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V.J. Davies, M. Boulton, M. Votruba; Expression Profile of Rat Primary Retinal Ganglion Cells and Retinal Cell Lines, RGC–5 and R28 and Mitochondrial Morphological Changes Following OPA1 Knockdown Mediated siRNA . Invest. Ophthalmol. Vis. Sci. 2006;47(13):169.
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© ARVO (1962-2015); The Authors (2016-present)
Autosomal Dominant Optic Atrophy (ADOA), due to mutations in the OPA1 gene, is characterised primarily by cell death confined to the retinal ganglion cell (RGC) layer. The purpose of this study was to examine how closely transformed rat RGC lines reflect the characteristics of primary RGCs in terms of the retinal cell markers expressed, and the effects on mitochondrial morphology of siRNA mediated knock–down of OPA1.
Primary RGCs were generated using immuno–magnetic selection of Thy–1 positive cells from P7 dissociated Wistar rat retinas, and grown on laminin coated 4–well plates. Cells from two retinal cell lines, RGC–5 and R28 were seeded on 4–well plates and grown in supplemented DMEM. Cells were fixed in 3.7% paraformaldehyde and immunostained. A battery of retinal cell markers was examined: Thy 1, Tuj 1, Brn–3b, neurofilament subunits, NF–L, M and H, Tau and MAP–2 and OPA1, which is expressed in RGCs and other cells of the inner retina. Short interfering (si)RNA–mediated transfection and downregulation of OPA1 using four de novo designed siRNAs to OPA1 and previously published anti–OPA1 siRNA was optimised in HeLa and RGC–5 cell lines and applied to primary RGCs at day 7.
Positive immunofluorescent staining of the cell body, membrane and axons was observed in rat RGC–5 and primary rat RGCs for Thy 1, Tuj 1 and OPA1. This is compatible with expression patterns seen with immunohistochemistry of rat, mouse and human retina sections. Primary RGCs were viable up to 17 days in culture. SiRNA–mediated affects on primary RGCs demonstrated loss of normal morphology and increasing fragmentation of the mitochondrial network.
The RGC–5 cell line and primary RGCs are valuable lines for examining the cytotoxic mechanisms behind RGC loss in ADOA. Si–RNA mediated downregulation of OPA1 leads to morphological changes in mitochondria and aggregation of the mitochondial network.
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