Organelles such as mitochondria, lysosomes, peroxisomes, and the endoplasmic reticulum form highly dynamic cellular networks and exchange information through sites of physical contact. While each organelle performs unique functions, this inter-organelle crosstalk helps maintain cell homeostasis. Age-related macular degeneration (AMD) is a devastating blinding disease strongly associated with mitochondrial dysfunction, oxidative stress, and decreased clearance of cellular debris in the retinal pigment epithelium (RPE). However, how these occur, and how they relate to organelle function both with the RPE and potentially the photoreceptors are fundamental, unresolved questions in AMD biology. Here, we report the discussions of the “Mitochondria, Lysosomes, and other Organelle Interactions” task group of the 2024 Ryan Initiative for Macular Research (RIMR). Our group focused on understanding the interplay between cellular organelles in maintaining homeostasis in the RPE and photoreceptors, how this could be derailed to promote AMD, and identifying where these pathways could potentially be targeted therapeutically.

THT is an additive for culture medium used to maintain retinal pigment epithelial (RPE) cells. The individual components of THT are taurine, hydrocortisone, and triiodothyronine. The effects of THT on RPE health had not been defined. To determine how THT affects RPE cell physiology, we grew induced pluripotent stem cell (iPSC)-derived RPE cells with a normal concentration of THT ("high" THT) or in a medium with a lower concentration of THT (low THT). We determined the impact of THT on the extracellular flux of glucose, lactate, palmitate, and VEGF. We assessed RPE function by measuring trans-epithelial electrical resistance (TEER). RPE cells grown in low THT are more glycolytic, oxidize fatty acids more slowly, have a higher TEER, and release more VEGF basally than RPE cells grown in high THT. Our findings show that THT impacts metabolism and function. Further studies will be needed to fully define the impact of each THT component on RPE metabolism, function, and health.

Neuroretinal degenerations including retinitis pigmentosa (RP) comprise a heterogeneous collection of pathogenic mutations that ultimately result in blindness. Despite recent advances in precision medicine, therapies for rarer mutations are hindered by burdensome developmental costs. To this end, Von Hippel-Lindau (VHL) is an attractive therapeutic target to treat RP. By ablating VHL in rod photoreceptors and elevating hypoxia-inducible factor (HIF) levels, we demonstrate a path to therapeutically enhancing glycolysis independent of the underlying genetic variant that slows degeneration of both rod and cone photoreceptors in a preclinical model of retinitis pigmentosa. This rod-specific intervention also resulted in reciprocal, decreased glycolytic activity within the retinal pigment epithelium (RPE) cells despite no direct genetic modifications to the RPE. Suppressing glycolysis in the RPE provided notable, non-cell-autonomous therapeutic benefits to the photoreceptors, indicative of metabolically sensitive crosstalk between different cellular compartments of the retina. Surprisingly, targeting HIF2A in RPE cells did not impact RPE glycolysis, potentially implicating HIF1A as a major regulator in mouse RPE and providing a rationale for future therapeutic efforts aimed at modulating RPE metabolism.

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate limiting enzyme for ketogenesis, 3-hydroxy-3 methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta hydroxybutyrate (β-HB). Prior work, based on detecting β-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export β-HB across the apical membrane. Here, we compare the accuracy of measuring β-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of β-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete β-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming β-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce β-HB. Altogether, we substantiate β-HB secretion in RPE but find that the secretion is equal apically and basally, RPE β-HB can derive from ketogenic amino acids or fatty acids, and accurate β-HB assessment requires mass spectrometric analysis.

The retina and its neighboring tissue, the retinal pigmented epithelium (RPE), require fuels for energy and anabolic activity. Each has unique metabolic features that they use synergistically to function together as a metabolic ecosystem. This article describes how nutrients reach the retina from the circulation. It then reviews the biochemistry of energy metabolism and describes specific metabolic interactions between the retina and RPE. Finally, the article reviews the relationships between energy metabolism and retinal diseases and it suggests how an understanding of those relationships may be used to develop treatments that slow the progression of several types of retinal degeneration.

The posterior eye contains two metabolic microenvironments, each occupied by a distinct tissue type. Retinal pigment epithelium (RPE)-choroid tissue is connected to the circulatory system and receives plentiful O 2 and nutrients. Tight junctions formed by the RPE regulate the diffusion of nutrients to the neural retina. Retina tissue consequently exists in an environment with low O 2 levels and a lower variety of fuels that can support its metabolic needs. Retina explants release lactate and succinate, presumably as an adaptation to a low O 2 environment where they cannot be fully oxidized. The RPE-choroid is thought to aid retina metabolism by oxidizing these ‘waste’ products. We tested these ideas in vivo by infusing 13 C-labeled fuels through jugular vein catheters into circulation. By probing the patterns of 13 C labeling in glycolysis and the TCA cycle from different fuels and genetic models, we found that the retina and RPE do form a cooperative metabolic relationship, where the RPE-choroid oxidizes retina derived succinate but not lactate. Rather than consuming lactate, mouse RPE-choroid may act as a conduit for retina-derived lactate to re-enter circulation and feed more distant tissues.

Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end-products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.

