Purine derivatives and pathophysiology of Alzheimer’s disease

La maladie d’Alzheimer (AD), pathologie neurodégénérative progressive, est caractérisée par des dépôts β-amyloïdes extracellulaires, des enchevêtrements neurofibrillaires intracellulaires de Tau et une dégénérescence neuronale. A travers les nombreux modèles transgéniques AD disponibles, les connaissances sur les peptides amyloïdes et la protéine Tau ne cessent de progresser. Mais contrairement aux cas génétiques, l’étiologie des cas sporadiques d’AD reste à ce jour idiopathique, rendant difficile d’établir une stratégie thérapeutique efficace. Au cours d’une étude sur l’implication des protéines kinases dans la pathogénèse d’AD, des collaborateurs ont fait une observation totalement inattendue, mais très intéressante: une molécule de faible poids moléculaire, serait capable d’induire une production spécifique d’Aβ1-42 sans altérer les niveaux d’Aβ1-40 dans un modèle de lignée cellulaire. Dans ce contexte, le projet de thèse portait sur l’utilisation de dérivé purique (PD1) pour développer des modèles AD induits chimiquement sur différents supports (culture primaire de neurones, culture organotypique d’hippocampe et souris) et en investiguer les mécanismes sous-jacents à l’augmentation des peptides A1-42 (issus du métabolisme de l’APP (Amyloid precursor protein)).La première partie du projet de thèse a permis de mettre en évidence dans un contexte in vitro (culture primaire de neurones et culture organotypique d’hippocampe) que PD1 à forte dose induisait une augmentation du ratio Aβ42/40 et de manière répétable. Fort de ces résultats, nous avons voulu étudier les mécanismes d’action de PD1 autour de deux hypothèses : interaction dans le métabolisme de l’APP et implication des cellules gliales. Contrairement à nos premières hypothèses, nous avons montré que PD1 aurait de potentiels effets anti-inflammatoires (i.e. IL-1β) in vitro et in vivo. La voie de signalisation de l’IL-1β étant de plus en plus incriminée dans la pathogenèse d’Alzheimer; nous nous sommes interrogés sur l’effet dual de PD1 : outil pharmacologique alzheimerigène ou candidat médicament pour le traitement d’AD?Alzheimer’s disease (AD), a progressive neurodegenerative disorder, appears to be associated with an increase in a particular form of β-amyloid deposits, intracellular Tau tangles and neuronal degeneration. Through many available transgenic AD models, knowledge about amyloid peptides and Tau protein continues to increase. However, in contrast to the genetic cases of AD, the etiology of sporadic AD cases remains unknown, making the establishment of an effective therapeutic strategy difficult.During the course of a study on the role of protein kinase involved in AD, our collaborators made an unexpected but very interesting observation. They identified a low molecular weight compound able to induce production of Aβ1-42 while the level of the much less toxic form Aβ1-40 remained constant. This selective induction of Aβ1-42 versus Aβ1-40 was observed in a cell line model. Therefore, the overall goal of the project thesis was based on the use of purine derivative (PD1) to understand the molecular mechanisms underlying the selective production of Aβ1-42. This would allow us to establish cellular assays and a chemically-induced animal AD model relevant to studies on the treatment and prevention of AD.The first part of this project allowed us to demonstrate in vitro that PD1, at high dose, repeatedly induced an increase in Aβ42/40 ratio in primary neurons and in neuronal hippocampal slice culture (OHSCs). Based on these facts, we analyzed the amyloid profile by focusing on APP metabolism and on glial cell activity. In contrary to our hypothesis, we highlighted whether PD1 exhibits potential anti-inflammatory properties (i.e. IL-1β) both in vitro and in vivo. The IL-1β pathway is more and more linked in the AD pathogen which leads us to consider that PD1 could have a dual effect : alzheimerogenic pharmacological tool or potential drug candidate for the treatment of AD

Evaluation de la sécurité d'un produit en développement en association avec le donepezil (pharmacologie de sécurité - Test d'Irwin)

PARIS-BIUP (751062107) / SudocSudocFranceF

P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach.

Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y) and macrophage (RAW 264.7) cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA) induces oxidative stress and apoptosis. Cultured cells were treated with 2-200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen) were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose) significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain

Cell viability and membrane integrity in SH-SY5Y cells exposed to oxaliplatin.

<p>Cells (2×10⁵ cells/well) were exposed 24 h to oxaliplatin (OXA) (2–200 µM). Cell viability (redox potential) was evaluated with Alamar blue and membrane integrity was evaluated with neutral red tests (A). Cell viability which is indicative of mitochondrial metabolism was evaluated with MTT test (B). Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. *: statistically different (p<0.05) from the mean values in control cells.</p

Cytokines and PGE2 release in oxaliplatin-treated cells after acetaminophen, ibuprofen or N-acetyl cysteine treatment.

