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W. Fan, N. G. F. Cooper; Characterization of Glutamate-Induced NFB Activation in Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):246.
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© ARVO (1962-2015); The Authors (2016-present)
NFΚB, a universal transcription factor, has been implicated in neuronal survival as well as in cell death mechanisms. Recently, it has been shown that NFΚB is activated in retina, especially in retinal ganglion cells (RGCs) in several scenarios, including NMDA-induced retinal neurotoxicity (p65), optic nerve transaction (p65 and p50) and retinal ischemia and reperfusion injury (p65). The mechanisms underlying distinct NFΚB protein activation and their role in regulating the cells death/survival following these types of injuries remain unclear. The purpose of this study is to investigate which NFΚB member(s) is activated in response to glutamate treatment in retina, specifically in RGCs.
Retinal explants and RGCs purified from postnatal day 6-8 SD rat retinas were used. Expression of NFΚB proteins was assessed in both retina and purified RGCs with the aid of immunohistochemical staining using the specific antibodies against distinct NFΚB members. Retinal explants or RGCs were treated with glutamate with or without the presence of the NDMA receptor antagonist, memantine, calcium chelator, EGTA, or the autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor for CaMKII, which has been shown to be neuroprotective against excitotoxic stress in retina in vivo. Activation and characterization of NFΚB proteins was determined by electrophoretic mobility shift assays, super shift assays and/or immunostaining.
All the five NFΚB proteins (p65, RelB, c-Rel, p50/p105, and p52/p100) were present in the retina and RGCs. P50, p52 and RelB were constitutively active in the retina. In response to a glutamate stimulus, all NFΚB proteins except c-Rel were activated. P65 only showed an inducible activation in retina and RGCs. Blocking NMDA receptor or chelation of extracelluar Ca2+ inhibited NFΚB activation. Inhibition of CaMKII activity with AIP also decreased NFΚB activation. Glutamate treatment induced an increase in IΚB-α phosphorylation and treatment with the AIP significantly reduced the level of glutamate-induced IΚB-α phosphorylation.
Our data indicate that glutamate activates distinct NFΚB proteins in the retina. P65 activation may be especially important in regulating RGC death/survival given that its activity can only be induced. The NMDA receptor-Ca 2+-CaMKII signaling pathway may be involved in glutamate-induced NFΚB activation. Since AIP blocks the phosphorylation of IΚB-α, the latter could either be a substrate for CaMKII or regulation of IΚB/NFΚB is mediated by some other factor downstream of CaMKII.
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