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R. R. Krishnamoorthy, V. R. Rao, M. Jiang, K. Tchedre, T. Yorio; Overexpression of Transcription Factor Brn3b Promotes Axon Elongation in Cultured Transformed Rat Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5374. doi: https://doi.org/.
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Recent studies indicate that there is a downregulation of the POU domain transcription factor, Brn3b, in animal models of glaucoma. This suggests the possibility that downregulation of Brn3b could be one mechanism responsible for the inability of retinal ganglion cells to regenerate axons after injury. The purpose of this study was to determine if overexpression of Brn3b could promote axon outgrowth in cultured transformed rat retinal ganglion cells.
Cultured transformed rat retinal ganglion cells (RGC-5 cells) were subjected to glaucomatous insults by treatment with 10, 100 and 200 µM glutamate. Another set of RGC-5 cells were treated with 1 and 10 nM endothelin-1 (ET-1). Nuclear extracts were isolated from these cells and analyzed for DNA binding activity of transcription factor Brn3b by electrophoretic mobility shift assays. In a separate set of experiments, RGC-5 cells were stably transformed with an expression vector for transcription factor Brn3b. Using immunocytochemistry, Brn3b expression was analyzed, in RGC-5 cells stably overexpressing transcription factor Brn3b and in control RGC-5 cells expressing the empty vector, pcDNA3.1+. Morphological changes were observed in these cells by microscopy and immunostaining for neurofilament H protein was carried out.
Treatment with glutamate and endothelin-1 produced a concentration-dependent decrease in the DNA binding activity of transcription factor Brn3b. Stable overexpression of transcription factor Brn3b in RGC-5 cells produced morphological changes including increased neurite outgrowth and a prominent axon elongation. An increased immunostaining for axon-specific neurofilament H was also observed in RGC-5 cells overexpressing Brn3b.
Glaucomatous stimuli including glutamate and endothelin-1 could be factors contributing to a decrease in transcription factor Brn3b as seen in animal models of glaucoma. Overexpressing transcription factor Brn3b could promote axon elongation in cultured rat retinal ganglion cells.
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