Characterization of the Retinal Phenotype In Methylene Tetrahydrofolate Reductase (Mthfr) Deficient Mice, A Model Of Mild Hyperhomocysteinemia

Abstract

Homocysteine (hcy), a sulfur containing amino acid, is an integral part of methionine metabolism. Elevated plasma level of hcy (Hhcy) is identified as a risk factor for cardiovascular disorders and implicated in various retinal diseases such diabetic retinopathy, glaucoma, age related macular degeneration and central retinal vein occlusion. Cystathionine β-synthase (CBS) and methylene tetrahydrofolate reductase (MTHFR) are key enzymes of hcy metabolism. CBS catalyzes the transsulfuration pathway yielding beneficial downstream products such as taurine, H2S and glutathione (GSH). MTHFR is required for methylation of hcy. Mutations in MTHFR are the most common genetic cause for Hhcy. Murine models of CBS and MTHFR are an invaluable tools to understand Hhcy pathophysiology in humans. Our lab has reported the retinal phenotype of CBS mutant mice. Depending upon the loss of one or both alleles, mild to marked retinal neurovascular and functional alterations are observed. The data from CBS mutant mice raise an important question: is the retinal neurovasculopathy observed in absence/deficiency of CBS attributed to excess hcy levels or is it due to decline in availability of taurine, H 2S and GSH? This can be addressed by studying the retinal phenotype of MTHFR mutant mice which have an intact CBS pathway. No information is available is currently available about the retinal expression of MTHFR and current data regarding CBS in the mouse retina is contentious. This thesis work tested the hypothesis that CBS and MTHFR are expressed in the mouse retina at gene and protein levels and that Hhcy would induce retinal functional and neurovascular alterations in MTHFR-deficient mice. For gene and protein expression studies, RNA and protein were isolated from retinas for analysis of Cbs and Mthfr gene expression by RT-PCR and protein expression by Western blotting. Eyes were harvested from C57BL6 mice and used for immunodetection of CBS and MTHFR in the retina. RT-PCR revealed robust Cbs and Mthfr expression in retina. Western blotting detected CBS and MTHFR protein in mouse retina. In immunohistochemical studies of the intact retina, CBS was present most abundantly in the ganglion cell layer of WT retina while MTHFR showed widespread retinal expression. Our immunofluorescence studies revealed presence of CBS and MTHFR in retinal ganglion, Müller and RPE cells. Taken together, we have compelling molecular evidence that CBS and MTHFR are expressed in mouse retina at gene and protein levels. These data indicate the underlying importance of hcy metabolism in the retina. For characterization of the retinal phenotype in MTHFR deficient mice, we employed tools such as ERG, Fundus and FA, OCT, HPLC, morphometric, immunohistochemistry (IHC) and PCR arrays. ERG revealed a significant decrease in positive scotopic threshold response in retinas of Mthfr+/- mice at 24 wks. FA revealed areas of focal vascular leakage in 20% of Mthfr+/- mice at 12-16 wks and 60% by 24 wks suggesting potential vascular damage mediated by Hhcy. SD-OCT revealed a significant decrease in NFL thickness at 24 wks in Mthfr+/- compared to Mthfr+/+ mice. There was a 2-fold elevation in retinal hcy at 24 wks in Mthfr+/- mice by HPLC and IHC. Morphometric analysis revealed ∼20% reduction in cells in the ganglion cell layer of Mthfr+/- mice at 24 wks. IHC indicated significantly-increased GFAP labeling suggestive of Müller cell activation. The similar loss of ganglion cells, focal vascular leakage, 2-fold increase in retinal hcy, gliosis and functional abnormities were reported in Cbs+/- mice. Taken together, these data support our hypothesis that Hhcy induces retinal neurovascular and functional alterations in MTHFR deficient mice. In addition, we explored retinal mitochondrial gene alteration as a possible mechanism of Hhcy mediated retinal alterations. PCR array data analysis revealed upregulation of pro-apoptotic genes and downregulation of genes associated with normal mitochondrial transport function. Future studies will validate these results at protein and functional levels. To conclude, our data support the hypothesis that Hhcy may be causative in certain retinal neurovasculopathies. These data contribute to our understanding of the potential effects of Hhcy on the retina and may prove useful in other disease model systems of Hhcy.