The alternative oxidase reconfigures the larval mitochondrial electron transport system to accelerate growth and development in Drosophila melanogaster
Curation statements for this article:-
Curated by eLife
eLife Assessment
The findings in this manuscript are important because they demonstrate the key role of metabolism in insect development. The data were collected and analyzed using solid and validated methodologies, but the evidence is incomplete, as the extent of the involvement of AOX activity in vivo and in physiological conditions is not addressed. This manuscript will be of interest for the fields of mitochondrial bioenergetics, metabolism and development.
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Abstract
Abstract
The alternative oxidase (AOX) is naturally present in the mitochondrial electron transfer system (ETS) of many organisms but absent in vertebrates and most insects. AOX oxidizes coenzyme Q and reduces O2 in H2O, partially replacing the ETS cytochrome c segment and alleviating the oxidative stress caused by ETS overload. As successfully demonstrated in animal models, AOX shows potential in mitigating mitochondrial diseases. However, its non-proton-pumping nature may uncouple mitochondria, leading to excessive heat generation and interference with normal metabolism and physiology. Here we show that AOX from the tunicate Ciona intestinalis accelerates development of Drosophila melanogaster, elevating larval biomass accumulation (primarily due to increased fat), mobility and food intake, without increasing body heat production. AOX intensifies Leak respiration and lowers oxidative phosphorylation efficiency through functional interactions with the mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH). This is associated with increased complex I (CI)-driven respiration and supercomplex formation, higher cellular NAD+/NADH ratios, and an enhanced flux through the central carbon metabolism. Chemical uncouplers and rotenone confirm the roles of mitochondrial uncoupling and CI in the development of AOX-expressing larvae. Thus, AOX appears to be promoting increased growth by reinforcing the larval proliferative metabolic program via an intricate mechanism that reconfigures the larval ETS.
Article activity feed
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eLife Assessment
The findings in this manuscript are important because they demonstrate the key role of metabolism in insect development. The data were collected and analyzed using solid and validated methodologies, but the evidence is incomplete, as the extent of the involvement of AOX activity in vivo and in physiological conditions is not addressed. This manuscript will be of interest for the fields of mitochondrial bioenergetics, metabolism and development.
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Reviewer #1 (Public review):
Summary:
The manuscript by Garcia et al. describes how the expression of a respiratory chain alternative oxidase (AOX) from the tunicate Ciona intestinalis, capable of transferring electrons directly from reduced coenzyme Q (CoQ) to oxygen, is able to induce an increase in the mass of Drosophila melanogaster larvae and an accelerated development, especially when the larvae are kept at low temperatures. In order to explain this phenomenon, the paper addresses the modifications in the activity and levels of the 'canonical' electron transfer system (ETS), i.e., complexes I-IV and of the ATP synthase. In addition, the abundance of different metabolites as well as the NAD+/NADH ratios are measured, finding significant differences between the larvae.
Strengths:
The observations of differences in growth, body mass …
Reviewer #1 (Public review):
Summary:
The manuscript by Garcia et al. describes how the expression of a respiratory chain alternative oxidase (AOX) from the tunicate Ciona intestinalis, capable of transferring electrons directly from reduced coenzyme Q (CoQ) to oxygen, is able to induce an increase in the mass of Drosophila melanogaster larvae and an accelerated development, especially when the larvae are kept at low temperatures. In order to explain this phenomenon, the paper addresses the modifications in the activity and levels of the 'canonical' electron transfer system (ETS), i.e., complexes I-IV and of the ATP synthase. In addition, the abundance of different metabolites as well as the NAD+/NADH ratios are measured, finding significant differences between the larvae.
Strengths:
The observations of differences in growth, body mass and food intake in the wt D. melanogaster larvae vs. those expressing the AOX transgene are solid. The evidence that mild uncoupling of the ETS might accelerate development of the fly larvae is convincing.
Weaknesses:
Some of the observations, especially those concerning the origin of the metabolic remodelling in AOX-expressing larvae, are left unexplained, and the argumentation is somewhat speculative. What the authors mean by "reconfiguration" of the mitochondrial electron transfer system is not clear. If this implies that there is an actual change in ETS function and/or structural organisation in the presence of AOX, this conclusion is not supported by the experimental data. In addition, the influence of AOX activity in the mitochondrial ETS system is tested in vitro in the presence of saturating concentrations of substrates. The real degree to which AOX activity is actually influencing ETS activity in vivo remains unknown.
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Reviewer #2 (Public review):
Summary:
This manuscript presents intriguing findings about the role of alternative oxidase (AOX) from the tunicate Ciona intestinalis in accelerating growth and development when expressed in Drosophila melanogaster.
Strengths:
The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. The present study provides valuable insights into AOX's role in Drosophila development. The paper attempts to explore a unique mechanism by which AOX influences Drosophila development, providing insights into mitochondrial respiration and its physiological effects. This is relevant for understanding mitochondrial dysfunction and potential therapeutic applications. The study employs a variety of approaches, including calorimetry, …
Reviewer #2 (Public review):
Summary:
This manuscript presents intriguing findings about the role of alternative oxidase (AOX) from the tunicate Ciona intestinalis in accelerating growth and development when expressed in Drosophila melanogaster.
