The retina uncouples glycolysis and oxidative phosphorylation via Cori-, Cahill-, and mini-Krebs-cycle

Reviewer #1 (Public Review):

Summary: In the manuscript by Chen et al. entitled, "The retina uncouples glycolysis and oxidative phosphorylation via Cori-, Cahill-, and mini-Krebs-cycle", the authors look to provide insight on retinal metabolism and substrate utilization by using a murine explant model with various pharmacological treatments in conjunction with metabolomics. The authors conclude that photoreceptors, a specific cell within the explant, which also includes retinal pigment epithelium (RPE) and many other types of cells, are able to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: 1) the mini-Krebs-cycle, fueled by glutamine and branched-chain amino acids; 2) the alanine-generating Cahill-cycle; and 3) the lactate-releasing Cori-cycle. While intriguing if determined to be true, these cell-specific conclusions are called into question due to the ex vivo experimental setup with the inclusion of RPE, the fact that the treatments were not cell-specific nor targeted at an enzyme specific to a certain cell within the retina, and no stable isotope tracing nor mitochondrial function assays were performed. Hence, without significant cell-specific methods and future experimentation, the primary claims are not supported.

Strengths: This study attempts to improve on the issues that have limited the results obtained from previous ex vivo retinal explant studies by culturing in the presence of the RPE, which is a major player in the outer retinal metabolic microenvironment. Additionally, the study utilizes multiple pharmacologic methods to define retinal metabolism and substrate utilization.

Weaknesses: A major weakness of this study is the lack of in vivo supporting data. Explant cultures remove the retina from its dual blood supply. Typically, retinal explant cultures are done without RPE. However, the authors included RPE in the majority of experimental conditions herein. However, it is unclear if the metabolomics samples included the RPE or not. The inclusion of the RPE, which is metabolically active and can be altered by the treatments investigated herein, further confounds the claims made regarding the neuroretina. Considering the pharmacologic treatments utilized with the explant cultures are not cell-specific and/or have significant off-target effects, it is difficult to ascertain that the metabolic changes are secondary to the effects on photoreceptors alone, which the authors claim. Additionally, the explants are taken at a very early age when photoreceptors are known to still be maturing. No mention or data is presented on how these metabolic changes are altered in retinal explants after photoreceptors have fully matured. Likewise, significant assumptions are made based on a single metabolomics experiment with no stable isotope tracing to support the pathways suggested. While the authors use immunofluorescence to support their claims at multiple points, demonstrating the presence of certain enzymes in the photoreceptors, many of these enzymes are present throughout the retina and likely the RPE. Finally, the claims presented here are in direction contradiction to recent in vivo studies that used cell-specific methods when examining retinal metabolism. No discussion of this difference in results is attempted.

Reviewer #2 (Public Review):

Summary: The authors aim to learn about retinal cell-specific metabolic pathways, which could substantially improve the way retinal diseases are understood and treated. They culture ex vivo mouse retinas for 6 days with 2 - 4 days of various drug treatments targeting different metabolic pathways or by removing the RPE/choroid tissue from the neural retina. They then look at photoreceptor survival, stain for various metabolic enzymes, and quantify a broad panel of metabolites. While this is an important question to address, the results are not sufficient to support the conclusions.

Strengths: The questions the authors are exploring at extremely valuable and I commend the authors and working to learn more about retina metabolism. The different sensitivity of the cones to various drugs is interesting and may suggest key differences between rods and cones. The authors also provide a thoughtful discussion of various metabolic pathways in the context of previous publications.

Weaknesses: As the authors point out, ex vivo culture models allow for control over multiple aspects of the environment (such as drug delivery) not available in vivo. Ex vivo cultures can provide good hints as to what pathways are available between interacting tissues. However, there are many limitations to ex vivo cultures, including shifting to a very artificial culture media condition that is extremely different than the native environment of the retina. It is well appreciated that cells have flexible metabolism and will adapt to the conditions provided. Therefore, observations of metabolic responses obtained under culture conditions need to be interpreted with caution, they indicate what the tissue is doing under those specific conditions (which include cells adapting and dying).

