Decreased diphthamide methyltransferase expression is associated with the process of Light–induced retinal degeneration and apoptosis.

Abstract

Abstract: : Purpose: Light–induced retinal degeneration (LIRD) in rats is known to involve oxidative–stress mediated loss of rod photoreceptor cells in response to intense green light exposure. This cell loss involves DNA fragmentation and the activation of the caspase cascade, hallmark signs of classical apoptosis. To better understand LIRD we have differentially cross–screened a LIRD derived cDNA library and identified 75 differentially expressed genes. Clone 28–2 defines a novel rat EST that encodes a putative diphthamide methyltransferase, the gene product of which plays a role in regulating cell death in response to an oxidative stress environment. Methods: Clone 28–2 was sequenced and the full gene structure was determined bioinformatically. In addition, Northern analysis was performed. Analysis of the clone 28–2 sequence revealed that it represents a novel rat gene with significant similarities to the yeast diphthamide methyltransferase (Dph5) gene. To assess the role of this gene in the face of an oxidative stress, wildtype and knockout mutant Dph5 yeast cells were treated with varying concentrations of acetic acid or hydrogen peroxide and cell survival was assayed. The treated cells were analyzed using Transmission Electron Microscopy (TEM) for morphological features of apoptosis. Results: The LIRD expression of 28–2 was analyzed by Northern analysis. The gene represented by clone 28–2 is expressed at high levels in untreated retinae and the levels decrease following light exposure. The effect of a dysfunctional Dph5 gene was studied using yeast Dph5 mutant and wildtype cells. Yeast cells were treated with varying concentrations of acetic acid and H₂O₂, in order to simulate an oxidative stress environment. We found that Dph5 mutant cells were more sensitive to acetic acid and H₂O₂ treatment than wildtype yeast cells. Analysis of treated yeast cells by TEM determined that the cells were undergoing apoptosis, as opposed to necrosis. Conclusions: The loss of Dph5 expression is associated with an increased sensitivity to oxidative stress–mediated apoptotsis. Yeast Dph5 facilitates the modification of elongation factor–2, a potent regulator of protein synthesis, and may therefore regulate translation in cells committed to undergo active cell death. Since the inhibition of translation is thought to be at the level of initiation of translation, this work represents the first evidence that regulation of translation at the level of elongation may be involved in the LIRD apoptotic program.

Keywords: gene/expression • apoptosis/cell death • oxidation/oxidative or free radical damage