Inducible Cell-Specific Mouse Model for Paired Epigenetic and Transcriptomic Studies of Müller Glia in Age-Related Macular Degeneration

Abstract

Purpose : Aging is the major risk factor for age-related macular degeneration (AMD) but how ‘normal’ aging contributes to AMD pathogenesis is unclear. Epigenetic mechanisms, mainly methylcytosine (mC) and hydroxymethylcytosine (hmC), affect DNA accessibility, regulate genomic organization and gene expression, and are altered with aging. DNA methylation analyses of AMD patient blood and retinas suggest differential methylation resulting in gene expression changes during development/progression of AMD. In addition, Müller glia are activated in AMD patient retinas. The aim of this study is the validation of a cre/ERT2-NuTRAP mouse model to allow the parallel interrogation of differential mC/hmC genome-wide and the transcriptome specifically in Müller glia with retinal aging.

Methods : Female NuTRAP ^flox/flox and male Aldh1l1-cre/ERT2^+/wt mice were bred to generate Aldh1l1-cre/ERT2^+/wt; NuTRAP^flox/wt (Aldh1l1-cre/ERT2⁺; NuTRAP⁺) mice. At 5 months old, mice received single intraperitoneal injections of tamoxifen (Tam) for 5 consecutive days (100 mg/kg) or were left untreated as controls. After Tam induction mice were euthanized and their retinas harvested and processed for: immunohistochemistry of mCherry, EGFP, GS, and GFAP expressions on retina sagittal sections via confocal microscopy; ribosome bound RNA isolation via TRAP protocol, qPCR validation and stranded RNA-Seq profile; and nuclei suspension preparation and DNA isolation via INTACT method for bisulfite amplicon sequencing (BSAS).

Results : Specific co-localization of EGFP, mCherry, and the Müller glia marker GS was observed in the Aldh1l1-cre/ERT2⁺; NuTRAP⁺ retinas. RNA-seq bioinformatic analysis revealed enrichment of Müller marker genes and pathways and depletion of other cell type markers in the positive TRAP fraction of Aldh1l1-cre/ERT2⁺; NuTRAP⁺ retinas, compared to input profiles. qPCR validated enrichment and depletion of selected transcript expressions. BSAS showed site-specific decrease of mC in the promoter region of selected Müller glia-specific genes in INTACT-DNA positive fraction relative to input.

Conclusions : The selective recombination in Müller glia, cell-specific transcriptome enrichments found via TRAP-RNAseq and qPCR, and BSAS findings, suggest the Aldh1l1-cre/ERT2; NUTRAP model is suitable for the study of Müller glia and its contributions to the transcriptome/epigenome of the aging retina.

This is a 2021 ARVO Annual Meeting abstract.