Age-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation. Epigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes. Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10⁻⁹), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10⁻⁷), and Tenascin X (TNXB) (p = 6.30 × 10⁻⁶) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors. We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-β (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

Age-related macular degeneration (AMD) predominantly affects the retina and retinal pigment epithelium in the posterior eye. While there are numerous studies investigating the non-coding transcriptome of retina and RPE, few significant differences between AMD and normal tissues have been reported. Strand specific RNA sequencing of both peripheral retina (PR) and RPE-Choroid-Sclera (PRCS), in both AMD and matched normal controls were generated. The transcriptome analysis reveals a highly significant and consistent impact on anti-sense transcription as well as moderate changes in the regulation of non-coding (sense) RNA. Hundreds of genes that do not express anti-sense transcripts in normal PR and PRCS demonstrate significant anti-sense expression in AMD in all patient samples. Several pathways are highly enriched in the upregulated anti-sense transcripts—in particular the EIF2 signaling pathway. These results call for a deeper exploration into anti-sense and noncoding RNA regulation in AMD and their potential as therapeutic targets.

Cholesterol-containing soft drusen and subretinal drusenoid deposits (SDD) occur at the basolateral and apical side of the retinal pigment epithelium (RPE), respectively, in the chorioretina and are independent risk factors for late age-related macular degeneration (AMD). Cholesterol in these deposits could originate from the RPE as nascent high-density lipoprotein (HDL) or apolipoprotein B (apoB)-lipoprotein. We characterized cholesterol efflux and apoB-lipoprotein secretion in RPE cells. Human RPE cells ARPE-19 formed nascent HDL that was similar in physicochemical properties to nascent HDL formed by other cell types. In highly-polarized primary human fetal RPE (phfRPE) monolayers grown in low-lipid conditions, cholesterol efflux to HDL was moderately directional to the apical side and much stronger than ATP-binding cassette transporter A1 (ABCA1)-mediated efflux to apolipoprotein A-I at both sides; ABCA1-mediated efflux was weak and equivalent between the two sides. Feeding phfRPE monolayers with oxidized or acetylated low-density lipoprotein (LDL) increased intracellular levels of free and esterified cholesterol and substantially raised ABCA1-mediated cholesterol efflux at the apical side. PhfRPE monolayers secreted apoB-lipoprotein preferentially to the apical side in low-lipid and oxidized LDL-feeding conditions. These findings together with evidence from human genetics and AMD pathology suggest that RPE-generated HDL may contribute lipid to SDD.

Glaucoma is an optic neuropathy characterized by permanent visual field loss caused by the death of retinal ganglion cells (RGCs) and it is the leading cause of irreversible blindness worldwide. Consequently, there is an unmet need for the development of new strategies for its treatment. We investigated RGC replacement therapy as a treatment for ganglion cell loss. Human-induced pluripotent stem cells (hiPSCs) were differentiated into mature, functional RGCs in vitro, labeled with AAV2.7m8-SNCG-eGFP, and transplanted intravitreally in wild-type 4-month-old C57BL/6J mice. Survival of the transplanted hiPSC-RGCs was assessed by color fundus photography and histological studies confirmed the localization of the transplanted hiPSC-RGCs within the retina. Two-photon live imaging of retinal explants and electrophysiological studies confirmed that the morphology and function of the transplanted hiPSC-RGCs were similar to native RGCs. These experiments will provide key strategies to enhance the efficiency of stem cell replacement therapy for neurodegenerative diseases, including glaucoma.