Loss of MPC1 reprograms retinal metabolism to impair visual function.

Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the "Warburg Effect"), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron-glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and 13C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization.

Impact of euthanasia, dissection and postmortem delay on metabolic profile in mouse retina and RPE/choroid.

Metabolomics studies in the retina and retinal pigment epithelium (RPE) in animal models or postmortem donors are essential to understanding the retinal metabolism and to revealing the underlying mechanisms of retinal degenerative diseases. We have studied how different methods of euthanasia (CO2 or cervical dislocation) different isolation procedures and postmortem delay affect metabolites in mouse retina and RPE/choroid using LC MS/MS and GC MS. Compared with cervical dislocation, CO2 exposure for 5 min dramatically degrades ATP and GTP into purine metabolites in the retina while raising intermediates in glucose metabolism and amino acids in the RPE/choroid. Isolation in cold buffer containing glucose has the least change in metabolites. Postmortem delay time-dependently and differentially impacts metabolites in the retina and RPE/choroid. In the postmortem retina, 18% of metabolites were changed at 0.5 h (h), 41% at 4 h and 51% at 8 h. However, only 6% of metabolites were changed in the postmortem RPE/choroid and it steadily increased to 20% at 8 h. Notably, both postmortem retina and RPE/choroid tissue showed increased purine metabolites. Storage of eyes in cold nutrient-rich medium substantially blocked the postmortem change in the retina and RPE/choroid. In conclusion, our study provides optimized methods to prepare fresh or postmortem retina and RPE/choroid tissue for metabolomics studies.

Quantitative proteomic profiling reveals sexual dimorphism in the retina and RPE of C57BL6 mice.

BACKGROUND: Sex as a biological variable is not a common consideration in molecular mechanistic or preclinical studies of retinal diseases. Understanding the sexual dimorphism of adult RPE and retina under physiological conditions is an important first step in improving our understanding of sex-based physio-pathological mechanisms. METHODS: Isobaric tags for relative and absolute quantitation (iTRAQ) were used for quantitative proteomics of male and female mouse retina and RPE (10 mice of each sex for each tissue type). Differentially expressed proteins were subjected to Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA). RESULTS: Differential expression analysis identified 21 differentially expressed proteins in the retina and 58 differentially expressed proteins in the RPE. Ingenuity pathway analysis identified the top canonical pathways differentially activated in the retina to be calcium transport I, nucleotide excision repair, molecular transport and cell death and survival. In the RPE, the top canonical pathways were calcium signaling, dilated cardiomyopathy signaling, actin cytoskeletal signaling and cellular assembly and organization. CONCLUSIONS: These results provide insights into sex differences in the retina and RPE proteome of mice and begin to shed clues into the sexual dimorphism seen in retinal diseases.

Tissue Inhibitor of Metalloproteinase 3 (TIMP3) mutations increase glycolytic activity and dysregulate glutamine metabolism in RPE cells.

OBJECTIVES: Mutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsby's Fundus Dystrophy (SFD), a dominantly inherited, rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells, which constitute the outer blood-retinal barrier. One major function of RPE is the synthesis and transport of vital nutrients, such as glucose, to the retina. Recently, metabolic dysfunction in RPE cells has emerged as an important contributing factor in retinal degenerations. We set out to determine if RPE metabolic dysfunction was contributing to SFD pathogenesis. METHODS: Quantitative proteomics was conducted on RPE of mice expressing the S179C variant of TIMP3, known to be causative of SFD in humans. Proteins found to be differentially expressed (P < 0.05) were analyzed using statistical overrepresentation analysis to determine enriched pathways, processes, and protein classes using g:profiler and PANTHER Gene Ontology. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. RPE metabolism was directly probed using isotopic tracing coupled with GC/MS analysis. Steady state [U-13C6] glucose isotopic tracing was preliminarily conducted on S179C ARPE-19 followed by [U-13C6] glucose and [U-13C5] glutamine isotopic tracing in SFD iRPE cells. RESULTS: Quantitative proteomics and enrichment analysis conducted on RPE of mice expressing mutant S179C TIMP3 identified differentially expressed proteins that were enriched for metabolism-related pathways and processes. Notably these results highlighted dysregulated glycolysis and glucose metabolism. Stable isotope tracing experiments with [U-13C6] glucose demonstrated enhanced glucose utilization and glycolytic activity in S179C TIMP3 APRE-19 cells. Similarly, [U-13C6] glucose tracing in SFD iRPE revealed increased glucose contribution to glycolysis and the TCA cycle. Additionally, [U-13C5] glutamine tracing found evidence of altered malic enzyme activity. CONCLUSIONS: This study provides important information on the dysregulation of RPE glucose metabolism in SFD and implicates a potential commonality with other retinal degenerative diseases, emphasizing RPE cellular metabolism as a therapeutic target.

Hyperoxia Inhibits Proliferation of Retinal Endothelial Cells in a Myc-Dependent Manner.

Oxygen supplementation is necessary to prevent mortality in severely premature infants. However, the supraphysiological concentration of oxygen utilized in these infants simultaneously creates retinovascular growth attenuation and vasoobliteration that induces the retinopathy of prematurity. Here, we report that hyperoxia regulates the cell cycle and retinal endothelial cell proliferation in a previously unknown Myc-dependent manner, which contributes to oxygen-induced retinopathy.

Metabolic signature of the aging eye in mice.

Aging is a major risk factor for age-related ocular diseases including age-related macular degeneration in the retina and retinal pigment epithelium (RPE), cataracts in the lens, glaucoma in the optic nerve, and dry eye syndrome in the cornea. We used targeted metabolomics to analyze metabolites from young (6 weeks) and old (73 weeks) eyes in C57 BL6/J mice. Old mice had diminished electroretinogram responses and decreased number of photoreceptors in their retinas. Among the 297 detected metabolites, 45-114 metabolites are significantly altered in aged eye tissues, mostly in the neuronal tissues (retina and optic nerve) and less in cornea, RPE/choroid, and lens. We noted that changes of metabolites in mitochondrial metabolism and glucose metabolism are common features in the aged retina, RPE/choroid, and optic nerve. The aging retina, cornea, and optic nerve also share similar changes in Nicotinamide adenine dinucleotide (NAD), 1-methylnicotinamides, 3-methylhistidine, and other methylated metabolites. Metabolites in taurine metabolism are strikingly influenced by aging in the cornea and lens. In conclusion, the aging eye has both common and tissue-specific metabolic signatures. These changes may be attributed to dysregulated mitochondrial metabolism, reprogrammed glucose metabolism and impaired methylation in the aging eye. Our findings provide biochemical insights into the mechanisms of age-related ocular changes.