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.

Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover. Daily cycles of light exposure and energy consumption suggest that mitochondrial turnover is important for cone health. We investigated the three-dimensional (3D) ultrastructure and metabolic function of zebrafish cone mitochondria throughout the day. At night retinas undergo a mitochondrial biogenesis event, corresponding to an increase in the number of smaller, simpler mitochondria and increased metabolic activity in cones. In the daytime, endoplasmic reticula (ER) and autophagosomes associate more with mitochondria, and mitochondrial size distribution across the cluster changes. We also report dense material shared between cone mitochondria that is extruded from the cell at night, sometimes forming extracellular structures. Our findings reveal an elaborate set of daily changes to cone mitochondrial structure and function.

Daily mitochondrial dynamics in cone photoreceptors.

Oxygen (O) and other dissolved gases such as the gasotransmitters HS, CO, and NO affect cell metabolism and function. To evaluate effects of dissolved gases on processes in tissue, we developed a fluidics system that controls dissolved gases while simultaneously measuring parameters of electron transport, metabolism, and secretory function. We use pancreatic islets, retina, and liver from rodents to highlight its ability to assess effects of O and HS. Protocols aimed at emulating hypoxia-reperfusion conditions resolved a previously unrecognized transient spike in O consumption rate (OCR) following replenishment of O, and tissue-specific recovery of OCR following hypoxia. The system revealed both inhibitory and stimulatory effects of HS on insulin secretion rate from isolated islets. The unique ability of this new system to quantify metabolic state and cell function in response to precise changes in dissolved gases provides a powerful platform for cell physiologists to study a wide range of disease states.

Fluidics system for resolving concentration-dependent effects of dissolved gases on tissue metabolism.

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.

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).

Article	Year
Succinate Can Shuttle Reducing Power from the Hypoxic Retina to the O-Rich Pigment Epithelium. Cell reports\| PubMed ID: 32375026	2020
Daily mitochondrial dynamics in cone photoreceptors. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 33144507	2020
Fluidics system for resolving concentration-dependent effects of dissolved gases on tissue metabolism. eLife\| PubMed ID: 34734803	2021
An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice. Investigative ophthalmology & visual science\| PubMed ID: 34797906	2021
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

Brian M. Robbings

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