Individual Protective Effects Of FK506 And NF-Bp50 Over-expression On Glutamate-inducible Neurotoxicity In Retinal Ganglion Cells

Abstract

Purpose: : Loss of retinal ganglion cells observed in glaucoma is the cause of characteristic glaucomatous cupping of the optic nerve head.To examine the neuroprotective effects of FK506 and NF-kBp50 over-expression on glutamate-inducible neurotoxicity in cultured retinal ganglion cells.

Methods: : RGC-5cells, which is an established rat retinal ganglion cell line were pre-treated with FK506 beginning 24 h before stimulation with glutamate or NMDA. The RGC-5 cells were transfected with NF-kBp50 expression vector, and cultured for 24 h before application of glutamate or NMDA. After stimulation with glutamate or NMDA, the RGC-5 cells were incubated for 24 h, and then the cell viabilities were quantitatively measured under microscope. Pro-apoptotic BAX protein markedly induces cell death. The pBAX-luciferase vector, which contained BAX promoter/enhancer element were transfected with and without NF-kBp50 expression vector. Glutamate was added to the culture media at 48 h after transfection. At 24 h after stimulation with glutamate, the whole cell lysates were prepared and then the luciferase activity was examined using the GloMax.

Results: : Brief exposure to glutamate or NMDA for 24 h markedly decreased the cell viability. On the other hand, NF-kBp50 over-expression and pre-treatment with FK 506 keep the cell viability after the stimulation with glutamate or NMDA. Western blot analysis showed that glutamate-induced NF-kB(p50/p65) expression and translocation to the nucleus were prevented by FK506 pre-treatement. Gel Shift Assay revealed that although glutamate-treatment markedly induced NF-kB activation, NF-kBp50 over-expression or FK506 pre-treatment inhibited the glutamate-induced translocation of NF-kB from the cytosol to nucleus in RGC-5 cells. As a result, glutamate-induced Bax expression was reduced by NF-kBp50 over-expression or FK506 pre-treatment. These experiments demonstrated the protective effects of FK506 or NF-kBp50 over-expression on glutamate-induceble neurotoxicity in retinal ganglion cells.

Conclusions: : FK506 pre-treatment or NF-kBp50 over-expression protected against glutamate-inducible neurotoxicity by BAX activation in cultured retinal neurons.

Keywords: cell survival • ganglion cells • retinal culture