Rescuable sleep and synaptogenesis phenotypes in a Drosophila model of O-GlcNAc transferase intellectual disability

  1. Ignacy Czajewski
  2. Bijayalaxmi Swain
  3. Jiawei Xu
  4. Laurin McDowall
  5. Andrew T Ferenbach
  6. Daan MF van Aalten

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

O-GlcNAcylation is an essential intracellular protein modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Recently, missense mutations in OGT have been linked to intellectual disability, indicating that this modification is important for the development and functioning of the nervous system. However, the processes that are most sensitive to perturbations in O-GlcNAcylation remain to be identified. Here, we uncover quantifiable phenotypes in the fruit fly Drosophila melanogaster carrying a patient-derived OGT mutation in the catalytic domain. Hypo-O-GlcNAcylation leads to defects in synaptogenesis and reduced sleep stability. Both these phenotypes can be partially rescued by genetically or chemically targeting OGA, suggesting that a balance of OGT/OGA activity is required for normal neuronal development and function.

Article activity feed

  1. Version published to 10.7554/elife.90376 on eLife
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    1. General Statements

    We would like to thank the reviewers for their careful reading of our manuscript and constructive comments.

    2. Description of the planned revisions

    Reviewer 1) “Indeed, the manuscript describe the alteration of total brain O-GlcNAc levels, but understanding pathways or protein specific changes would allow to identify the mechanisms potentially at the basis of the development of intellectual disability.”

    Response: While finding the pathways involved in phenotypes described here is beyond the scope of the present manuscript, we plan to include RNAi experiments elucidating cell types responsible for the sleep phenotype observed in sxc mutant flies.

    Reviewer 3) “2) Lacing fly food with compounds can sometimes lead to phenotypes not actually caused by the drug. There are reports I have previously seen where the compound can make the food more aversive or attractive, both leading to results not due to the drug. Specifically, it has been previously reported that starved flies (if the compound leads to aversion from the food and causes starvation) will reduce the bouts of sleep in Drosophila ( Masek et al J Exp Biol 2014; Figure 4). Do the authors know if the TMG treated food eaten at the same level as normal food? Is there the potential for a starvation phenotype?”

    Response: We appreciate this insight and we plan to perform this control experiment. Briefly, this will entail measuring male adult ingestion of Thiamet G laced food by adding Blue No. 1 dye and measuring absorbance of lysed flies, as previously described in Wong et al. 2009 (PMID: 19557170).

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer 1) “In figure 1C the blot show a different MW range compared to blots 1A and 1B, author should correct. “ and “For figure 1 and 2 the dot graph are too small and difficult to read”

    Response: The figures have been amended to address this.

    Reviewer 3) “In the methods - Neuromuscular Junction Immunohistochemistry - which muscles and which types of boutons were imaged was not denoted in this section - it is described in results (lines 210-211) but should be in methods for ease to the reader.”

    Response: The methods section has been amended.

    “The statistics and data analyses are some of the best I have seen to date. One concern is the removal of a single outlier data point described at line 575. Was this necessary? Does it change the data? If not, I would recommend leaving it in. If it does, I would further recommend additionally biasing toward the alternative hypothesis by additionally removing the data point that lies furthest from the outlier. This would reduce bias.”

    Response: Removal of an outlier does indeed change the results of the data. Following the suggestions of the reviewer, we re-analyzed our data removing the minimum for the group for which we previously removed an outlier (the maximum).

    “1) line 391 mentions that feeding higher doses of TMG results in a non-rescue phenotype. Is there any data to support this statement (maybe supplementally) to give the reader the full picture of the availability of this compound? For example, how far above 250 uM does this happen?”

    Response: This statement refers to adult Thiamet G feeding experiments, and the data to support this statement can be found in figures 2B and S2A. This statement has been amended for clarity and to include the caveat that even higher doses of TMG were not trialed.

    4. Description of analyses that authors prefer not to carry out

    Reviewer 1) “Authors employed RL2 antibody for O-GlcNac detection, however it recognized mainly high MW proteins and it would be nice to obtain the alteration profile of low MW proteins at the same conditions.”

    Response: We agree that the use of a single method for detecting O-GlcNAcylation is limited, however, there is no reason to believe that immunoblotting using this antibody would bias the interpretation of the effects of mutations studied here on global O-GlcNAcylation. Specifically, there is no reason to believe low molecular weight proteins are recognized and modified by OGT differently to high molecular weight proteins. While gaining insight into substrate specific alterations in O-GlcNAcylation is of great interest to us, this is technically very challenging and beyond the scope of this study.

