Involvment of cytosolic and mitochondrial GSK-3beta 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-3beta (GSK-3beta), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3beta 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-3beta, evidenced by the increased level of the active form of the kinase, i.e. GSK-3beta phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3beta partially localized within mitochondria in both neuronal cell models. Moreover, MPP(+) treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3beta 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-3beta 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-3beta is a critical mediator of MPTP/MPP(+)-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3beta activity might provide protection against mitochondrial stress-induced cell death.
Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6097, Valbonne, France. petit@ipmc.cnrs.fr
Agnès
A
Petit-Paitel
Frédéric
F
Brau
Julie
J
Cazareth
Joëlle
J
Chabry
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000008:administration & dosage; D015655:1-Methyl-4-phenylpyridinium / Q000008:administration & dosage / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D002458:Cell Fractionation; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D005434:Flow Cytometry; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D007150:Immunohistochemistry; D007274:Injections, Intraperitoneal; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000201:enzymology / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D015655:1-Methyl-4-phenylpyridinium