Lipid-rich deposits called drusen accumulate under the retinal pigment epithelium (RPE) in the eyes of patients with age-related macular degeneration and Sorsby's fundus dystrophy (SFD). Drusen may contribute to photoreceptor degeneration in these blinding diseases. Stimulating β-oxidation of fatty acids could decrease the availability of lipid with which RPE cells generate drusen. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate β-oxidation and diminish lipid accumulation in fatty liver disease. In this report, we test the hypothesis that an ACC inhibitor, Firsocostat, can diminish lipid deposition by RPE cells. We probed metabolism and cellular function in mouse RPE-choroid tissue and human RPE cells. We used 13C6-glucose, 13C16-palmitate, and gas chromatography-linked mass spectrometry to monitor effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. We quantified lipid abundance, apolipoprotein E levels, and vascular endothelial growth factor release using liquid chromatography-mass spectrometry, ELISAs, and immunostaining. RPE barrier function was assessed by trans-epithelial electrical resistance (TEER). Firsocostat-mediated ACC inhibition increases β-oxidation, decreases intracellular lipid levels, diminishes lipoprotein release, and increases TEER. When human serum or outer segments are used to stimulate lipoprotein release, fewer lipoproteins are released in the presence of Firsocostat. In a culture model of SFD, Firsocostat stimulates fatty acid oxidation, increases TEER, and decreases apolipoprotein E release. We conclude that Firsocostat remodels RPE metabolism and can limit lipid deposition. This suggests that ACC inhibition could be an effective strategy for diminishing pathologic drusen in the eyes of patients with age-related macular degeneration or SFD.

It is known that metabolic defects in the retinal pigment epithelium (RPE) can cause degeneration of its neighboring photoreceptors in the retina, leading to retinal degenerative diseases such as age-related macular degeneration. However, how RPE metabolism supports the health of the neural retina remains unclear. The retina requires exogenous nitrogen sources for protein synthesis, neurotransmission, and energy metabolism. Using 15N tracing coupled with mass spectrometry, we found human RPE can utilize the nitrogen in proline to produce and export 13 amino acids, including glutamate, aspartate, glutamine, alanine and serine. Similarly, we found this proline nitrogen utilization in the mouse RPE/choroid but not in the neural retina of explant cultures. Co-culture of human RPE with the retina showed that the retina can take up the amino acids, especially glutamate, aspartate and glutamine, generated from proline nitrogen in the RPE. Intravenous delivery of 15N proline in vivo demonstrated 15N-derived amino acids appear earlier in the RPE before the retina. We also found proline dehydrogenase (PRODH), the key enzyme in proline catabolism is highly enriched in the RPE but not the retina. The deletion of PRODH blocks proline nitrogen utilization in RPE and the import of proline nitrogen-derived amino acids in the retina. Our findings highlight the importance of RPE metabolism in supporting nitrogen sources for the retina, providing insight into understanding the mechanisms of the retinal metabolic ecosystem and RPE-initiated retinal degenerative diseases.

Purpose: Retinal pigment epithelium (RPE) oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. Methods: We altered the availability of O2 to human primary and induced pluripotent stem cell–derived RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCRs), glucose consumption, lactate production, 13C6-glucose and 13C5-glutamine flux, hypoxia inducible factor 1α (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, transepithelial electrical resistance, cell morphology, and pigmentation. Results: Medium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis and reductive carboxylation, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. Conclusions: Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood." As RPE-centric diseases like age-related macular degeneration involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2 availability seen with smaller media volumes.

Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low-frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function. G→A/C→T substitutions, indicative of replication errors and/or cytidine deamination, were the predominant mutation type across all tissues and increased with age, whereas G→T/C→A substitutions, indicative of oxidative damage, were the second most common mutation type, but did not increase with age regardless of tissue. We also show that clonal expansions of mtDNA mutations with age is tissue- and mutation type-dependent. Unexpectedly, mutations associated with oxidative damage rarely formed clones in any tissue and were significantly reduced in the hearts and kidneys of aged mice treated at late age with elamipretide or nicotinamide mononucleotide. Thus, the lack of accumulation of oxidative damage-linked mutations with age suggests a life-long dynamic clearance of either the oxidative lesions or mtDNA genomes harboring oxidative damage.

This protocol is designed to extract glycogen from samples and hydrolyze glycogen to glucose monomers that can be detected using an enzymatic system or using liquid chromatography-linked mass spectrometry. It has not been directly compared to other systems for measuring glycogen

Metabolism is adapted to meet energetic needs. Based on the amount of ATP required to maintain plasma membrane potential, photoreceptor energy demands must be high. The available evidence suggests that photoreceptors primarily generate metabolic energy through aerobic glycolysis, though this evidence is based primarily on protein expression and not measurement of metabolic flux. Aerobic glycolysis can be validated by measuring flux of glucose to lactate. Aerobic glycolysis is also inefficient and thus an unexpected adaptation for photoreceptors to make. We measured metabolic rates to determine the energy-generating pathways that support photoreceptor metabolism. We found that photoreceptors indeed perform aerobic glycolysis and this is associated with mitochondrial uncoupling.

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.