The Ketogenic Diet Metabolite β-Hydroxybutyrate Promotes Mitochondrial Elongation via Deacetylation and Improves Autism-like Behavior in Zebrafish

  1. Golam M. Uddin
  2. Rachel Lacroix
  3. Mashiat Zaman
  4. Iman Al Khatib
  5. Amit Jaiswal
  6. Jong M. Rho
  7. Deborah M. Kurrasch
  8. Timothy E. Shutt

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Abstract

The ketogenic diet (KD) is clinically beneficial and has therapeutic potential across a growing list of neurological disorders, including autism spectrum disorder (ASD). However, the underlying mechanisms mediating the benefits of the KD, which can also have undesirable side effects, remain undefined. To this end, improvements in mitochondrial morphology and function correlate with improved ASD behaviours in response to the KD, though how the KD influences mitochondrial morphology, and whether this is sufficient to improve behaviour remains unknown. Here, we investigate how beta-hydroxybutyrate (BHB), a key metabolite produced by the KD regulates mitochondrial morphology, and whether this pathway could be exploited to alter phenotypes in a zebrafish model of ASD. We found that β-oxidation of BHB promotes mitochondrial elongation by increasing NAD + levels, which in turn activates SIRT deacetylases that act on key regulators of both mitochondrial fusion and fission. Our data suggest that increasing NAD + levels with its precursor, nicotinamide nucleotide (NMN), is sufficient to promote mitochondrial hyperfusion. Finally, both BHB and NMN impact neurodevelopment in the shank3b +/- zebrafish model of ASD. Together, our findings elucidate a mechanism by which the ketogenic diet promotes mitochondrial elongation. Moreover, manipulation of this pathway may provide a novel avenue for the treatment of neurological disorders such as ASD that also may obviate potential complications of the KD in clinical practice.

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  1. Review Commons

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    Reply to the reviewers

    1. General Statements

    We thank the reviewer for their positive comments regarding the research article titled "The Ketogenic Diet Metabolite 1 β-Hydroxybutyrate Promotes Mitochondrial Elongation via Deacetylation and Improves Autism-like Behaviour in Zebrafish" by Uddin GM and colleagues. We appreciate your input, and we will address these comments as indicated below with specific responses to each point raised by reviewers.

    The main changes in the updated manuscript are as follows:

    We have revised the introduction to now incorporate additional background information on mitochondria, NAD, and mitochondrial dynamics and function. This addition aims to provide readers with a broader understanding of the mitochondrial context in relation to our study.

    Furthermore, we recognize that previous studies have explored mitochondrial function in the context of the ketogenic diet. While our specific investigation centered on mitochondrial morphology, we acknowledge the importance of comprehensively investigating mitochondrial function. To this end, we have added new data showing how BHB impacts mitochondrial oxidative phosphorylation in HeLa cells (Sup Fig 2), and how both BHB and NMN impact oxygen consumption/glycolysis in zebrafish (Fig 7).

    We have also added new behaviour analysis of the zebrafish (Fig 6), and have re-framed the discussion around neurodevelopment generally, rather than ASD specifically.

    Finally, we have now included a section in our manuscript that discusses the limitations of our study. These limitations can be further investigated to explore and characterize the full mechanistic potential behind the effects of the ketogenic diet and/or NMN on mitochondrial dynamics.

    2. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Uddin GM and colleagues presented a research article entitled 'The Ketogenic Diet Metabolite 1 β-Hydroxybutyrate Promotes Mitochondrial Elongation via Deacetylation and Improves Autism-like Behaviour in Zebrafish'. Roles of ketogenic diet (KD) and NAD+ precursors in health promotion and longevity, as well as on the alleviation of a broad range of diseases are evident. However, their roles in autism are not well done, which is the novelty of the current study. Addressing below questions will improve the quality of the paper.

