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-3 beta (GSK-3 beta), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3 beta 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-3b, evidenced by the increased level of the active form of the kinase, i.e. GSK-3 beta phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3 beta partially localized within mitochondria in both neuronal cell models. Moreover, MPP+ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3 beta 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-3 beta 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-3 beta is a critical mediator of MPTP/MPP+-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3 beta activity might provide protection against mitochondrial stress-induced cell death.