Comparison of Serine metabolism in Müller Cells from the Macula and Peripheral Retina

Abstract

Purpose : The macula is preferentially affected by several common blinding conditions such as age-related macular degeneration and diabetic macular edema. A better understanding of this vulnerability will provide a foundation for the development of better treatments. We believe that a key difference between the macula and the peripheral retina is the nature of their Müller cells. The aim of this study was to explore the molecular differences between macular and peripheral Müller cells.

Methods : mRNA and non-coding RNA transcripts of primary Müller cells from the human macular (M-hPMCs) or peripheral (P-hPMCs) retina were analysed by Transcriptome Deep Sequencing. Western blot was used to validate differential expression of proteins related to serine synthesis. ¹³C labelled serine was used to trace utilization of serine byM-hPMCs and P-hPMCs. AlamarBlue and lactate dehydrogenase assays were used to determine the susceptibility of M-hPMCs and P-hPMCs to oxidative stress following inhibition of serine synthesis. The redox balance from M-hPMCs and P-hPMCs under stress was measured using a CM-H2DCFDA reactive oxygen species (ROS) detection kit and ThiolTracker Violet Glutathione (GSH) detection kit by flow cytometry. The in vitro human primary cell results were further validated by ex vivo human retinal explant experiments.

Results : There were significant differences in gene transcription between M-hPMCs and P-hPMCs, notably in the de novo serine synthesis pathway. The upstream regulator (Nrf2) of the de novo serine synthesis pathway and its rate limiting enzyme (PHGDH) had significantly higher protein expression levels in M-hPMCs than in P-hPMCs. Most ¹³C-labeled serine converted to glycine but not to other glucose intermediates. More serine was converted to glycine in M-hPMCs than in P-hPMCs and there was a lower rate of reverse serine/glycine reactions occurring in M-hPMCs. The intracellular level of ROS and GSH are significantly higher in M-hPMCs. Inhibiting serine synthesis in Müller cells in vitro under oxidative stress led to more pronounced cytotoxicity in M-hPMCs compared with P-hPMCs.

Conclusions : There are different rates of de novo serine metabolism in M-hPMCs and P-hPMCs. Alterations in this pathway can disturb the redox balance in the macula, increasing susceptibility to oxidative stress. This may be a mechanism through which the macula is more susceptible to oxidative stress than the peripheral retina.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.