This application will determine the effects of decreased folate/elevated homocysteine (Hey) on retinal function. Folate, a water-soluble vitamin, is essential for DNA, RNA and protein synthesis. Decreased levels of folate lead to elevation of Hey. In humans, folate deficiency is associated with optic neuropathy and nutritional amblyopia;hyperhomocysteinemia is associated with glaucoma, age-related maculopathy and diabetic retinopathy. In the previous funding period, we analyzed the mechanisms by which retinal pigment epithelial (RPE) cells acquire folate and transfer it to the neural retina. We discovered the polarized distribution of two folate transport proteins in RPE, reduced-folate transporter-1 (RFT-1) and folate receptor a (FRa). Our studies focused on RFT-1. In this proposal we focus on folate receptors (FRs). We predict that they are the sole mediators of folate uptake in retinal ganglion (RGC) and Muller cells (RMC). We will study RGCs because intravitreal exposure to high levels of Hey in mice leads to marked degeneration of these cells;we will study RMCs because of their key role in maintaining RGC function. There have been no studies of the impact of sustained elevation of endogenous Hey on retinal function. We predict that sustained elevation of Hey in retina will compromise retinal function leading to degenerative retinopathy.
Aim 1 will test the hypothesis that FRs mediate folate uptake in RGC and RMCs;that FR subtypes are expressed differentially in RGC, RMC and RPE cells;and that expression of these subtypes is subject to differential regulation in a cell-type specific manner. Biochemical, functional and molecular methods will be used to characterize folate transport proteins in these cells.
Aim 2 will test the hypothesis that sustained elevation of Hey, due either to genetic diseases and/or to diminished folate uptake in RGC, RMC or RPE cells, will compromise retinal function leading to degenerative retinopathy. We will systematically analyze retinas of heterozygous and homozygous cystathionine-fc-synthase (cbs) knockout mice, in which plasma Hey levels are, respectively, 2-4-fold or 40-fold greater than normal, for alterations in morphology, electrophysiological function, amino acid profile, folate transport, and gene and protein expression. We will determine whether the Hcy-induced retinopathy in cbs mutant mice is due solely to lack of adequate folate or is due to a complexity of cellular insults including oxidative stress, excitotoxic damage, ER stress, and DNA damage.
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