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E.A. Seidler, L.A. Levin; Functional Consequences of Mitochondrial Stress in mtDNA–Depleted RGC–5 Cells for the Study of Mitochondrial Optic Neuropathies . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2894.
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© ARVO (1962-2015); The Authors (2016-present)
Leber’s hereditary optic neuropathy (LHON) is a maternally inherited optic neuropathy characterized by spontaneous bilateral central vision loss. Most patients have one of three primary mutations in mitochondrial DNA (mtDNA) coding for complex I subunits. Currently there exists no effective treatment for LHON, as its pathogenesis is not well understood. As a first step in studying this disease, we have created an mtDNA–depleted line of RGC–5 cells that can be differentiated into retinal ganglion cell (RGC)–like cells. The cellular response of these cells under mitochondrial stress was compared to that of nondepleted RGC–5 cells.
Mitotic RGC–5 cells, a transformed RGC precursor cell line, were treated with varying doses of ethidium bromide (EtBr) supplemented with pyruvate and uridine (P/U). Both mtDNA and nuclear DNA levels of the EtBr–treated and untreated RGC–5 cells were assessed using PCR. Cells were plated and treated in duplicate with four different media preparations (all mtDNA–depleted cell conditions supplemented with P/U): plain media, rotenone (10 µM), potassium cyanide (KCN; 3 mM), and glucose–free media. Microscopic images of all conditions were obtained 24 hours after treatment to qualitatively assess cellular responses.
mtDNA–deficient RGC–5 cells responded differently to rotenone, KCN and glucose deprivation than control RGC–5 cells. Both rotenone and KCN induced cell death in control RGC–5 cells, but did not significantly affect mtDNA–deficient cells. Conversely, glucose deprivation did not significantly affect control RGC–5 cells, but induced massive cell death in mtDNA–depleted cells.
mtDNA–depleted RGC–5 cells are highly sensitive to glucose deprivation, but are resistant to cell death induced by rotenone, a complex I inhibitor, and KCN, a cytochrome aa3 inhibitor. Mechanisms by which differentiated mtDNA–deficient RGC–5 cells undergo cell death could correlate with their sensitivity in LHON.
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