Age-related macular degeneration (AMD) is the leading cause of blindness among older adults. It has been suggested that mitochondrial defects in the retinal pigment epithelium (RPE) underlies AMD pathology. To test this idea, we developed primary cultures of RPE to ask whether RPE from donors with AMD differ in their metabolic profile compared with healthy age-matched donors. Analysis of gene expression, protein content, and RPE function showed that these cultured cells replicated many of the cardinal features of RPE in vivo. Using the Seahorse Extracellular Flux Analyzer to measure bioenergetics, we observed RPE from donors with AMD exhibited reduced mitochondrial and glycolytic function compared with healthy donors. RPE from AMD donors were also more resistant to oxidative inactivation of these two energy-producing pathways and were less susceptible to oxidation-induced cell death compared with cells from healthy donors. Investigation of the potential mechanism responsible for differences in bioenergetics and resistance to oxidative stress showed RPE from AMD donors had increased PGC1α protein as well as differential expression of multiple genes in response to an oxidative challenge. Based on our data, we propose that cultured RPE from donors phenotyped for the presence or absence of AMD provides an excellent model system for studying "AMD in a dish". Our results are consistent with the ideas that (i) a bioenergetics crisis in the RPE contributes to AMD pathology, and (ii) the diseased environment in vivo causes changes in the cellular profile that are retained in vitro.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is characterized by loss of the retinal pigment epithelium (RPE). While the disease mechanism remains unclear, prior studies have linked AMD with RPE mitochondrial defects and genetic polymorphisms in the complement pathway. This study used RPE generated from induced pluripotent stem cells (iPSC-RPE), which were derived from human donors with or without AMD and genotyped for the complement factor H (CFH) AMD high-risk allele (rs1061170, Y402H) to investigate whether donor disease state or genotype had a detrimental effect on mitochondrial function and inflammation. Results show that cells derived from donors with AMD display decreased mitochondrial function under conditions of stress and elevated expression of inflammatory markers compared to iPSC-RPE from individuals without AMD. A more pronounced reduction in mitochondrial function and increased inflammatory markers was observed in CFH high-risk cells, irrespective of disease state. These results provide evidence for a previously unrecognized link between CFH and mitochondrial function that could contribute to RPE loss in AMD patients harboring the CFH high-risk genotype.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. No universally effective treatments exist for atrophic or “dry” AMD, which results from loss of the retinal pigment epithelium (RPE) and photoreceptors and accounts for ≈80% of all AMD patients. Prior studies provide evidence for the involvement of mitochondrial dysfunction in AMD pathology. This study used induced pluripotent stem cell (iPSC) RPE derived from five AMD patients to test the efficacy of three drugs (AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), Metformin, trehalose) that target key processes in maintaining optimal mitochondrial function. The patient iPSC-RPE lines were used in a proof-of-concept drug screen, utilizing an analysis of RPE mitochondrial function following acute and extended drug exposure. Results show considerable variability in drug response across patient cell lines, supporting the need for a personalized medicine approach for treating AMD. Furthermore, our results demonstrate the feasibility of using iPSC-RPE from AMD patients to develop a personalized drug treatment regime and provide a roadmap for the future clinical management of AMD.

The systemic transport of dietary vitamin A/all-trans retinol bound to RBP4 into peripheral tissues for storage is an essential physiological process that continuously provides visual chromophore precursors to the retina under fasting conditions. This mechanism is critical for phototransduction, photoreceptor cell maintenance and survival, and in the support of visual function. While the membrane receptor STRA6 facilitates the blood transport of lipophilic vitamin A into the eye, it is not expressed in most peripheral organs, which are proposed to express a second membrane receptor for the uptake of vitamin A from circulating RBP4. The discovery of a novel vitamin A receptor, RBPR2, which is expressed in the liver and intestine, but not in the eye, alluded to this long-sort non-ocular membrane receptor for systemic RBP4-ROL uptake and transport. We have previously shown in zebrafish that the retinol-binding protein receptor 2 (Rbpr2) plays an important role in the transport of yolk vitamin A to the eye. Mutant rbpr2 zebrafish lines manifested in decreased ocular retinoid concentrations and retinal phenotypes. To investigate a physiological role for the second vitamin A receptor, RBPR2, in mammals and to analyze the metabolic basis of systemic vitamin A transport for retinoid homeostasis, we established a whole-body Rbpr2 knockout mouse (Rbpr2^−/−) model. These mice were viable on both vitamin A-sufficient and -deficient diets. Rbpr2^−/− mice that were fed a vitamin A-sufficient diet displayed lower ocular retinoid levels, decreased opsins, and manifested in decrease visual function, as measured by electroretinography. Interestingly, when Rbpr2^−/− mice were fed a vitamin A-deficient diet, they additionally showed shorter photoreceptor outer segment phenotypes, altogether manifesting in a significant loss of visual function. Thus, under conditions replicating vitamin A sufficiency and deficiency, our analyses revealed that RBPR2-mediated systemic vitamin A transport is a regulated process that is important for vitamin A delivery to the eye when RBP4-bound ROL is the only transport pathway in the fasting condition or under vitamin A deficiency conditions.

