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W. Fan, N.G. F. Cooper; Regulation of Brain–Derived Growth Factor in Retinal Ganglion Cells (RGC5): Expression, Secretion and Signaling Pathway . Invest. Ophthalmol. Vis. Sci. 2005;46(13):172.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Brain–derived neurotrophic factor (BDNF) plays an important role in neuronal development, survival and synaptic plasticity. BDNF expression has been shown to vary following a number of injuries in retinal ganglion cells (RGCs). Understanding how BDNF is regulated and how it functions will enhance our knowledge of its diverse effects and may lead to more effective treatments for neurodegenerative disorders, including glaucoma. The purpose of this study was to investigate the mode of BDNF expression and secretion, and to deduce the possible signaling pathway involved during excitotoxic stress by using RGC cell line, RGC5. Methods:RGC5 cells were exposed to glutamate for various periods of time, in the presence or absence of prospective regulatory molecules. BDNF mRNA and protein expression was assessed with the aid of real–time PCR and immunoblots, respectively. BDNF secretion into the culture medium was determined with the aid of ELISA. Nuclear translocation of NFΚB was evaluated at single cell level with the aid of immunofluorescence microscopy. To determine the function of endogenous BDNF, neutralizing antibody to BDNF or its TrKB receptor were used and cell viability was assessed. NFΚB inhibitor, N–α–tosyl–L–phenylalanine chloromethyl ketone (TLCK) was used to study the prospective association of NFΚB and BDNF expression in RGC5 cells. The effect of myristoylated autocamtide–2–related inhibitory peptide (m–AIP), a specific calcium/ calmodulin–dependent protein kinase–II (CaMKII) inhibitor on BDNF expression and secretion was also investigated. Results:Glutamate stimulated a transient increase in BDNF mRNA and protein in RGC5 cells. Also glutamate initiated an early and small but significant release of BDNF into culture media. Application of BDNF neutralizing antibody or TrkB blocking antibody enhanced glutamate–induced excitotoxicity. NFΚB nuclear translocation was revealed in response to glutamate treatment. Application of TLCK inhibited glutamate–induced BDNF expression and secretion. Inhibition of CaMKII by m–AIP significantly enhanced the release of BDNF from glutamate challenged cells. Conclusions: Our data suggest that glutamate treatment may stimulate expression of BDNF in RGC5 cells through NFΚB activation. A novel neuroprotective role is proposed for the CaMKII inhibitor, AIP, which may protect RGC5 cells from excitotoxicity by enhancing release of BDNF from glutamate challenged cells.
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