets antidépressifs de la spadine sur deux modèles animaux, un modèle génétique, les souris Rouen, et un modèle induit par un traitement à la coticostérone. La dernière partie de ma thèse a porté sur l’identification d’analogues de la spadine ayant une meilleure affinité et une meilleure efficacité in vivo, et ne présentant pas d’effet secondaire. Deux peptides synthétisés par la technique de retro-inverso ont présenté ces propriétés. Parallèlement, nous avons également recherché, en collaboration avec la société MedinCell, une formulation de polymère permettant la libération constante et prolongée de l’analogue sélectionné. Cette formulation a pour but de résoudre les problèmes engendrés par le non-respect de la prise de médicaments par les patients en administrant en une seule injection le traitement pour trois voire quatre semaines

Pharmacological activation of h-TREK-1 current in whole cell patch clamp configuration.

<p>Currents were recorded in the presence of a cocktail of potassium channel inhibitors (K⁺ blockers). <b>A</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by AA 10 µM (n = 118). <b>B</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by DHA 10 µM (n = 10). <b>C</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by ALA 10 µM (n = 10). <b>D</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after current activation by riluzole 100 µM (n = 12) perfused during 30 s (open circle) or 90 s (closed triangle). Each pharmacological activator was tested on HEK-293 native cells (n = 10) and current/potential curves were shown in the inset of each curve. <b>E</b>) Current density values measured at 0 mV after different times of perfusion of 100 µM riluzole (n = 10 at each time value).</p

Stretch and pH activation of h-TREK-1 current in cell attached (C.A.) and in inside out (I.O.) patch clamp configurations.

<p>Currents were recorded by application of negative pressure and by internal acidification. <b>A, B</b>) Current/pressure relationship and typical current traces obtained on h-TREK-1/HEK cells (closed circle, n = 30) and on HEK cells (closed square, n = 5) in cell-attached patch clamp configuration. <b>C, D</b>) Current/pressure relationship and typical current traces obtained on h-TREK-1/HEK cells (closed circle, n = 30) and on HEK cells (closed square, n = 4) in inside-out patch clamp configuration. Inset: negative pressure step protocol, increase −10 mmHg. <b>E, F</b>) Current/potential curves and corresponding histograms obtained after internal acidification on h-TREK-1/HEK cells (n = 8) and on HEK cells (n = 3).</p

GFP expression and functional h-TREK-1 channel activity on h-TREK-1/HEK cells.

<p><b>A</b>) Typical pictures of cells observed in transmission and in fluorescence at round three of cell culture. Functional channel activity was evaluated by current activation with 10 µM AA. <b>B</b>) Typical pictures of cells observed in transmission and in fluorescence at round fourteenth of cell culture. Functional channel activity was evaluated by current activation with 10 µM AA. <b>C</b>) Real time q-PCR. Levels of TREK-1 or sortlin expression were normalized with the cyclophillin D expression.</p

Pharmacological inhibition of h-TREK-1 current in whole cell patch clamp configuration by spadin (n = 12 for each dose) and fluoxetine (n = 12 for each dose).

<p>Currents were recorded in the presence of a cocktail of potassium channel inhibitors (K⁺ blockers). The inhibition was obtained after the pre-activation of the current by 10 µM AA. <b>A)</b> Current/potential curves obtained in presence of K⁺ blockers (closed circles), K⁺ blockers + AA (open circles) and K⁺ blockers + AA + spadin 1 µM (closed triangles). The absence of voltage dependence of spadin inhibition (100 nM) was shown in the inset. <b>B)</b> Spadin dose dependent inhibition at 0 mV potential. <b>C)</b> Typical traces of hTREK-1 current pre-activated by 10 µM AA and inhibited by 1 µM spadin. <b>D)</b> Current/potential curves obtained in the presence of K⁺ blockers (closed circles), K⁺ blockers + 10 µM AA (open circles) and K⁺ blockers + 10 µM AA + 30 µM fluoxetine (closed triangles). The absence of voltage dependence of fluoxetine inhibition (10 µM) was shown in the inset. <b>E)</b> Fluoxetine dose-dependent inhibition at 0 mV potential. <b>F)</b> Typical traces of h-TREK-1 current pre-activated by 10 µM AA and inhibited by 30 µM fluoxetine. <b>G, H)</b> Current/potential curves and representative traces of spadin inhibition (1 µM) on 100 µM riluzole activated hTREK-1 current (n = 10). <b>I, J)</b> Current/potential curves and representative traces of fluoxetine inhibition (30 µM) on 100 µM riluzole activated hTREK-1 current (n = 10).</p

Oxygen deprivation glucose experiments (OGD) on h-TREK-1/HEK cells and on HEK cells (A, B).

<p>After two hours of OGD in control conditions or with different treatments, survival cells were fixed, labeled and counted (n = 8 for each groups). <b>A)</b> Typical pictures of cells from each tested condition. <b>B)</b> Histograms of number of survival cells (Tukey test, F_7,568 = 38.65; *** or ### or $$$ p<0.001) <b>c)</b> The different tested conditions and their corresponding number.</p

Additional file 1: of Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation

ST1. Genome features and annotations. 1a. Genome resources and nomenclature used in this study. 1b. BUSCO analysis. 1c. TE analysis. 1d. Orthology analysis (OrthoMCL) summary (nine oomycetes and Ae, Aa). 1e. OrthoMCL, group ID (11 oomycete proteomes). 1f. Ae, Aa: predicted secreted genes ID. 1g. Ae, Aa: GO analysis (secretome). 1h. Ae, Aa: specific secretomes analysis. 1i. CAZyome analysis. 1j. Fungal and oomycete pectate lyase sequences. 1k. CRN effectors predicted in Ae and Aa genomes. (XLSX 2962 kb

Additional file 6: of Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation

ST3. A. euteiches small secreted proteins (AeSSPs). 3a. AeSSP classification and expression. 3b. Primers used in this study. (XLSX 37 kb