Introducing a novel Mthfr^677C>T mouse to model a common risk variant for glaucoma

Abstract

Purpose : Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate/methionine/homocysteine pathway. Variants in MTHFR, notably 677C>T, have been associated with glaucoma as well as Alzheimer’s disease and vascular dementia, suggesting an overlapping mechanism in brain and eye. However, mechanisms driving increased risk are not known, hindering the development of new treatments. Approximately 30% of individuals carry at least one copy of MTHFR^677C>T, causing a 50% decrease in MTHFR enzyme efficiency. Reduced efficiency can lead to high levels of homocysteine in blood, resulting in vascular inflammation and increased risk for vascular damage. We hypothesize that vascular-specific expression of MTHFR^677C>T drives damaging effects in the retinal vasculature, priming the environment for additional risk.

Methods : CRISPR technology was employed to engineer the Mthfr^677C>T allele into C57BL/6J (B6) mice. Characterization of the Mthfr^677C>T retina includes: RNAscope to determine MTHFR expression, assays to examine MTHFR enzyme activity and relevant metabolite levels, retinal wholemount immunostaining to evaluate vascular abnormalities and inflammatory markers, retinal cross-section immunostaining to examine neuronal layer thickness and glial reactivity, and in vivo OCT and Micron IV imaging as well as pERG to assess retinal morphology and ganglion cell function, respectively.

Results : Previous data show Mthfr^677C>T mice phenocopy the human condition — mice show a 50% reduction in MTHFR enzyme activity in liver and brain, elevated levels of plasma homocysteine, altered brain metabolites associated with the methionine cycle, as well as morphological and functional changes in cerebrovasculature. We anticipate similar phenotypes will arise in retinas of our young (6 month) and aged (18 month) cohorts of Mthfr^677C>T mice, and are focusing on examining blood retinal barrier integrity using leakage assays and assessing endothelium, vascular smooth muscle, pericytes, basement membrane and tight junctions by immunostaining. Current data support our hypothesis with Mthfr^677C>T mice showing evidence of retinal arterial tortuosity.

Conclusions : We have created a novel mouse strain to determine the precise mechanisms by which MTHFR^677C>T increases risk for vascular dysfunction with age and in neurodegenerative retinal disease.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.