    Major concerns

    1. In the introduction section, a broad overview of the roles of ketogenic diet (KD) in neurodegenerative disease (and ageing, if possible) should be provided. E.g., the authors should summarize exciting progress on the use of KD to treat Alzheimer's disease in animal models (PMID: 23276384). *

    Response: Thank you for your valuable suggestion. While it is true that the KD appears to be beneficial in neurodegenerative (and other disease) models, our focus in this paper is looking at neurodevelopment, rather than all potential benefits of the KD. Nonetheless, we have addressed this comment by incorporating a brief overview of the roles of the KD in neurodegenerative diseases, including Alzheimer's disease (AD), in the introduction section of the manuscript. Specifically, we have summarized the exciting progress made in utilizing KD to treat AD in animal models, as highlighted in the suggested study. This addition helps to provide a better overview of the potential therapeutic effects of KD in neurodegenerative diseases and strengthens the introduction section of the manuscript.

    • Roles of high fat diet to treat diseases could be extended to rare premature ageing diseases. In such scenario, high fat and NAD+ boosting shared some joint mechanisms (PMID: 25440059 ). *

    Response: This information and the reference are now added to the discussion.

    *In the introduction, a more detailed introduction of NAD+ and its roles in mitochondrial homeostasis (especially mitophagy and the mitochondrial fusion-fission balance) should be included (PMID: 24813611; PMID: 30742114; PMID: 31577933). *

    Response: Although our paper focused primarily on mitochondrial fission and fusion, we have incorporated a new paragraph in the introduction to provide a more detailed introduction detailing NAD+ and its roles in mitochondrial homeostasis, specifically highlighting mitophagy. We have included the suggested references.

    • In regarding to the statement of KD increases NAD+, was it due to increased generation (to check protein levels and activities of different NAD+ synthetic enzymes, such as iNAMPT, NMNAT1-3, and NRK) and/or reduced consumption (in addition to reduced glycolysis, does KD inhibit the activities of CD38 and PARPs? In this paper, Sirtuins' activities is (are increased)). Detailed exploration of the activities of these proteins will unveil a clear molecular mechanisms on how KD affects/regulates NAD+. *

    Response: Thank you for the comment. We agree that exploring the detailed mechanism of how the ketogenic diet (KD) affects NAD+ is an interesting question that will have important implications once answered. However, fully elucidating the mechanism of action would require a more comprehensive investigation, which is beyond the scope of this current project. We have now added this as a future direction in the manuscript.

    *Fig. 1: in the NAD+ field, the normal used NR/NMN concentrations are normally high like to use 500 µM to 2-5 mM (as the NAD+ levels in cells are high). In addition to use 50 µM, the authors are strongly to have a dose-dependent study (50 µM, 500µM, 1, 2, 5 mM), and see changes of mitochondrial funciton and parameters. In this condition, NAD+ levels should be also checked. *

    Response: We have added new supplemental data showing the initial dose response of the effects of BHB and NMN on mitochondrial morphology, which led us to choosing the relevant doses for the remainder of the paper. Our objective was not to investigate the broad impacts of different NMN concentrations on mitochondrial function and parameters, or NAD+ levels. As such, we have only focused on doses where we see effects on mitochondrial morphology.

    *Fig. 2: a comprehensive characterization of mitochondrial fusion-fission should be performed. In addition to the protein evaluated, changes on other key fusion-fission proteins, like Bax, Bak, Mfn-1, Mfn-2, etc should be performed (PMID: 17035996; PMID: 24813611). *

    Response: We agree that looking at other key proteins involved in mediating mitochondrial fission and fusion could provide additional insight. Indeed, given the changes in global acetylation that we see, it is expected that some other proteins may also be regulated in this way. However, there are at least a dozen proteins involved in mediating mitochondrial fusion and fission, not to mention many more proteins that regulate these proteins. Unfortunately, it is not feasible to analyze all the proteins involved in mitochondrial fusion-fission. Moreover, looking only at protein levels, doesn't necessarily inform about the activity of any protein. Instead, we concentrated in this paper on investigating known links between protein acetylation and mitochondrial dynamics, particularly focusing on the proteins that have known links to acetylation (i.e., DRP1, OPA1, MFNs). We have added a note in the discussion acknowledging that other means of regulation could also be occurring in parallel.