<p>SH-SY5Y cells and RAW 264.7 cells were exposed 24 h to oxaliplatin (OXA) (200 µM) with a 30-min pre-treatment either with acetaminophen (AAP, 50 µM), ibuprofen (IBU, 1 µM) or N-acetyl cysteine (NAC, 1 mM). SH-SY5Y cell supernatant was collected and TNF-α, IL-1β and IL-6 levels (pg/mL) were assessed using ELISA kits according to the manufacturer's instructions. Prostaglandin E2 (PGE2) levels were determined in RAW 264.7 supernatant using a PGE2 Enzyme-Immuno-Assay kit. Values are the mean ± S.E.M. levels in pg/ml, five different assays per group. Significance of differences: OXA alone versus control, *p<0.05; AAP, or IBU, or NAC versus OXA alone: ^$ p<0.05.</p

Cell viability and oxidative stress in oxaliplatin-treated SH-SY5Y cells pre-treated by protective drugs.

<p>Cells (2×10⁵ cells/well) were exposed for 24 h to oxaliplatin (OXA) (50, 100 or 200 µM) after a 30-min pre-treatment either with acetaminophen (AAP, 50 µM), ibuprofen (IBU, 1 µM) or N-acetyl cysteine (NAC, 1 mM). Cell viability (redox potential) was evaluated with Alamar blue (A). Oxidative stress was evaluated by ROS production using dihydroethidium (B) and DCF-DA (C) tests and nitric oxide (NO) production evaluated as nitrite content (D) by the Griess reaction. Lipid peroxidation (E) was determined using the thiobarbituric acid (TBA) method at 200 µM OXA. Mitochondrial activity was evaluated by determining mitochondrial membrane potential (Δφ_m) (F) using JC-1 test and mitochondrial levels of negatively charged phospholipids, mainly cardiolipin, using nonyl acridine orange test (G). Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. Significance of differences: OXA alone versus control, *p<0.05; AAP, or IBU, or NAC versus OXA alone: $ p<0.05.</p

Oxidative stress and mitochondrial activity in SH-SY5Y cells exposed to oxaliplatin.

<p>Cells (2×10⁵ cells/well) were exposed 24 h to oxaliplatin (OXA) (2–200 µM). Oxidative stress was evaluated by Reactive Oxygen Species (ROS) production using dihydroethidium and DCF-DA tests (A) and Nitric Oxide (NO) production evaluated as nitrite content (B) by the Griess reaction. Mitochondrial activity was evaluated by determining mitochondrial membrane potential (Δφ_m) (C) using JC-1 test and mitochondrial levels of negatively charged phospholipids, mainly cardiolipin, using nonyl acridine orange test (D). Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. *: statistically different (p<0.05) from the mean values in control cells.</p

Chromatin condensation, P2X7 receptor activation and caspase-3 activity in oxaliplatin-treated C57BL/6 mice.

<p>Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose; n = 10). Mitochondrial activity was evaluated by determining mitochondrial membrane potential (A) using JC-1 test and mitochondrial levels of negatively charged phospholipids (B) using nonyl acridine orange test. Chromatin condensation (C) was evaluated using Hoechst 33342 test and P2X7 receptor activation (D) using YOPRO-1 test. The apoTarget^TM Caspase-3 Protease assay was used for the <i>in vitro</i> determination of caspase-3 proteolytic activity (E) in lysates of brain mitochondrial homogenates as described by the manufacturer's instructions. Values are the mean ± S.E.M. expressed as percentage of the control (n = 8). *: statistically different (p<0.05) from the mean values in control mice.</p

Chromatin condensation, P2X7R activation and caspase-3 activity in oxaliplatin-treated SH-SY5Y cells pre-treated by protective drugs.

<p>Cells (2×10⁵ cells/well) were exposed for 24 h to oxaliplatin (OXA) (50, 100 or 200 µM) after a 30-min pre-treatment either with acetaminophen (AAP, 50 µM), ibuprofen (IBU, 1 µM) or N-acetyl cysteine (NAC, 1 mM). Chromatin condensation (A) was evaluated using Hoechst 33342 test and P2X7 receptor (P2X7R) activation (B) using YOPRO-1 test. The apoTarget^TM Caspase-3 Protease assay was used for the <i>in vitro</i> determination of caspase-3 proteolytic activity (C) in lysates of SH-SY5Y cells as described by the manufacturer's instructions. Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. Significance of differences: OXA alone versus control, *p<0.05; AAP, or IBU, or NAC versus OXA alone: $ p<0.05.</p