Comparison of Serine Biosynthesis in Human Retinal Pigment Epithelium from the Macula and Periphery

Abstract

Purpose : Degeneration of retinal pigment epithelium (RPE) is strongly associated with the pathogenesis of macular diseases such as age-related macular degeneration. Chronic oxidative stress and inflammation are primary contributing factors to degeneration of the RPE. Serine biosynthesis is a major metabolic pathway that protects cells from oxidative stress. The purpose of this study was to explore the role of the serine biosynthesis pathway in human RPE and its different activity in the macular and peripheral RPE.

Methods : Western Blot was carried out to assess the expressional changes of phosphoglycerate dehydrogenase (PHGDH, the rate limiting enzyme in the serine biosynthesis pathway) and its regulator-Nrf2 in primary cultured human RPE cells after exposed to mild oxidative stress (hydrogen peroxide) overnight. We then inhibited PHDGH with a specific inhibitor, CBR-5884. AlamarBlue Cell Viability and LDH Cytotoxicity assays were used to determine any differences in the susceptibility of human RPE cells to oxidative stress. The JC-1 assay was used to assess changes in mitochondrial membrane potential under oxidative stress. Immunostaining of serine biosynthesis-related enzymes was done on flat-mounted tissue and cross sections of human RPE from the macula and retinal periphery.

Results : We found all serine biosynthesis-related enzymes were expressed in human RPE. The expression of PHGDH and Nrf2 were significantly upregulated in RPE cells under oxidative stress. Inhibiting serine synthesis in primary cultured human RPE cells under oxidative stress led to a pronounced reduction in cell viability and mitochondrial membrane potentials. The expression of PHGDH in human RPE is patchy with significant higher number of PHGDH expressed RPE cells in macula than in retina periphery.

Conclusions : Serine biosynthesis plays a pivotal role in maintaining both the redox balance and normal mitochondria function in human RPE cells. Nrf2 is associated with serine biosynthesis in RPE cells to resist oxidative stress. The higher expression of PHGDH in macular RPE suggests more active serine biosynthesis compared with peripheral retina. This may be due to the higher oxidative stress caused by stronger metabolic activity in macula.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.