    *Figs. 1-5 were focused on mitochondrial morphology, whether KD and NMN changed mitochondrial funciton should be explored, such as to use seahorse to check ECR and OCR. *

    Response: Although our question was focused on morphology, we agree that mitochondrial function is important. We have added new data showing that BHB increases basal oxygen consumption in HeLa cells (Sup Fig 2), as well as new data showing that BHB and NMN influence oxygen consumption and glycolysis in our zebrafish model (Fig 7)

    • Fig. 6: NR/NMN used in animal studies (via gavage or in drinking water in mice, and on plate for worms and flies) are normally high (e.g., in drinking water for mice could be 4-12 mM; for worms and flies are normally 1-5 mM); for zebrafish, while they are swimming in water, this reviewer concerned whether it was true that 50 µM of NMN was sufficient to show the benefit presented.*

    Response: Our data show that these doses are indeed sufficient. We did look at some higher doses for NMN, but these were toxic, leading to poor survival and were not studied further.

    *Minor concerns

    1. Line 26: For 'a growing list of neurological disorders, including autism spectrum disorder (ASD)', please add AD in. *

    Response: Line 26 is part of the abstract, which we feel should be focused more on the main message of the paper, which does not involve AD. As addressed above, we have added AD as an example in the introduction.

    *Line 57: For 'with side effects such as gastrointestinal disturbances, nausea/vomiting, diarrhea, constipation, and hypertriglyceridemia being reported', rate of frequency shall be provided if any. *

    Response: We have modified the statement to indicate the relative percent of patients suffering the various side effects.

    *Reviewer #1 (Significance (Required)):

    The novelty of the current study was to investigate effects of KD and NAD+ on autism. This investigation was not performed before and thus is the novelty.

    Weakness, effects of KD and NAD+/NMN on mitochondrial function were not well-investigated and should be done. Introduction was not well done, many key information in the fields were not provided which may mislead the readers an over-evaluation of the novelty of the current study.*

    Response: As outlined above, we have edited the introduction to include additional information requested by the reviewer. Moreover, our focus in this manuscript was to look at the mechanisms underlying changes in mitochondrial morphology, not mitochondrial function per se, though this is clearly important and related. Nonetheless, as discussed above, we have also added new data showing how BHB impacts mitochondrial function.

    *My expertise lies in NAD+, mitochondria, and brain health.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The study examined the effect of beta-hydroxybutyrate and nicotinamide nucleotide on mitochondrial morphology and the molecular pathways which mitigate this effect as well as the effect of these treatments on behavior in zebrafish. The study is well done and well written. The only thing I think that could be improved are the bar in the graph some the significant comparisons. It is sometimes difficult to see which groups are being compared.*

    Response: We're happy to adjust how the data is displayed in the relevant bar graphs, but it is not clear exactly what changes the reviewer would like. To some degree this will depend on the specific guideline of the final journal where we hope the manuscript will be published. As such, we have not made changes at this point.

    ***Referees cross-commenting**

    The other reviewers do have some fair comments. Multiple doses would be helpful and showing bioenergetic data would complement the morphological measurements. Additionally, behavioral assays showing changes in social behavior in the Zebrafish would provide a stronger link to ASD. *

    Response: As discussed above, we have added new information on doses and mitochondrial bioenergetics. With respect to behaviour, we have added thigmotaxis data and reworked the discussion around behaviour and neurodevelopment so that it is less specific to ASD.

    *Reviewer #2 (Significance (Required)):

    As beta-hydroxybutyrate is an important substrate for the ketogenic diet, this study helps explain the potential mechanisms in which the ketogenic diet may enhance mitochondrial function.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this paper, Uddin and colleagues have investigated components of the ketogenic diet to understand changes in both mitochondrial morphology and protein expression, and zebrafish locomotor behaviour. They investigate whether beta-hydroxybutyrate (BHB) or nicotinamide nucleotide (NMN) application can later human mitochondria in HeLA cell lines, and also recue a locomotion defect in shank3b+/- zebrafish larvae that have previously been proposed as a model for autism. This study is strengthened by showing data from two species; however the link between the HeLA cell line data and larval zebrafish is not strong. The study would be improved by assessing zebrafish mitochondrial changes after drug application, and testing more than one concentration of BH and NMN in the behavioural assay. This is an interesting study, and it is nicely written and presented. I have made some comments to strengthen the study below.

