Mitochondrial redox adaptations enable alternative aspartate synthesis in SDH-deficient cells
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Evaluation Summary:
Hart et al show that loss of mitochondrial complex I rescues succinate dehydrogenase deficient (SDH) cells. The experiments are well performed and the phenotype is potentially very interesting to researchers of cancer metabolism. The authors propose that rescue of SDH deficiency by complex I inhibition is caused by an increase in mitochondrial NADH which leads to a restoration of aspartate levels, which in turn rescues proliferation. To support the model, the authors do demonstrate that there are possible correlations of this phenotype to restored aspartate biosynthesis. However, they do not unambiguously establish a mechanism that fully defines how complex I inhibition rescues the proliferation of SDH deficient cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD +to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss-of-function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC), but mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports human cell proliferation through aspartate synthesis but, unlike other ETC impairments, the effects of SDH inhibition are not ameliorated by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH-impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context depend on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production through pyruvate carboxylation and reductive carboxylation of glutamine. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. These data therefore identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal how compartmentalized redox changes can impact cellular fitness.
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Evaluation Summary:
Hart et al show that loss of mitochondrial complex I rescues succinate dehydrogenase deficient (SDH) cells. The experiments are well performed and the phenotype is potentially very interesting to researchers of cancer metabolism. The authors propose that rescue of SDH deficiency by complex I inhibition is caused by an increase in mitochondrial NADH which leads to a restoration of aspartate levels, which in turn rescues proliferation. To support the model, the authors do demonstrate that there are possible correlations of this phenotype to restored aspartate biosynthesis. However, they do not unambiguously establish a mechanism that fully defines how complex I inhibition rescues the proliferation of SDH deficient cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; …
Evaluation Summary:
Hart et al show that loss of mitochondrial complex I rescues succinate dehydrogenase deficient (SDH) cells. The experiments are well performed and the phenotype is potentially very interesting to researchers of cancer metabolism. The authors propose that rescue of SDH deficiency by complex I inhibition is caused by an increase in mitochondrial NADH which leads to a restoration of aspartate levels, which in turn rescues proliferation. To support the model, the authors do demonstrate that there are possible correlations of this phenotype to restored aspartate biosynthesis. However, they do not unambiguously establish a mechanism that fully defines how complex I inhibition rescues the proliferation of SDH deficient cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Hart et al set out to determine how cancer cells lacking succinate dehydrogenase are able to proliferate despite having dysfunctional mitochondria. They use cell-line based models to show that inhibition of mitochondrial complex I rescues succinate dehydrogenase (SDH) deficient cells and that cells adapt to loss of SDH by decreasing complex I levels. The authors propose that lack of complex I causes an increase in mitochondrial NADH and that increased mitochondrial NADH leads to a restoration of Asp levels. The study confirms and complements previous work addressing these relationships and the experiments are expertly performed. The authors do a nice job of using genetic tools, for example, to modulate mitochondrial NADH levels, and the metabolomics analysis is of a high quality. The conclusions of this …
Reviewer #1 (Public Review):
Hart et al set out to determine how cancer cells lacking succinate dehydrogenase are able to proliferate despite having dysfunctional mitochondria. They use cell-line based models to show that inhibition of mitochondrial complex I rescues succinate dehydrogenase (SDH) deficient cells and that cells adapt to loss of SDH by decreasing complex I levels. The authors propose that lack of complex I causes an increase in mitochondrial NADH and that increased mitochondrial NADH leads to a restoration of Asp levels. The study confirms and complements previous work addressing these relationships and the experiments are expertly performed. The authors do a nice job of using genetic tools, for example, to modulate mitochondrial NADH levels, and the metabolomics analysis is of a high quality. The conclusions of this paper are mostly supported by data, but some aspects of the analysis of Asp levels and the NAD+/NADH ratio need to be clarified. Extensive previous work has detailed a complex relationship between mitochondrial dysfunction, Asp levels and proliferation. In this version of the manuscript, the authors advance our understanding, but do not unambiguously demonstrate a molecular mechanism that fully explains how complex I inhibition rescues loss of SDH.
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Reviewer #2 (Public Review):
Hart et al. examine how cells maintain proliferation in the context of SDH inhibition. A major strength of this study is the interesting observation that ETC complex I inhibition restores proliferation in SDH-deficient cells, which could have relevance for evolution of SDH-deficient tumors. The authors also demonstrate that SDH-inhibition is not coupled to a decrease in NAD+/NADH ratio. For the most part, the authors rigorously tested these observations using a number of genetic and chemical perturbations. However, a major weakness of the paper is the unclear physiological relevance of the findings and the tendency to overstate results, especially with regards to the dependence on aspartate biosynthesis.
