Mitochondrial alterations in humanized ARMS2^A69S variant mouse model of age-related macular degeneration (AMD)

Abstract

Purpose : A humanized ARMS2 mouse model was developed to examine the contribution of the ARMS2 rs10490924, pAla69Ser,risk allele to age-related macular degeneration (AMD). ARMS2^A69S and its wild-type ARMS2^A69 counterpart were generated on the C57BL/6J background using CRISPR/Cas9 technology. The ARMS2 gene was inserted into a non-coding site that is homologous between humans and mice, upstream of the mouse Htra1 gene. A native promoter was used to preserve hARMS2 regulatory regions. The aim of this study was to identify the early changes in ARMS2^A69S mice that ultimately may result in AMD-like phenotypes with aging.

Methods : RNA-sequencing was performed to compare the transcriptome of 14-day old ARMS2^A69S mutant mice to that of C57BL/6J (B6J) controls. A69S allele specific changes were verified by performing qRT-PCR on selected genes on ARMS2^A69S, wild-type ARMS2^A69 and B6 mice. Altered mitochondrial function was assessed by measuring NAD/NADH ratios using a calorimetric assay in 6-month-old mice.

Results : Transcriptomic analysis showed significant downregulation of multiple mitochondria-associated genes including Nd2, Nd4, Cytb, Cox1, Atp5a1 and Ndufa7 in retina and RPE enriched tissue fractions from ARMS2^A69S mice as compared to B6J controls. Further, qRT-PCR confirmed that the expression of mitochondria-associated genes was significantly lower in ARMS2^A69S mice as compared to both B6J and wild-type ARMS2^A69 WT mice. To test if the altered mitochondrial gene expression had a functional effect, NAD+ levels and NAD+/NADH ratios were obtained and compared. The results confirmed that the eyecups isolated from ARMS2^A69S mutants had significantly lower total NAD+ levels than B6J controls and lower NAD+/NADH ratios than both B6J and ARMS2^A69 WT mice.

Conclusions : Our initial analysis suggests early mitochondrial alterations may contribute to AMD-like phenotypes observed in aged ARMS2^A69S mice. A complete characterization of structural and functional changes in mitochondria are underway to identify the major mitochondrial pathways affected by the ARMS2^A69S AMD risk allele. Unravelling such networks will provide a basis for furthering our understanding of the role of mitochondrial dysfunction leading to AMD-associated phenotypes.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.