Abstract
Purpose :
Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate/homocysteine pathways. Variants in MTHFR, notably 677C>T, have been associated with glaucoma and central retinal vein occlusion 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 MTHFR677C>T, causing a 50% decrease in MTHFR enzyme efficiency. Reduced efficiency can cause high levels of homocysteine in blood, resulting in vascular inflammation and increased risk for damage. We hypothesize that vascular-specific expression of MTHFR677C>T drives damaging effects in the retinal vasculature, priming the environment for additional risk.
Methods :
CRISPR technology was employed to engineer the Mthfr677C>T allele into C57BL/6J (B6) mice. Characterization of the Mthfr677C>T retina includes: RNAscope to determine MTHFR expression, examining how the variant affects the retinal proteome and metabolome, 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 :
Like the human condition, the Mthfr677C>T variant results in a reduction in MTHFR enzyme activity (~50%) in liver and brain, and elevated levels of plasma homocysteine in mice. By six months of age, Mthfr677C>T mice show altered protein and metabolite profiles in brain and retina and display evidence of retinal arterial tortuosity, decreased arterial and increased venous diameter as well as altered microglial states compared to controls. We anticipate additional phenotypes in retinas of aged Mthfr677C>T mice and will measure blood retinal barrier integrity using leakage assays and will further assess changes in endothelial cells, vascular smooth muscle cells, pericytes, and tight junctions by immunostaining.
Conclusions :
We reveal baseline differences within retinal vasculature in the novel Mthfr677C>T mouse. These data provide a foundation for future studies to determine the precise mechanisms by which MTHFR677C>T increases risk for vascular dysfunction with age and in neurodegenerative retinal disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.