Chen et al use pharmacological interventions to the impact of various metabolic pathways on photoreceptor survival and "long term" metabolic changes. The dose and timing of these drug treatments are not examined though. It is also hard to know how these drugs penetrate the tissue and it needs to be validated that the intended targets are being accurately hit. These relatively long-term treatments should be causing numerous downstream changes to metabolism, cell function, and survival, which makes looking at a snapshot of metabolite levels hard to interpret. It would be more valuable to look at multiple time points after drug treatment, especially easy time points (closer to 1 hr). The authors use metabolite ratios to make conclusions about pathway activity. It would be more valuable to directly measure pathway activity by looking a metabolite production rates in the media and/or with metabolic tracers again in time scales closer to minutes and hours instead of days.

It is not clear from the text if the ex vivo samples with RPE/choroid intact are analyzed for metabolomics with the RPE/choroid still intact or if this is removed. If it is not removed, the comparison to the retina without RPE/choroid needs to be re-interpreted for the contribution of metabolites from the added tissue. The composition of the tissue is different and cannot be disentangled from the changes to the neural retina specifically.

While the data is interesting and may give insights into some rod and cone-specific metabolic susceptibility, more work is needed to validate these conclusions. Given the limitations of the model the authors have over-interpreted their findings and the conclusions are not supported by the results. They need to either dramatically limit the scope of their conclusions or validate these hypotheses with additional models and tools.

Reviewer #3 (Public Review):

Summary:
The neural retina is one of the most energetically active tissues in the body and research into retinal metabolism has a rich history. Prevailing dogma in the field is that the photoreceptors of the neural retina (rods and cones) are heavily reliant on glycolysis, and as oxygen tension at the level of photoreceptors is very low, these specialized sensory neurons carry out aerobic glycolysis, akin to the Warburg effect in cancer cells. It has been found that this unique metabolism changes in many retinal diseases, and targeting retinal metabolism may be a viable treatment strategy. The neural retina is composed of 11 different cell types, and many research groups over the past century have contributed to our current understanding of cell-specific metabolism of retinal cells. More recently, it has been shown in mouse models and co-culture of the mouse neural retina with human RPE cultures that photoreceptors are reliant on the underlying retinal pigment epithelium for supplying nutrients. Chen and colleagues add to this body of work by studying an ex vivo culture of the developing mouse retina that maintained contact with the retinal pigment epithelium. They exposed such ex vivo cultures to small molecule inhibitors of specific metabolic pathways, performing targeted metabolomics on the tissue and staining the tissue with key metabolic enzymes to lay the groundwork for what metabolic pathways may be active in particular cell types of the retina. The authors conclude that rod and cone photoreceptors are reliant on different metabolic pathways to maintain their cell viability - in particular, that rods rely on oxidative phosphorylation and cones rely on glycolysis. Further, their data support multiple mechanisms whereby glycolysis may occur simultaneously with anapleurosis to provide abundant energy to photoreceptors. The data from metabolomics revealed several novel findings in retinal metabolism, including the use of glutamine to fuel the mini-Krebs cycle, the utilization of the Cahill cycle in photoreceptors, and a taurine/hypotaurine shuttle between the underlying retinal pigment epithelium and photoreceptors to transfer reducing equivalents from the RPE to photoreceptors. In addition, this study provides robust quantitative metabolomics datasets that can be compared across experiments and groups. The use of this platform will allow for rapid testing of novel hypotheses regarding the metabolic ecosystem in the neural retina.

Strengths:
The data on differences in the susceptibility of rods and cones to mitochondrial dysfunction versus glycolysis provides novel hypothesis-generating conjectures that can be tested in animal models. The multiple mechanisms that allow anapleurosis and glycolysis to run side-by-side add significant novelty to the field of retinal metabolism, setting the stage for further testing of these hypotheses as well.

Weaknesses:
Almost all of the conclusions from the paper are preliminary, based on data showing enzymes necessary for a metabolic process are present and the metabolites for that process are also present. However, to truly prove whether these processes are happening, C13 labeling or knock-out or over-expression experiments are necessary. Further, while there is good data that RPE cultures in vitro strongly recapitulate RPE phenotypes in vivo, ex vivo neural retina cultures undergo rapid death. Thus, conclusions about metabolism from explants should either be well correlated with existing literature or lead to targeted in vivo studies. This paper currently lacks both.