engleski

Neuroprotective effect of zolpidem against glutamate-induced toxicity in P19 neurons

Excitotoxicity is the pathological process in which neurons are damaged by excessive glutamate spillover, the major fast excitatory neurotransmitter in the mammalian central nervous system, and consequent prolonged stimulation of N-methyl-D-aspartate (NMDA) glutamate receptors. Excitotoxicity-related neuronal degeneration contributes to ischemia-induced brain damage, traumatic brain injury, epilepsy, and various chronic neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Although intracellular pathways responsible for the glutamate-induced neuronal death are not completely understood, it is known that glutamate enhances accumulation of reactive oxygen species (ROS) and induces oxidative stress. The aim of our study was to investigate a potential neuroprotective effect of zolpidem, a drug that acts via GABAA receptor complex and possess structural analogy with melatonin, an endogenous compound with antioxidative properties, against glutamate-induced toxicity in the culture of differentiated P19 neurons that express both GABAA and NMDA receptors. P19 neurons were obtained from the mouse embryonal carcinoma cells in the presence of retinoic acid. They were exposed to glutamate (300 µM, 3 hours) and then to zolpidem (10 µM, 2 hours). 24 hours later, the survival of P19 neurons was determined by the colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method and trypan blue assay. ROS accumulation was measured by the assay that utilizes the cell permeable substrate, 2’, 7’- dichlorofluorescin diacetate. Effect on plasma membrane integrity was determined by CytoTox-ONETM Homogeneous Membrane Integrity Assay (Promega). Changes in the nuclear condensation that are characteristic for the programmed type of cell death were assessed by Hoechst staining. Measurement of the activities of key apoptotic markers caspase-3 and -7 was performed with Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega). Glutamate- and zolpidem-induced changes in the protein expression of transcription factor p53 and activated (phosphorylated) Akt were determined by the Western blot method, while changes in the expression of apoptosis-related genes (Bax, Bcl-2, p53 and fos) were monitored by the semiquantitative RT-PCR method. Zolpidem post-treatment diminished glutamate-induced decrease in viability of P19 neurons. Although zolpidem prevented glutamate-induced ROS increase, it failed to prevent neuronal death induced by exposure to hydrogen peroxide and excess copper ions that trigger much higher accumulation of ROS, thus indicating that neuroprotective effect of zolpidem against glutamate toxicity was not achieved solely by direct antioxidant activity. Zolpidem also prevented induction of caspase-independent programmed cell death, as well as the glutamate-induced p53 and Bax increase at the transcriptional and translational level. The neuroprotective effect of zolpidem was prevented in the presence of 100 nM wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K)/Akt signalling pathway. The obtained results indicate that neuroprotective effect of zolpidem against glutamate-induced neuronal injury is predominantly achieved via PI3K/Akt signalling. Although further studies are required to better elucidate mechanisms of neuroprotective action of zolpidem at the cellular and molecular level, the presented findings are promising regarding the potential use of zolpidem in the reduction of excitotoxicity-related neuronal dysfunction and degeneration.

Zolpidem ; Excitotoxicity ; Neuroprotection ; Akt signalling ; P19 neurons

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