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Thomas Wubben, Heather Knupp, Kevin Toolan, Cagri G Besirli; Regulatory roles of glycolytic enzymes in photoreceptor survival. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5378.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the regulatory roles of hexokinase II (HKII) and pyruvate kinase muscle isozyme 2 (PKM2) in pro- and anti-apoptotic pathways in photoreceptors following apoptotic stress.
Retina-retinal pigment epithelium (RPE) separation was created in Brown-Norway rats by subretinal injection of 1% hyaluronic acid. Retinas were harvested and assayed with Western blot analysis and immunohistochemistry. Cultured 661W photoreceptor cells were subjected to hypoxic and glucose-deprived conditions and assayed with Western blot analysis and quantitative PCR.
Retina-RPE separation resulted in an increase in the expression of HKII in the outer retina with HKII predominantly localized in the inner segments of photoreceptors at 24 hours post-detachment. Similarly, retinal detachment led to increased PKM2 expression in the outer nuclear layer and a global decrease in PKM2 phosphorylation. We developed an in vitro cell culture model to mimic hypoxic and hypoglycemic stress induced by photoreceptor-RPE separation. Cultured 661W photoreceptor cells subjected to hypoxic and glucose-deprived conditions showed increased protein levels of HKII as well as increased gene expression. These conditions were also observed to regulate PKM2 phosphorylation in 661W cells.
Our results demonstrate that the expression of glycolytic proteins HKII and PKM2, which are enriched in the outer nuclear layer of the retina, are regulated during apoptotic stress. We developed an in vitro system using 661W cone-like cells to replicate the hypoxic and hypoglycemic stress experienced by photoreceptors after retina-RPE separation. Similar to in vivo changes, we showed that hypoxic and hypoglycemic stress leads to changes in the expression and post-translational modification of glycolytic proteins, providing an in vitro platform to investigate the regulatory roles of glycolytic proteins in photoreceptor survival.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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