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J. Kogo, Jr., Y. Takeba, T. Kumai, Y. Kitaoka, K. Kuribayashi, Y. Hayashi, H. Takeda, H. Fujino, S. Ueno, S. Kobayashi; Bax Activation-Mediated Apoptosis in Glutamate-Induced Neurotoxicity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3681. doi: https://doi.org/.
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Glutamate excitotoxicity has been implicated in a number of neurological disorders, such as stroke, Alzheimer’s disease and glaucoma. Bax, a proapoptotic member of the Bcl-2 family, has a major role in mitochondria-mediated apoptosis in different neuronal cell types. Recently, it has been reported that Bax deficiency prevents retinal ganglion cell death in glaucoma model mice. The object of this study is to investigate the involvement of Bax expression in glutamate-induced neurotoxicity in vitro.
We used nerve growth factor (NGF)-differentiated PC12h cells for neuronal model. NGF-treated PC12h resembled neuronal cells with neurite outgrowth as previously reported (Kogo et al., 2006). For differentiation, the cells were cultured in the presence of 100 ng/ml NGF for three days in medium contained 1% FCS. Neuronal cells were incubated with or without glutamate (0.1 mM, 1 mM, 5 mM) for 24 hours, subsequently apoptotic cells were detected by TUNEL assay. We examined the expression of Bax and caspase-9 protein which activate caspase-3 leading to DNA fragmentation, using Western blotting analysis. Furthermore, neuronal cells were pretreated with or without furosemide, a chloride channel inhibitor thought to block the Bax translocation to mitochondria prior to glutamate exposure.
Treatment of glutamate resulted in dose-dependent apoptosis in neuronal cells. Glutamate increased the levels of Bax and caspase-9 proteins. Furthermore, pretreatment of furosemide significantly inhibited the increase of caspase-9 protein induced by glutamate. However, the increase of Bax protein induced by glutamate was not significantly altered by furosemide.
These results indicate glutamate induces apoptosis through activation of Bax and caspase-9, which represent the classical pathway to apoptosis. Preventing increase of caspase-9 by furosemide may lead to be anti-apoptotic. Since furosemide showed no effect on Bax induction, it is assumed that the translocation of Bax to mitochondria may occur but not suppression of Bax synthesis.
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