    Major comments My expertise is in modelling some aspects of autism in zebrafish. To this end I have focussed on the zebrafish part of this manuscript more fully. I have several comments related to the zebrafish experiments.

    1. The changes in mitochondrial morphology, peroxisome number and mitochondrial protein levels were measured in HeLA cells and not comparable data is shown for zebrafish. The same experiments should be repeated using larval zebrafish or a zebrafish cell line. *

    Response: We chose to use HeLa cells for the mechanistic studies due to practical reasons. Cell lines offer a controlled and well-established system for investigating cellular processes and molecular mechanisms. Measuring these parameters in tissues is significantly more challenging and requires different reagents (e.g., antibodies) and methodology (electron microscopy) that are not feasible in the current study.

    On the other hand, zebrafish larvae were employed for the behavior studies, which cannot be conducted using cell lines. By utilizing zebrafish, we were able to examine the effects of beta-hydroxybutyrate (BHB) and nicotinamide nucleotide (NMN) on locomotor behavior, providing valuable insights into potential therapeutic implications for autism.

    While we acknowledge the limitations of not directly measuring mitochondrial morphology, peroxisome number, and mitochondrial protein levels in zebrafish, we believe that our study provides significant contributions to understanding the effects of BHB and NMN in zebrafish behavior. Future studies could certainly consider incorporating zebrafish-specific experiments to complement the findings in HeLa cells.

    • How did you choose the concentration of BHB and NMN to use in behavioural experiments? And the timing of application - I don't really understand why you waited 3 days after drug application to measure locomotion. *

    Response: These doses chosen initially as they were similar the doses that induced mitochondrial elongation in HeLa cells and were tolerated by the fish larvae. As we saw promising effects at these initial doses, we decided to explore them in more detail. While we agree that it would be worth comparing the effects of additional doses, as well as looking at their effects at other timepoints, such work would be a major endeavour and is beyond the scope of our initial investigations, which we feel are worth reporting in their current state.

    With respect to the treatment paradigm, fish larvae were treated 10-48 hours post fertilization, as this is a critical neurogenic developmental timepoint that is often used for exposure studies. Fish do not fully hatch until 3-4 days post fertilization, and display only minimal movement before 5 days, which is why we waited until 5 days to look at movement.

    • Do the shank3b+/- larvae show any morphological deficits? Their decrease in locomotion is striking. Is the morphology also rescued by drug application? Can you tie this to the mitochondrial changes that you observed in HeLA cells?*

    Response: We do not observe any gross changes in fish morphology that might explain a decrease in locomotion. Unfortunately, it is not feasible to look at mitochondrial morphology in the fish at this time. However, based on previous published work showing that the ketogenic diet promotes mitochondrial elongation in mouse brains (PMID:32380723), we would expect mitochondrial morphology also to be changed in the fish. Nonetheless, as we have not examined this directly in fish, we are not making this specific claim in this manuscript.

    • In figure 6A you use time spent swimming as a readout of distance. This doesn't really make sense, because without also showing speed of swimming it is not possible to know whether time and distance correlate in the same way across genotypes. This figure could be improved by showing more detail - speed of swimming, time spent immobile etc. This can easily be extracted from the films that you have already made using the ViewPoint software. *

    Response: As requested, we have reanalyzed the zebrafish movement data for a more refined analysis. In the revised version (Fig 6), we include analysis of both speed and distance travelled within a defined time. Importantly, these findings still support differences between WT and shank3b+/- fish that are restored by BHB and NMN to varying degrees.