Microglia are essential to the development of the CNS and its homeostasis. Our prior findings suggested a niche model to describe the behaviors of retinal microglia. Here, we ask whether new myeloid cells recruited to the retina are constrained to resemble endogenous microglia morphologically and functionally. Use of CD11cDTR/GFP transgenic mouse allowed identification of two niches of retinal microglia distinguished by being GFPlo or GFPhi. We also used transgenic mice in which CX3CR1+ cells expressed YFP and were depletable following tamoxifen-induced expression of diphtheria toxin subunit A. We employed several ablation and injury stimulation protocols to examine the origin and fate of myeloid cells repopulating the retina. Analysis of retinal myeloid cells was done by microscopy, flow cytometry, and qRT-PCR. We found that the origin of new GFPhi and GFPlo myeloid cells in the retina of CD11cDTR/GFP mice, whether recruited or local, depended on the ablation and stimulation protocols. Regardless of origin, new GFPlo and GFPhi retinal myeloid cells were CD45medCD11b+Ly6G−Ly6CloIba1+F4/80+, similar to endogenous microglia. Following tamoxifen-induced diphtheria toxin ablation, myeloid cell repopulation differed in the retina compared to the brain and optic nerve. Stimulation of replacement GFPhi cells was substantially attenuated in repopulating retinas after tamoxifen-induced diphtheria toxin ablation compared to control or radiation-ablated mice. In radiation bone marrow chimeric mice, replacement GFPhi myeloid cells from the circulation were slow to repopulate the retina unless stimulated by an optic nerve crush injury. However, once stimulated, recruited GFPhi cells were found to concentrate on injured retinal ganglion cells and were morphologically similar to GFPhi cells in non-ablated control CD11cDTR/GFP mice. The results support the idea that GFPhi cells in the CD11cDTR/GFP mouse, whether recruited or from resident microglia, mark a unique niche of activated retinal myeloid cells. We conclude that the retinal environment has a potent influence on the function, morphology, and proliferative capacity of new myeloid cells regardless of their origin, compelling them to be equivalent to the endogenous microglia.

Primary cultures of retinal pigment epithelium (RPE) from human adult donors (haRPE) and induced pluripotent stem cell derived-RPE (iPSC-RPE) are valuable model systems for gaining mechanistic insight and for testing potential therapies for age-related macular degeneration (AMD). This study evaluated the treatment response of haRPE and iPSC-RPE to oxidative stress and potential therapeutics addressing mitochondrial defects. haRPE and iSPC-RPE were derived from donors with or without AMD. Mitochondrial function was measured after treatment with menadione, AICAR, or trehalose and the response to treatment was compared between cell models and by disease status. In a subset of samples, haRPE and iPSC-RPE were generated from the same human donor to make a side-by-side comparison of the two cell models’ response to treatment. Disease-specific responses to all three treatments was observed in the haRPE. In contrast, iPSC-RPE had a similar response to all treatments irrespective of disease status. Analysis of haRPE and iPSC-RPE generated from the same human donor showed a similar response for donors without AMD, but there were significant differences in treatment response between cell models generated from AMD donors. These results support the use of iPSC-RPE and haRPE when investigating AMD mechanisms and new therapeutics but indicates that attention to experimental conditions is required.