maintaining-assessing-various-tissue-cell-types-eye-using-novel

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell ...

University of Washington

When O is plentiful, the mitochondrial electron transport chain uses it as a terminal electron acceptor. However, the mammalian retina thrives in a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by reversing the succinate dehydrogenase reaction to use fumarate to accept electrons instead of O. Reverse succinate dehydrogenase activity produces succinate and is enhanced by hypoxia-induced downregulation of cytochrome oxidase. Retinas can export the succinate they produce to the neighboring O-rich retinal pigment epithelium-choroid complex. There, succinate enhances O consumption by severalfold. Malate made from succinate in the pigment epithelium can then be imported into the retina, where it is converted to fumarate to again accept electrons in the reverse succinate dehydrogenase reaction. This malate-succinate shuttle can sustain these two tissues by transferring reducing power from an O-poor tissue (retina) to an O-rich one (retinal pigment epithelium-choroid).

Succinate Can Shuttle Reducing Power from the Hypoxic Retina to the O-Rich Pigment Epithelium.

Oxidative stress is a major component of most major retinal diseases. Many extrinsic anti-oxidative strategies have been insufficient at counteracting one of the predominant intrinsic sources of reactive oxygen species (ROS), mitochondria. The proton gradient across the inner mitochondrial membrane is a key driving force for mitochondrial ROS production, and this gradient can be modulated by members of the mitochondrial uncoupling protein (UCP) family. Of the UCPs, UCP2 shows a widespread distribution and has been shown to uncouple oxidative phosphorylation, with concomitant decreases in ROS production. Genetic studies using transgenic and knockout mice have documented the ability of increased UCP2 activity to provide neuroprotection in models of a number of diseases, including retinal diseases, indicating that it is a strong candidate for a therapeutic target. Molecular studies have identified the structural mechanism of action of UCP2 and have detailed the ways in which its expression and activity can be controlled at the transcriptional, translational and posttranslational levels. These studies suggest a number of ways in control of UCP2 expression and activity can be used therapeutically for both acute and chronic conditions. The development of such therapeutic approaches will greatly increase the tools available to combat a broad range of serious retinal diseases.

Uncoupling proteins in the mitochondrial defense against oxidative stress.

The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light.

An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice.

Succinate is exported by the retina and imported by eyecup tissue. The transporters mediating this process have not yet been identified. Recent studies showed that monocarboxylate transporter 1 (MCT1) can transport succinate across plasma membranes in cardiac and skeletal muscle. Retina and retinal pigment epithelium (RPE) both express multiple MCT isoforms including MCT1. We tested the hypothesis that MCTs facilitate retinal succinate export and RPE succinate import.

Monocarboxylate Transporter 1 (MCT1) Mediates Succinate Export in the Retina.

Fumarate can be a surrogate for O as a terminal electron acceptor in the electron transport chain. Reduction of fumarate produces succinate, which can be exported. It is debated whether intact tissues can import and oxidize succinate produced by other tissues. In a previous report, we showed that mitochondria in retinal pigment epithelium (RPE)-choroid preparations can use succinate to reduce O to HO. However, cells in that preparation could have been disrupted during tissue isolation. We now use multiple strategies to quantify intactness of the isolated RPE-choroid tissue. We find that exogenous C-succinate is oxidized by intact cells then exported as fumarate or malate. Unexpectedly, we also find that oxidation of succinate is different from oxidation of other substrates because it uncouples electron transport from ATP synthesis. Retinas produce and export succinate. Our findings imply that retina succinate may substantially increase O consumption by uncoupling adjacent RPE mitochondria.

Succinate metabolism in the retinal pigment epithelium uncouples respiration from ATP synthesis.

To test the hypothesis that rod energy biomarkers in light and dark are similar in mice without functional rod transducin (Gnat1rd17).

Transducin-Deficient Rod Photoreceptors Evaluated With Optical Coherence Tomography and Oxygen Consumption Rate Energy Biomarkers.

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.

Article	Year
Succinate Can Shuttle Reducing Power from the Hypoxic Retina to the O-Rich Pigment Epithelium. Cell reports\| PubMed ID: 32375026	2020
Uncoupling proteins in the mitochondrial defense against oxidative stress. Progress in retinal and eye research\| PubMed ID: 33422637	2021
An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice. Investigative ophthalmology & visual science\| PubMed ID: 34797906	2021
Monocarboxylate Transporter 1 (MCT1) Mediates Succinate Export in the Retina. Investigative ophthalmology & visual science\| PubMed ID: 35363247	2022
Succinate metabolism in the retinal pigment epithelium uncouples respiration from ATP synthesis. Cell reports\| PubMed ID: 35675773	2022
Transducin-Deficient Rod Photoreceptors Evaluated With Optical Coherence Tomography and Oxygen Consumption Rate Energy Biomarkers. Investigative ophthalmology & visual science\| PubMed ID: 36576748	2022
Aerobic Glycolysis in Photoreceptors Supports Energy Demand in the Absence of Mitochondrial Coupling. Advances in experimental medicine and biology\| PubMed ID: 37440069	2023

Daniel T. Hass

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