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Reviewer #3 (Public Review):
The authors demonstrate a fascinating phenotype wherein combined disruption of both complex I and II activity led to a restoration of proliferative capacity compared to when only Complex II is disrupted. While this phenomenon is interesting, we were unable to decipher a coherent mechanistic explanation for these observations. In addition, the authors would need to address a few points to strengthen the claims they make within this work.
• In Fig. 1D, the authors state that a 1.9x increase is not noteworthy, while in Fig. 1B, a 2.3x increase is noteworthy. Can the authors more clearly describe how they are determining their thresholds?
o On a related note, more evidence is needed to conclude that the small changes/restoration in aspartate abundance is sufficient to completely, or almost completely, restore …Reviewer #3 (Public Review):
The authors demonstrate a fascinating phenotype wherein combined disruption of both complex I and II activity led to a restoration of proliferative capacity compared to when only Complex II is disrupted. While this phenomenon is interesting, we were unable to decipher a coherent mechanistic explanation for these observations. In addition, the authors would need to address a few points to strengthen the claims they make within this work.
• In Fig. 1D, the authors state that a 1.9x increase is not noteworthy, while in Fig. 1B, a 2.3x increase is noteworthy. Can the authors more clearly describe how they are determining their thresholds?
o On a related note, more evidence is needed to conclude that the small changes/restoration in aspartate abundance is sufficient to completely, or almost completely, restore proliferation. This seems surprising and the data must be strengthened.
• The data presented between Fig. 1F and 3A appear to be inconsistent. It is our understanding that the conditions are identical (same media, cell type, etc.), yet in 1F, addition of AA5 without PYR leads to minimal change in the NAD+/NADH ratio, while in 3A, sole addition of AA5 roughly doubles the NAD+/NADH ratio.
• While we appreciate the different approaches of utilizing both pharmacological and genetic inhibition of SDH, we are not sufficiently convinced that AA5 acts fully on-target and is not causing additional off-target effects. Perhaps this has been sufficiently described in previous literature but based on the data contained within this work and the citations provided, this wasn't evident. It would also be helpful to include the source of AA5 used - we were unable to find such details in the main text nor in the methods section. In many cases, there seem to be discordant results between the AA5 treatment and SDH inhibition or knockout. For example:
o Fig. 2F,G: Based on the proposed model, if the authors were to add AA5 in the SDHB KO, this would presumably not effect proliferation, aspartate levels, or NAD+/NADH balance and would implicate that AA5 is not effecting anything other than SDH. This experiment should be done to test for off-target effects of AA5.
• There are several sub-figures where we feel there are missing controls. Examples include:
o Fig 3A: Why is a rotenone-only control not provided in this sub-panel?
o Fig 3D,E: What does the no-cyto & no-mitoLbNOX look like under these different treatment conditions?
o Fig 4A,B,F: Why is the add-back not included in these panels, but it is in several others within this figure? We would be concerned if they did not show the expected CI activity, etc. that is being assumed by the authors.
o Fig S3D: Why is a AZD7545-only control not provided?
• We feel like there are several instances where the connections to NAD+/NADH balance need to be improved to support the claims being made by the authors:
o Fig 3: It is clear from the data that the NAD+/NADH pools are compartmentalized, but their connection to proliferation and aspartate synthesis is not clear from the presented data.
• Presumably the authors have the NAD+ and NADH ion counts for the LbNOX experiments since they have the aspartate levels. Are cyto- and mito-LbNox causing the same level of reversal of the NAD+/NADH balance? Is the LbNOX construct just less functional when in the mitochondrial versus the cytosolic form?
• Fig 3G,H: The change in proliferation seems to be relatively minor compared to other proliferation changes presented in this work. Can the authors speak to the correlation between the NAD+/NADH ratio and proliferation?
o Why would NADH levels, or the NAD+/NADH ratio influence proliferation rates? Similarly, why would AA5 increase NAD+/NADH ratio (Fig. 3A)? This is not particularly clear in the text.
o There seem to be many claims that NAD+/NADH ratio is tied to reductive carboxylation, yet this connection is not clear from the data and seems to just be pure conjecture. If this is essentially the only mechanistic hypothesis of the manuscript, it is important to show it.We feel the current manuscript lacks a coherent explanation for the restoration of proliferation. We encourage the authors to take one step further to demonstrate some of the possible mechanisms. Mito NAD+/NADH ratio seems to be important, but how does this explain the proliferation phenotype? Aspartate levels indeed seem to be correlated to some extent with the restoration of proliferation, yet this alone does not appear sufficient to explain the phenomenon. This is perhaps beyond the scope of this work, but we feel some sort of CRISPR screen using proliferation as a read-out would be beneficial to this work. The authors may choose to use some other methods to address this problem. For example, at the end of the first paragraph in the discussion section, the authors suggested two modalities that can potentially be tested to make sense of the current observations.
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