    Reviewer 2) “… would it be possible for the authors to overexpress specifically in neurons wildtype OGT postnatally on a mutant background and quantify the effects on neuro-muscular synapse number and morphology? It would be interesting to compare these data with a similar experiment where they overexpress wildtype OGT in the corresponding muscle.”

    Response: While temporal control of transgene expression is possible in Drosophila, it is not a technique that we routinely use and would require extensive optimization to include in the present manuscript.

    _Reviewer 3) “In Figure 3D the authors show sxcWT compared with OgaKO with no significant difference at ~20 boutons in the count. Other work done by [47] in their reference list (ref 47: Figure 2D) shows an increase in OgaKO boutons vs WT and also shown in [50] (ref 50; Figure 4B) where # of boutons in 1B muscle 4 is increased in OgaKO significantly. There appears to be a difference in what was found with OgaKO vs controls in the authors' results vs these two manuscripts and it should be noted and explained to the reader.” _

    Response: This is indeed an inconsistency we have observed, however, looking at reference Fenckova et al. 2022 (47 in our manuscript) we find that in figure legend 2 the following is stated: “None of the parameters is significantly affected in the OgaKO larvae (N = 30, in purple; OgaKO experiments were performed simultaneously and first published here [53] with significantly increased bouton counts (p <0.05) without multiple testing correction)” Reference [53] in the quote refers to Muha et al. 2020 (reference 50 in our manuscript). Therefore, it appears that this effect is too weak to withstand multiple correction testing, which we employ in our analysis.

  3. 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 #3

    Evidence, reproducibility and clarity

    The manuscript written by Czajewski et al. "Rescuable sleep and synaptogenesis phenotypes in a Drosophila model of O-GlcNAc transferase intellectual disability" is a novel approach to examining genetic missense mutations representing a patient derived OGT mutation in quantifiable phenotypes coupled with genetic and pharmacological manipulation. The authors find novel contradictory results in synaptic bouton parameters than previous work leading to increased interest in these results. The authors also use pharmacological intervention to reverse the phenotypes derived from the OGT mutations creating an interesting path forward for these types of studies. The manuscript was well written, the experiments are sounds, and the analyses are extremely well done. The manuscript would benefit from addressing a few concerns:

    Minor:

    In the methods - Neuromuscular Junction Immunohistochemistry - which muscles and which types of boutons were imaged was not denoted in this section - it is described in results (lines 210-211) but should be in methods for ease to the reader.

    The statistics and data analyses are some of the best I have seen to date. One concern is the removal of a single outlier data point described at line 575. Was this necessary? Does it change the data? If not, I would recommend leaving it in. If it does, I would further recommend additionally biasing toward the alternative hypothesis by additionally removing the data point that lies furthest from the outlier. This would reduce bias.

    Major:

    In Figure 3D the authors show sxcWT compared with OgaKO with no significant difference at ~20 boutons in the count. Other work done by [47] in their reference list (ref 47: Figure 2D) shows an increase in OgaKO boutons vs WT and also shown in [50] (ref 50; Figure 4B) where # of boutons in 1B muscle 4 is increased in OgaKO significantly. There appears to be a difference in what was found with OgaKO vs controls in the authors' results vs these two manuscripts and it should be noted and explained to the reader.

    The results working with Thiamet G (TMG) is very interesting and needs a bit more clarification. I tried to find other research where TMG is fed to Drosophila, and could not find this, and I suspect this is novel and very interesting, especially as a tool. However, I do have concerns about the details for this feeding and would like to further understand a few things that came up in the manuscript that need to be addressed:

    1. line 391 mentions that feeding higher doses of TMG results in a non-rescue phenotype. Is there any data to support this statement (maybe supplementally) to give the reader the full picture of the availability of this compound? For example, how far above 250 uM does this happen?
    2. Lacing fly food with compounds can sometimes lead to phenotypes not actually caused by the drug. There are reports I have previously seen where the compound can make the food more aversive or attractive, both leading to results not due to the drug. Specifically, it has been previously reported that starved flies (if the compound leads to aversion from the food and causes starvation) will reduce the bouts of sleep in Drosophila ( Masek et al J Exp Biol 2014; Figure 4). Do the authors know if the TMG treated food eaten at the same level as normal food? Is there the potential for a starvation phenotype?