    • Showing a change in locomotion is not enough to claim that a model is autism-like. At a minimum I think that you need to show changes in social behaviour - likely using older fish (more than three weeks) that interact with each other. Changes in locomotion can be caused by so many factors, many of which are not indicative of autism. It is important that as a field we do not simply claim that locomotion can be used as a proxy for more complex disease phenotypes. This recent review may help you with this point:* https://www.frontiersin.org/articles/10.3389/fnmol.2020.575575/full.

    Response: The reviewer makes an important point that the movement behaviour phenotypes that we see do not necessarily represent classic ASD phenotypes (i.e., repetitive behaviour, reduced sociability, and reduced communication). To begin to address this issue, we analyzed thigmotaxis, which can be a measure of anxiety. Notably, we also see differences that are reversed by BHB and NMN. However, we cannot model all ASD behaviours in a fish model, and we are not set up to look at social behaviour, especially in the young fish that we were studying. As such, even though Shank3 is a recognized ASD gene, and the shank3b+/- model we are studying is a validated ASD model (PMID: 29619162), we have re-phrased the manuscript in the context of neurodevelopment generally, rather than with respect to ASD specifically. As such, we ascribe the movement and thigmotaxis phenotypes as neurodevelopmental phenotypes that are improved by BHB and NMN.

    *For the statistics, as far as I can tell, all of the data should be analysed by ANOVA or the non-parametric equivalent followed by a post-hoc test. Please check this and add information about normality in. *

    Response: As requested, we have clarified our statistical methodology throughout the manuscript.

    For the mechanistic data, we used t-tests for direct comparisons between two groups (e.g., vehicle vs. treatment). While multiple conditions such as vehicles, NMN, BHB, or etomoxir were tested, statistical comparisons were only conducted comparisons between the vehicle and each treatment group individually. As we are not also making comparisons between treatments this is not a multiple comparison, and ANOVA is not applicable in this context. We have clarified this rationale in the manuscript to avoid any confusion.

    For the zebrafish study, where multiple factors were involved (e.g., treatments across different time points or conditions), we performed a two-way ANOVA followed by Tukey's post-hoc test to identify specific group differences. This approach was appropriate for analyzing these datasets and ensures robust conclusion.

    With respect to normality testing, all datasets were assessed for normality using the Shapiro-Wilk test, and no violations of normality were observed. The updated text now includes these details.

    *Minor comments

    1. Make sure that you refer to the fish line as shank3b+/- throughout - see abstract.*

    This has bee corrected.

    • Please add a space between all numbers and units (e.g. 5 Mm). *

    This has bee corrected.

    • There is a spelling error on line 340 page 16: finings instead of findings. *

    This has bee corrected.

    • In figure 1, if each dot represents a different sample, then there appear to be many fewer samples analysed in 1D compared to 1B. Can you comment upon this please*

    __Response: __A total of 80-150 cells were counted per condition, and the analyses were performed on 3 independent replicates with 2 independent technical replicates for each treatment condition. The quantification of mean mitochondrial branch length in Figure 1B was measured using Image-J and the MiNA plugin. The measurements were taken from three independent replicates using a standard region of interest (ROI) and randomly selected areas from each image.

    In Figure 1D, NAD+ levels were measured 24 hours after treatment of vehicle, βHB, NMN, or Eto+βHB in HeLa cells (n=3-6/group). Each sample lysate represents an independent experimental dish from which coverslips were collected for image analysis.

    The difference in sample numbers between Figure 1B and 1D arises because image analysis involves individual cells fixed and stained on coverslips, whereas the NAD assay requires the whole lysate from the entire cell culture dish. Therefore, the higher cell count in Figure 1B represents the number of cells analyzed on coverslips, while Figure 1D represents NAD levels from the lysate normalized to the protein concentration.

    *Reviewer #3 (Significance (Required)):

    I think that this will be interesting to autism researchers and it could lead to more investigation of the ketogenic diet. Some more work is needed, likely in other model organisms, before this research can be translated to human patients. *

    __Response: __We agree that the findings of our study could be of interest to autism researchers and have implications for further investigation of the ketogenic diet (KD). It is important to note that further work, including studies in other model organisms, would be beneficial before translating this research to human patients.

