Abstract
Purpose :
Glucose metabolism has been central in the study of photoreceptor cell physiology. However, recent evidence shows that fuel sources besides glucose can be utilized to meet the metabolic needs of photoreceptors. Therefore, we sought fundamental insight into the contribution of glutamine (Gln) as a potential alternative fuel source to photoreceptor metabolism, function, and survival.
Methods :
Targeted metabolomic analyses was performed in 661W cells grown in uniformly labeled 13C-Gln in the presence or absence of the glutaminase (GLS) inhibitor, CB-839. 661W cell viability and redox status were measured with luminescent-based assays (Promega). To study the role of Gln catabolism in vivo in photoreceptors, GLS was deleted from rod photoreceptors (Gls cKO) in animals expressing cre-recombinase under the control of the rhodopsin promoter and a floxed Gls gene. The effect of GLS deletion on the survival of the photoreceptors was examined using in vivo and ex vivo analyses.
Results :
Gls expression was over 20 times greater than its paralog, Gls2, in 661W cells and predominantly segregated to mitochondria. Treatment with CB-839 in 661W cells increased cellular Gln and decreased Gln-derived Glu. Furthermore, α-ketoglutarate, succinate, aspartate and malate were severely depleted underscoring that Gln contributes to TCA cycle. Additionally, inhibiting GLS reduced the biosynthesis of glutathione (GSH), altered redox homeostasis (GSH/GSSG ratio) and increased reactive oxygen species. Concomitantly, GLS inhibition reduced cell survival. In the mouse retina IHC, RT-PCR, and western blot analyses demonstrated that GLS is the predominant isoenzyme, enriched in photoreceptor inner segments, and segregated more to the mitochondrial than cytosolic fraction. IHC and western blot validated the rod-specific knockdown of GLS protein levels of the Gls cKO mouse. Gls cKO mice demonstrated comparable outer retinal thickness to wild-type animals at P14 with rapid photoreceptor degeneration evident by P21.
Conclusions :
This study demonstrates that Gln catabolism via GLS is critical for redox balance, mitochondrial metabolism, and photoreceptor survival. Insight into the contribution of Gln to photoreceptor metabolism, function and survival may provide a framework for developing novel therapeutic approaches to prevent blindness in retinal degenerations.
This is a 2021 ARVO Annual Meeting abstract.