The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that requires an active metabolism to maintain outer retinal homeostasis and compensate for oxidative stress. Using 13 C metabolic flux analysis in human RPE cells, we found that RPE has an exceptionally high capacity for reductive carboxylation, a metabolic pathway that has recently garnered significant interest because of its role in cancer cell survival. The capacity for reductive carboxylation in RPE exceeds that of all other cells tested, including retina, neural tissue, glial cells, and a cancer cell line. Loss of reductive carboxylation disrupts redox balance and increases RPE sensitivity to oxidative damage, suggesting that deficiencies of reductive carboxylation may contribute to RPE cell death. Supporting reductive carboxylation by supplementation with an NAD + precursor or its substrate α-ketoglutarate or treatment with a poly(ADP ribose) polymerase inhibitor protects reductive carboxylation and RPE viability from excessive oxidative stress. The ability of these treatments to rescue RPE could be the basis for an effective strategy to treat blinding diseases caused by RPE dysfunction.reductive carboxylation | RPE | metabolism | age-related macular degeneration | oxidative stress T he retinal pigment epithelium (RPE) is a monolayer of postmitotic cells situated between the photoreceptors of the retina and the choroidal blood supply. The interaction of the RPE and photoreceptors is critical to maintaining vision. Functions of the RPE include phagocytosis of shed photoreceptor outer segments, recycling of retinoids, production and secretion of cytokines and chemokines, and mediating the exchange of nutrients and metabolites between the choroid and photoreceptors (1, 2). RPE cells provide crucial metabolic support for the retina.RPE dysfunction can lead to photoreceptor death and retinal degenerative disease, such as age-related macular degeneration (AMD), which is the leading cause of irreversible vision loss in the elderly human population (3-5). RPE cells are exposed to ongoing oxidative stress from the combined effects of light, choroidal O 2 , polyunsaturated fatty acids, and retinoids (6). The resulting impairment in RPE energy metabolism and function by oxidative stress is one likely mechanism for the pathogenesis of AMD (3, 7-11).Mitochondria support the active energy metabolism of the RPE (10-12). A recent report showed that RPE is less stable and less able to support the retina when it is forced to rely on glycolytic rather than mitochondrial metabolism (13). Another recent report supports the importance of mitochondria in RPE by showing that bolstering mitochondrial activity makes these cells more resilient to oxidative damage (14).In mitochondria, citrate can be generated from acetyl CoA and oxaloacetate as part of the TCA cycle. However, under hypoxic conditions, some cells also produce citrate via reductive carboxylation of α-ketoglutarate (αKG) through the action of NADPH-dependent isocitrate dehydrogenases (IDH) (15-17)....
reducing activity of the mtorC1/S6K1 pathway has been shown to extend lifespan in both vertebrate and invertebrate models. For instance, both pharmacological inhibition of mtorC1 with the drug rapamycin or S6K1 knockout extends lifespan in mice. Since studies with invertebrate models suggest that reducing translational activity can increase lifespan, we reasoned that the benefits of decreased mtorC1 or S6K1 activity might be due, at least in part, to a reduction of general translational activity. Here, we report that mice given a single dose of rapamycin have reduced translational activity, while mice receiving multiple injections of rapamycin over 4 weeks show no difference in translational activity compared with vehicle-injected controls. Furthermore, mice lacking S6K1 have no difference in global translational activity compared with wild-type littermates as measured by the percentage of ribosomes that are active in multiple tissues. translational activity is reduced in S6K1-knockout mice following single injection of rapamycin, demonstrating that rapamycin's effects on translation can occur independently of S6K1. taken together, these data suggest that benefits of chronic rapamycin treatment or lack of S6K1 are dissociable from potential benefits of reduced translational activity, instead pointing to a model whereby changes in translation of specific subsets of mrNas and/or translation-independent effects of reduced mtor signaling underlie the longevity benefits.
Sorsby Fundus Dystrophy (SFD) is a rare form of macular degeneration that is clinically similar to age-related macular degeneration (AMD). SFD results from mutations found in the tissue inhibitor of metalloproteinase 3 (TIMP3) gene. TIMP3 is secreted by the retinal pigmented epithelium (RPE) into the underlying Bruch’s membrane (BrM), and it plays a critical role in maintaining extracellular matrix (ECM) homeostasis. A characteristic feature of post-mortem SFD globes is a thick layer of sub-RPE deposits overlying a disorganized BrM. Although likely central to the pathogenesis of SFD, no animal models have reproduced this phenotype. We generated induced pluripotent stem cell (iPSC)-derived RPE lines from SFD family members with the S204C TIMP3 mutation and observed that SFD RPE have highly dysregulated ECM and form large basal deposits by ~30 days in culture. The sub-RPE deposits are similar in ultrastructure and composition when compared to SFD family member globes. Mutant TIMP3 correction by CRISPR-Cas9 gene editing in SFD iPSC RPE cells resulted in the reversal of sub-RPE calcium deposition. We found that SFD TIMP3 has decreased inhibition of secreted matrix metalloproteinases. ECM dysfunction substantially impacts cellular metabolism. Targeted metabolomics data showed that intracellular 4-hydroxyproline, a major breakdown product of collagen, is significantly elevated in SFD RPE. Further, SFD RPE also has decreased intracellular reduced glutathione and is more vulnerable to oxidative stress. These findings suggest that key elements of SFD pathology can be recapitulated in culture which may lead to insights into disease mechanisms and potential treatments.Significance StatementThis study demonstrates that retinal pigmented epithelial (RPE) cells generated from patients with Sorsby Fundus Dystrophy (SFD) produce highly dysregulated extracellular matrices. SFD RPE form large basal deposits in culture that are similar in composition to what is observed in donated SFD post-mortem globes from family members. Further, SFD RPE demonstrate high levels of 4-hydroxyproline, a major breakdown product of collagen. SFD RPE are also more vulnerable to oxidative stress. Our studies indicate that key elements of SFD pathology can be recapitulated in culture, and ECM dysregulation may lead to metabolic changes detrimental to RPE health.
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