Vision Research

Our vision research is focused on how changes in specific cell types of the retina with aging contribute to diseases such as age related macular degeneration, glaucoma, and diabetic retinopathy.

Müller Glia

Analysis of retina cell type-specific epigenetic and transcriptomic signatures is crucial to understanding the pathophysiology of retinal degenerations such as age-related macular degeneration (AMD) and delineating cell autonomous and cell-non-autonomous mechanisms. We have validated through a variety of approaches, the Aldh1l1cre/ERT2-NuTRAP model for generation of Müller glia specific translatomic and epigenomic profiles without the need to isolate whole cells. Application of this approach to models of acute injury (optic nerve crush) and chronic stress (aging) uncovered few common Müller glia-specific transcriptome changes in inflammatory pathways, and mostly differential signatures for each stimulus. Unique changes in neuroinflammation and fibrosis signaling pathways are observed in response to aging but not with optic nerve crush. The Aldh1l1cre/ERT2-NuTRAP model allows focused molecular analyses of a single, minority cell type within the retina, providing more substantial effect sizes than whole tissue analyses. The NuTRAP model, nucleic acid isolation, and validation approaches presented here can be applied to any retina cell type for which a cell type-specific cre is available.

NuTRAP model system. Breeding of NuTRAP^flox/flox females with Aldh1l1^cre/ERT2 males (I) produces viable and phenotypically normal offspring (II) that when induced with tamoxifen (Tam) (III) express EGFP on the ribosomal protein L10a and Biotin and mCherry on the nuclear envelope protein RanGap1 of Müller glia. Translating Ribosome Affinity Purification (TRAP) isolates ribosome bound RNA in cell-type specific fashion while INTACT (Isolation of Nuclei Tagged in specific Cell Types) isolates nuclei, and thus gDNA, from only Aldh1l1⁺ cells. This enables translatome and/or epigenome analyses of Müller glia in a wide variety of experimental conditions using one mouse model and without the need for cell sorting.

From: Translatomic response of retinal Müller glia to acute and chronic stress. Chucair-Elliott AJ, Ocañas SR, Pham K, Van Der Veldt M, Cheyney A, Stanford D, Gurley J, Elliott MH, Freeman WM. Neurobiol Dis. 2022; 175:105931.

Retinal Pigmented Epithelium and Aging

Aging is the main risk factor for age-related macular degeneration (AMD), a retinal neurodegenerative disease that leads to irreversible blindness, particularly in people over 60 years old. Retinal pigmented epithelium (RPE) atrophy is an AMD hallmark. Genome-wide chromatin accessibility, DNA methylation, and gene expression studies of AMD and control RPE demonstrate epigenomic/transcriptomic changes occur during AMD onset and progression. However, mechanisms by which molecular alterations of normal aging impair RPE function and contribute to AMD pathogenesis are unclear. We are interrogating the RPE translatome with advanced age and across sexes in a novel RPE reporter mouse model. We find differential age- and sex- associated transcript expression with overrepresentation of pathways related to inflammation in the RPE. Concordant with impaired RPE function, the phenotypic changes in the aged translatome suggest that aged RPE becomes immunologically active, in both males and females, with some sex-specific signatures, which supports the need for sex representation for in vivo studies.

Retinal function changes with age. At chronological ages recognized as “old” (24–25 mo), “adult” (11–12 mo), and “young” (6–7 mo) (simplified in the figure as 24mo, 12 mo, and 24 mo, respectively), Aldh1l1-NuTRAP mice were overnight dark-adapted and subjected to ERG recordings of scotopic a- and b-wave, as well as c-wave responses to flash light intensities. (b) a: Representative dark-adapted flash waveforms elicited by increasing luminance flashes (0.01, 0.1, and 1.0 cd.s/m²) for each age group. Line graphs depict the averaged (b) photoreceptor-driven response (a-wave amplitude) and (c) bipolar/Müller cell-driven response (b-wave amplitude) at the different intensity flashes in young, adult, and old groups. d: Averaged implicit time for a-waves and (e) averaged implicit time for b-waves are represented for each intensity flash in young, adult, and old mice. f: Representative waveforms of RPE-driven c-wave responses to a single light flash (0.9 cd.s/m²) at young, adult, and old ages. g: Bar graphs depict the averaged c-wave amplitudes at young, adult, and old ages. h: Averaged implicit time c-wave is represented for all age groups. *, **, *** p<0.05, p<0.01 and p<0.001, by 2-way ANOVA (b and c) or ordinary one-way ANOVA (d-e-g-h) followed by Tukey multi-comparison test (n=6–10/group, Mean ± SEM).

From: Age- and sex- divergent translatomic responses of the mouse retinal pigmented epithelium. Chucair-Elliott AJ, Ocañas SR, Pham K, Machalinski A, Plafker S, Stout MB, Elliott MH, Freeman WM. Neurobiol Aging. 2024; 140:41-59.