    Our study aimed to provide mechanistic insights into the effects of the KD on mitochondrial morphology and behavior. We recognize that the translation of research findings to human patients requires rigorous investigation, including preclinical and clinical studies. Our study contributes to the understanding of the underlying mechanisms involved in the KD's effects, laying the groundwork for future research and potential therapeutic avenues.

    We appreciate your perspective and emphasize that our intention is to provide valuable insights into the mechanisms underlying the KD's effects rather than suggesting immediate translation to human patients. Further investigation and validation in diverse models and clinical settings will be necessary before considering clinical applications.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    In this paper, Uddin and colleagues have investigated components of the ketogenic diet to understand changes in both mitochondrial morphology and protein expression, and zebrafish locomotor behaviour. They investigate whether beta-hydroxybutyrate (BHB) or nicotinamide nucleotide (NMN) application can later human mitochondria in HeLA cell lines, and also recue a locomotion defect in shank3b+/- zebrafish larvae that have previously been proposed as a model for autism. This study is strengthened by showing data from two species; however the link between the HeLA cell line data and larval zebrafish is not strong. The study would be improved by assessing zebrafish mitochondrial changes after drug application, and testing more than one concentration of BH and NMN in the behavioural assay.

    This is an interesting study, and it is nicely written and presented. I have made some comments to strengthen the study below.

    Major comments

    My expertise is in modelling some aspects of autism in zebrafish. To this end I have focussed on the zebrafish part of this manuscript more fully. I have several comments related to the zebrafish experiments.

    1. The changes in mitochondrial morphology, peroxisome number and mitochondrial protein levels were measured in HeLA cells and not comparable data is shown for zebrafish. The same experiments should be repeated using larval zebrafish or a zebrafish cell line.
    2. How did you choose the concentration of BHB and NMN to use in behavioural experiments? And the timing of application - I don't really understand why you waited 3 days after drug application to measure locomotion.
    3. Do the shank3b+/- larvae show any morphological deficits? Their decrease in locomotion is striking. Is the morphology also rescued by drug application? Can you tie this to the mitochondrial changes that you observed in HeLA cells?
    4. In figure 6A you use time spent swimming as a readout of distance. This doesn't really make sense, because without also showing speed of swimming it is not possible to know whether time and distance correlate in the same way across genotypes. This figure could be improved by showing more detail - speed of swimming, time spent immobile etc. This can easily be extracted from the films that you have already made using the ViewPoint software.
    5. Showing a change in locomotion is not enough to claim that a model is autism-like. At a minimum I think that you need to show changes in social behaviour - likely using older fish (more than three weeks) that interact with each other. Changes in locomotion can be caused by so many factors, many of which are not indicative of autism. It is important that as a field we do not simply claim that locomotion can be used as a proxy for more complex disease phenotypes. This recent review may help you with this point: https://www.frontiersin.org/articles/10.3389/fnmol.2020.575575/full.
    6. For the statistics, as far as I can tell, all of the data should be analysed by ANOVA or the non-parametric equivalent followed by a post-hoc test. Please check this and add information about normality in.

    Minor comments

    1. Make sure that you refer to the fish line as shank3b+/- throughout - see abstract.
    2. Please add a space between all numbers and units (e.g. 5 Mm).
    3. There is a spelling error on line 340 page 16: finings instead of findings.
    4. In figure 1, if each dot represents a different sample, then there appear to be many fewer samples analysed in 1D compared to 1B. Can you comment upon this please?

    Significance

    I think that this will be interesting to autism researchers and it could lead to more investigation of the ketogenic diet. Some more work is needed, likely in other model organisms, before this research can be translated to human patients.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The study examined the effect of beta-hydroxybutyrate and nicotinamide nucleotide on mitochondrial morphology and the molecular pathways which mitigate this effect as well as the effect of these treatments on behavior in zebrafish. The study is well done and well written. The only thing I think that could be improved are the bar in the graph some the significant comparisons. It is sometimes difficult to see which groups are being compared.