    Significance

    There is a novel technique in this manuscript that could enhance OGT research in Drosophila, which is significant.

  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 #2

    Evidence, reproducibility and clarity

    The authors of this manuscript describe the effect on neuronal development and function in drosophila of OGT mutations derived from patients with intellectual disability. OGT is an enzyme that adds the posttranslational modification O-GlcNAc to proteins. Once added, O-GlcNAc can be removed by the enzyme OGA. O-GlcNAc cycling on and off proteins has been associated not only with intellectual disability but a range of other brain-dependent disorders. However, molecular mechanisms by which O-GlcNAc cycling may affect brain development and function are largely unclear. It is equally unclear whether and how disorders associated with OGT mutations may be treated. This manuscript presents evidence that it is possible to rescue neurological phenotypes dependent on patient-derived OGT mutations using genetic or pharmacological manipulations of OGA. These results strongly suggest that at least some aspects of the phenotype of patient-derived OGT mutations depend on O-GlcNAc cycling rather than other mechanisms. Excitingly, they also suggest that it may be possible to treat patients suffering from OGT mutations with drugs that target OGA after the baby has been born. This last point is critical not only for the field of OGT-associated disorders but for the whole field of intellectual disability.

    Minor comment:

    While the manuscript delivers its message clearly with a simple and concise language, the manuscript would become even stronger if the observation that some aspects of intellectual disability can be treated postnatally is substantiated with additional methods that are more specific. The current data are also somewhat difficult to interpret because the pharmacological and genetic manipulations of OGA used so far may not be a direct rescue of the OGT mutations, which the authors also point out. For example, would it be possible for the authors to overexpress specifically in neurons wildtype OGT postnatally on a mutant background and quantify the effects on neuro-muscular synapse number and morphology? It would be interesting to compare these data with a similar experiment where they overexpress wildtype OGT in the corresponding muscle. These experiments would both strengthen their finding that it is possible to rescue neurodevelopmental conditions postnatally and give further evidence to the molecular mechanism by which OGT affects neurodevelopment.

    Significance

    In summary, while it is a short manuscript and on a topic studied previously, its data are novel, clearly presented and would appeal to researchers within and outside the field of O-GlcNAc. It is ready for publication as it has been submitted but including more experiments along the lines suggested above would help it reach one level higher.

  5. 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

    The manuscript from Czajewski and colleagues demonstrates that patients-derived OGT mutation can lead to reduced O-GlcNAc levels, which can be rescued by genetic OGA ablation or pharmacological OGA inhibition. Several studies in the last decade demonstrated that O-GlcNac homeostasis is crucial for brain development and function and that its alteration is deeply involved in neurodegeneration and cognitive decline. Therefore rescuing protein O-GlcNAcylation by targeting OGT/OGA cycling could represent a valuable therapeutic approach for intellectual disability. Results obtained by the authors are very promising and support the notion of a mutual interplay between OGT and OGA in regulating brain O-GlcNAc levels. Furthermore, the partial rescue of synaptogenesis and sleep stability support the efficacy of OGA reduction in rescuing O-GlcNAc levels.
    I do not find Major flaws in the manuscript structure or in the experimental approach, however I believe that the manuscript would benefit of the analysis of the molecular target that lead to brain defects under OGT mutation and that are rescued after OGA inhibition. Indeed, the manuscript describe the alteration of total brain O-GlcNAc levels, but understanding pathways or protein specific changes would allow to identify the mechanisms potentially at the basis of the development of intellectual disability . Furthermore, it would be also interesting to understand if the mutation of OGT has direct or indirect effects on Ser/Thr phosphorylation levels.

    Minor comments:

    Authors employed RL2 antibody for O-GlcNac detection, however it recognized mainly high MW proteins and it would be nice to obtain the alteration profile of low MW proteins at the same conditions.

    In figure 1C the blot show a different MW range compared to blots 1A and 1B, author should correct.

    For figure 1 and 2 the dot graph are too small and difficult to read

    Significance

    General assessment: I believe that the study is well executed and interesting since it nicely demonstrate the influence of OGT mutation on O-GlcNAc levels and the efficacy of OGA reduction in rescuing the process and in improving synaptogenesis and sleep stability. However, I also believe that a better understanding of the molecular mechanisms involved could substantially improve the study.

    Advance: the present study provide further knowledge about the physio/pathological role of OGT/OGA cycling in the brain.

    Audience: basic researchers

  6. Version published to 10.1101/2023.06.28.546900v1 on bioRxiv