TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance

Abstract Ligands addressed to the mitochondrial Translocator Protein (TSPO) have been suggested as cell death/life and steroidogenesis modulators. Thus, TSPO ligands have been proposed as drug candidates in several diseases; nevertheless, a correlation between their binding affinity and in vitro efficacy has not been demonstrated yet, questioning the specificity of the observed effects. Since drug-target residence time is an emerging parameter able to influence drug pharmacological features, herein, the interaction between TSPO and irDE-MPIGA, a covalent TSPO ligand, was investigated in order to explore TSPO control on death/life processes in a standardized glioblastoma cell setting. After 90 min irDE-MPIGA cell treatment, 25 nM ligand concentration saturated irreversibly all TSPO binding sites; after 24 h, TSPO de-novo synthesis occurred and about 40 % TSPO binding sites resulted covalently bound to irDE-MPIGA. During cell culture treatments, several dynamic events were observed: (a) early apoptotic markers appeared, such as mitochondrial membrane potential collapse (at 3 h) and externalization of phosphatidylserine (at 6 h); (b) cell viability was reduced (at 6 h), without cell cycle arrest. After digitonin-permeabilized cell suspension treatment, a modulation of mitochondrial permeability transition pore was evidenced. Similar effects were elicited by the reversible TSPO ligand PIGA only when applied at micromolar dose. Interestingly, after 6 h, irDE-MPIGA cell exposure restored cell survival parameters. These results highlighted the ligand-target residence time and the cellular setting are crucial parameters that should be taken into account to understand the drug binding affinity and efficacy correlation and, above all, to translate efficiently cellular drug responses from bench to bedside

Involvment of Cytosolic and Mitochondrial GSK-3β in Mitochondrial Dysfunction and Neuronal Cell Death of MPTP/MPP+-Treated Neurons

Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson's disease (PD). 1-methyl-4-phenylpyridinium iodide (MPP+), the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3β (GSK-3β), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3β in modulating MPP+-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP+ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3β, evidenced by the increased level of the active form of the kinase, i.e. GSK-3β phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3β partially localized within mitochondria in both neuronal cell models. Moreover, MPP+ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3β labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP+ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3β activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP+-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3β is a critical mediator of MPTP/MPP+-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3β activity might provide protection against mitochondrial stress-induced cell death

Tracking zoonotic pathogens using bloodsucking flies as 'flying syringes'

About 60% of emerging infectious diseases in humans are of zoonotic origin. Their increasing number requires the development of new methods for early detection and monitoring of infectious agents in wildlife. Here, we investigated whether blood meals from hematophagous flies could be used to identify the infectious agents circulating in wild vertebrates. To this aim, 1230 blood-engorged flies were caught in the forests of Gabon. Identified blood meals (30%) were from 20 vertebrate species including mammals, birds and reptiles. Among them, 9% were infected by different extant malaria parasites among which some belonged to known parasite species, others to new parasite species or to parasite lineages for which only the vector was known. This study demonstrates that using hematophagous flies as 'flying syringes' constitutes an interesting approach to investigate blood-borne pathogen diversity in wild vertebrates and could be used as an early detection tool of zoonotic pathogens

Plusieurs épidémies de fièvre hémorragique due au virus Ebola au Gabon, d'octobre 2001 à avril 2002

Des épidémies de fièvre hémorragique virale à virus Ebola (FHVE) ont été rapportées de 1994 à 1996 dans la province de l’Ogooué Ivindo, une zone forestière située au Nord Est du Gabon. Chaque fois, les grands primates avaient été reconnus comme la source initiale de l’infection humaine. Fin novembre 2001 une nouvelle alerte est venue de cette province qui a rapidement était confirmée comme étant une épidémie de FHVE. La riposte a été organisée par le ministère de la santé publique avec l’aide d’une équipe internationale sous l’égide de l’OMS. Un système de surveillance active a été mis en place dans les trois districts touchés par l’épidémie (Zadié, Ivindo and Mpassa) afin d’organiser la détection des cas et le suivi de leur contacts. Une définition de cas a été adoptée, les cas suspects étaient isolés à l’hôpital, à domicile ou dans des lazarets, et des tests sérologiques étaient réalisés. Ces tests comportaient la détection de l’antigène ou des IgG spécifiques, et la RT-PCR. Un classement des cas était réalisé en fonction des résultats des tests biologiques et des données cliniques et épidémiologiques. Les sujets contacts étaient surveillés durant 21 jours. On a dénombré 65 cas dont 53 décès. Le premier cas humain, un chasseur, était décédé le 28 octobre 2001. L’épidémie s’est propagée grâce une transmission familiale et une contamination nosocomiale. Quatre foyers primaires distincts ont été mis en évidence ainsi qu’un cas isolé situé dans le Sud est du Gabon, à 580 km de l’épicentre, Les décès sont survenus dans un délai de 6 jours. Le dernier décès a été enregistré le 22 mars 2002 et la fin de l’épidémie déclarée le 6 mai 2002. L’épidémie s’est étendue au Congo voisin. Des morts inexpliquées d’animaux avaient été signalées dans les forêts environnantes dès le mois d’août 2001 : des grands primates et des céphalophes. Les prélèvements réalisés sur leurs carcasses confirmaient une épidémie animale concomitante