    Referees cross-commenting

    The other reviewers do have some fair comments. Multiple doses would be helpful and showing bioenergetic data would complement the morphological measurements. Additionally, behavioral assays showing changes in social behavior in the Zebrafish would provide a stronger link to ASD.

    Significance

    As beta-hydroxybutyrate is an important substrate for the ketogenic diet, this study helps explain the potential mechanisms in which the ketogenic diet may enhance mitochondrial function.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Uddin GM and colleagues presented a research article entitled 'The Ketogenic Diet Metabolite 1 β-Hydroxybutyrate Promotes Mitochondrial Elongation via Deacetylation and Improves Autism-like Behaviour in Zebrafish'. Roles of ketogenic diet (KD) and NAD+ precursors in health promotion and longevity, as well as on the alleviation of a broad range of diseases are evident. However, their roles in autism are not well done, which is the novelty of the current study. Addressing below questions will improve the quality of the paper.

    Major concerns

    1. In the introduction section, a broad overview of the roles of ketogenic diet (KD) in neurodegenerative disease (and ageing, if possible) should be provided. E.g., the authors should summarize exciting progress on the use of KD to treat Alzheimer's disease in animal models (PMID: 23276384).
    2. Roles of high fat diet to treat diseases could be extended to rare premature ageing diseases. In such scenario, high fat and NAD+ boosting shared some joint mechanisms (PMID: 25440059 ).
    3. In the introduction, a more detailed introduction of NAD+ and its roles in mitochondrial homeostasis (especially mitophagy and the mitochondrial fusion-fission balance) should be included (PMID: 24813611; PMID: 30742114; PMID: 31577933).
    4. In regarding to the statement of KD increases NAD+, was it due to increased generation (to check protein levels and activities of different NAD+ synthetic enzymes, such as iNAMPT, NMNAT1-3, and NRK) and/or reduced consumption (in addition to reduced glycolysis, does KD inhibit the activities of CD38 and PARPs? In this paper, Sirtuins' activities is (are increased)). Detailed exploration of the activities of these proteins will unveil a clear molecular mechanisms on how KD affects/regulates NAD+.
    5. Fig. 1: in the NAD+ field, the normal used NR/NMN concentrations are normally high like to use 500 µM to 2-5 mM (as the NAD+ levels in cells are high). In addition to use 50 µM, the authors are strongly to have a dose-dependent study (50 µM, 500µM, 1, 2, 5 mM), and see changes of mitochondrial funciton and parameters. In this condition, NAD+ levels should be also checked.
    6. Fig. 2: a comprehensive characterization of mitochondrial fusion-fission should be performed. In addition to the protein evaluated, changes on other key fusion-fission proteins, like Bax, Bak, Mfn-1, Mfn-2, etc should be performed (PMID: 17035996; PMID: 24813611).
    7. Figs. 1-5 were focused on mitochondrial morphology, whether KD and NMN changed mitochondrial funciton should be explored, such as to use seahorse to check ECR and OCR.
    8. Fig. 6: NR/NMN used in animal studies (via gavage or in drinking water in mice, and on plate for worms and flies) are normally high (e.g., in drinking water for mice could be 4-12 mM; for worms and flies are normally 1-5 mM); for zebra fish, while they are swimming in water, this reviewer concerned whether it was true that 50 µM of NMN was sufficient to show the benefit presented.

    Minor concerns

    1. Line 26: For 'a growing list of neurological disorders, including autism spectrum disorder (ASD)', please add AD in.
    2. Line 57: For 'with side effects such as gastrointestinal disturbances, nausea/vomiting, diarrhea, constipation, and hypertriglyceridemia being reported', rate of frequency shall be provided if any.

    Significance

    The novelty of the current study was to investigate effects of KD and NAD+ on autism. This investigation was not performed before and thus is the novelty.

    Weakness, effects of KD and NAD+/NMN on mitochondrial function were not well-investigated and should be done. Introduction was not well done, many key information in the fields were not provided which may mislead the readers an over-evaluation of the novelty of the current study.

    My expertise lies in NAD+, mitochondria, and brain health.

  5. Version published to 10.1101/2022.10.03.510695 on bioRxiv