Strengths:
The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. The present study provides valuable insights into AOX's role in Drosophila development. The paper attempts to explore a unique mechanism by which AOX influences Drosophila development, providing insights into mitochondrial respiration and its physiological effects. This is relevant for understanding mitochondrial dysfunction and potential therapeutic applications. The study employs a variety of approaches, including calorimetry, infrared thermography, and genetic analyses, to investigate AOX's impact on metabolism and development.
Weaknesses:
There are a number of methodological limitations and substantial gaps in the interpretation of the data presented, which reduces the strength of its conclusions. For instance, there is a misunderstanding of the non-proton motive nature of the AOX - it does not uncouple respiration, merely decouple it as it neither contributes to nor dissipates the proton motive force, in contrast to chemical uncouplers or proton uncouplers such as UCPs. The authors need to reassess their data in light of the above.
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Author response:
Reviewer #1 (Public review):
Summary:
The manuscript by Garcia et al. describes how the expression of a respiratory chain alternative oxidase (AOX) from the tunicate Ciona intestinalis, capable of transferring electrons directly from reduced coenzyme Q (CoQ) to oxygen, is able to induce an increase in the mass of Drosophila melanogaster larvae and an accelerated development, especially when the larvae are kept at low temperatures. In order to explain this phenomenon, the paper addresses the modifications in the activity and levels of the 'canonical' electron transfer system (ETS), i.e., complexes I-IV and of the ATP synthase. In addition, the abundance of different metabolites as well as the NAD+/NADH ratios are measured, finding significant differences between the larvae.
Strengths:
The observations of differences in …
Author response:
Reviewer #1 (Public review):
Summary:
The manuscript by Garcia et al. describes how the expression of a respiratory chain alternative oxidase (AOX) from the tunicate Ciona intestinalis, capable of transferring electrons directly from reduced coenzyme Q (CoQ) to oxygen, is able to induce an increase in the mass of Drosophila melanogaster larvae and an accelerated development, especially when the larvae are kept at low temperatures. In order to explain this phenomenon, the paper addresses the modifications in the activity and levels of the 'canonical' electron transfer system (ETS), i.e., complexes I-IV and of the ATP synthase. In addition, the abundance of different metabolites as well as the NAD+/NADH ratios are measured, finding significant differences between the larvae.
Strengths:
The observations of differences in growth, body mass and food intake in the wt D. melanogaster larvae vs. those expressing the AOX transgene are solid. The evidence that mild uncoupling of the ETS might accelerate development of the fly larvae is convincing."
We appreciate the reviewer’s attention to our results and hope we can improve the manuscript to address all criticism appropriately.
Weaknesses:
Some of the observations, especially those concerning the origin of the metabolic remodelling in AOX-expressing larvae, are left unexplained, and the argumentation is somewhat speculative. What the authors mean by "reconfiguration" of the mitochondrial electron transfer system is not clear. If this implies that there is an actual change in ETS function and/or structural organisation in the presence of AOX, this conclusion is not supported by the experimental data. In addition, the influence of AOX activity in the mitochondrial ETS system is tested in vitro in the presence of saturating concentrations of substrates. The real degree to which AOX activity is actually influencing ETS activity in vivo remains unknown.
Indeed, the term “reconfiguration” may seem a little too strong. However, we do have preliminary structural data on larval mitochondria indicating that the term is adequate in this context. We plan to work on obtaining concrete data to sustain our claims that AOX imparts significant functional and structural remodeling of the organelle, which would be consistent with our respirometry and BN-PAGE data. If the data turns out not to be robust enough, we will consider replacing the term with one that better reflects our findings.
We also realize that the in vivo data we are presenting (body mass, mobility, food intake) are indirect measurements of metabolism and that a more direct approach is necessary to assess the real degree to which AOX influences ETS activity in vivo. To address this issue, we plan to expand our pharmacological treatments of the larval development and to measure whole larval oxygen consumption.
Reviewer #2 (Public review):
Summary:
This manuscript presents intriguing findings about the role of alternative oxidase (AOX) from the tunicate Ciona intestinalis in accelerating growth and development when expressed in Drosophila melanogaster.
Strengths:
The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. The present study provides valuable insights into AOX's role in Drosophila development. The paper attempts to explore a unique mechanism by which AOX influences Drosophila development, providing insights into mitochondrial respiration and its physiological effects. This is relevant for understanding mitochondrial dysfunction and potential therapeutic applications. The study employs a variety of approaches, including calorimetry, infrared thermography, and genetic analyses, to investigate AOX's impact on metabolism and development.
We sincerely thank the reviewer for recognizing the strengths and acknowledging the novelty of our study.
Weaknesses:
There are a number of methodological limitations and substantial gaps in the interpretation of the data presented, which reduces the strength of its conclusions. For instance, there is a misunderstanding of the non-proton motive nature of the AOX - it does not uncouple respiration, merely decouple it as it neither contributes to nor dissipates the proton motive force, in contrast to chemical uncouplers or proton uncouplers such as UCPs. The authors need to reassess their data in light of the above.
The reviewer is absolutely right about the non-proton motive nature of AOX. We will reassess our data considering that AOX decouples respiration and, if necessary and possible, we will add new experiments to address the methodological limitations raised by the reviewer.
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