Abstract
Purpose :
AMD is the leading cause of blindness among the elderly in the western world with no efficient treatment. Elevated reactive oxygen species (ROS) levels in the retinal pigment epithelial cells (RPE) are a hallmark feature of AMD and ROS scavenging is an attractive therapeutic option. Mitochondria are the major source of cellular ROS and we have earlier shown loss of Pink1, the master regulator of mitophagy causes glycolytic perturbations and elevated ROS levels. Since serine synthesis through PHGDH is tightly linked to glycolysis, here we investigate the effect of serine metabolism on ROS levels.
Methods :
Following experiments were conducted in ARPE-19 cells treated with scramble or Pink1 siRNA and on the RPE extracted from C57 and Pink1-/- male mice aged 8 weeks (n=3) (i) RT-qPCR and immunoblots for PHGDH (ii) Colorimetric or fluorometric quantification of NAD+/NADH NADP+/NADPH and GSH/GSSG ratios. (iii) Fluorometric quantification of serine levels. (iv) Measuring ROS levels in vitro and in vivo (DHE and MitoROS) with 200µM serine supplementation. (v) Further, to provide disease relevance, PHGDH levels were assessed in AMD and healthy control eye sections using immunohistochemistry.
Results :
(i) PHGDH was significantly decreased in areas of drusen in AMD eye sections. (ii) PHGDH was significantly reduced in both mRNA and protein expression in the RPE of Pink1-/- mice (p<0.0005; n=3) and in siPink1 treated ARPE-19 cells (p<0.0005; n=6). (iii) In ARPE-19 treated with siPink1, NAD+/NADH ratio was increased by 40% (p<0.005; n=3), NADP+/NADPH ratio was reduced by 20% (p<0.05; n=3), GSH/GSSG ratio was reduced by 10% (p<0.005; n=3). (iv) Serine levels were reduced by 40% in Pink1-/- mice (p<0.0005; n=3), however they were reduced by 80% (p<0.0005; n=3) in siPink1 cells. (v) ROS levels were increased by 1.5-fold (p<0.0005; n=3) in ARPE-19 cells upon siPink1 treatment (p<0.005; n=3) and decreased by 1.5-fold (p<0.005; n=3) when supplemented with serine.
Conclusions :
Our results show that: (i) Both serine synthesis and cellular serine levels are affected by mitophagic impairment (ii) Redox mechanisms like NADH and NADPH which are directly downstream of serine metabolism are also affected (iii) Ultimate effect of these is elevated ROS levels and role of serine metabolism in ROS management is further established by rescue of ROS following serine supplementation, which can also be used as a potential therapeutic target in AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.