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Reply to the reviewers
We would like to thank the editors and the four reviewers for their careful consideration of our manuscript. We are very grateful for their positive appreciation of our work and we believe that their suggestions, which have been included in the preliminary revised version of the manuscript whenever possible, have greatly improved the quality of the paper and have helped us deepen our understanding of the results.
We were happy to note that all the reviewers found value in our work, as stated in their general comments: “This is certainly a useful contribution to our understanding of neuronal V-ATPase functions in vivo” (…)” (Reviewer 1) – “Dulac et al report the very interesting discovery of a previously uncharacterized neuronal specific regulator of the V-ATPase. (…) The experiments are very well performed, the data presented very convincing and the paper is well written.” (Reviewer 2) – “The discovery of a neuronal specific regulator of the V-ATPase is very interesting (…) The work is therefore of great interest to researchers working on synaptic function in general and on synaptic vesicle biology in particular.” (Reviewer 3) – “The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.” (Reviewer 4).
In their remarks, the reviewers suggested additional experiments that could be done to improve our understanding of the role of this new V-ATPase regulator, as well as several minor issues. We have addressed all their comments in our answers below, in which the full text of the reviews is included in blue type, and the responses in black. The line numbers refer to the revised version of the manuscript.
Reviewer #1
Dulac et al. present a first in vivo characterization of the 'accessory' v-ATPase subunit vhaAC45L in Drosophila. The key findings are localization and association of the protein with v-ATPase complexes at synapses and a functional requirement based on lethality and reduced synaptic function. This is certainly a useful contribution to our understanding of neuronal v-ATPase functions in vivo. The main weakness of the study is a lack of depth. The study focuses on localization, co-IP of associated proteins, an analysis of acidification and reduced synaptic function in fly larvae, thus providing a baseline for mechanistic study. However, the mechanism of vhaAC45L is not addressed in this short report. How does is vhaAC45L function different from its homolog vhaAC45? Is it required for v-ATPase assembly? Is it required to localize the full v-ATPase complex (or just V0) to the synapse? Is the defect really due to partial loading of synaptic vesicles or does loss of vhaAC45L also affect endosomal and lysosomal function at synapses? The work as is certainly represents a publishable contribution without answering any of these questions - more as an invite for the community to study the role of vhaAC45L; however, I feel this is a bit of a missed opportunity to put the function of a new potential regulator of specific synaptic v-ATPase functions in the context of the most basic functions obvious in this field.
My main concerns are:
- clearly, vhaAC45L is required for SOME function of v-ATPase in neurons - but it remains entirely unclear which one. It is not even clear what compartments are affected. Reduced quantal size of single vesicle exocytosis events can be a direct or indirect consequence of problems in SV biogenesis and recycling.
Is exo- /endocytosis unaffected? (FM1-43 uptake!).
We agree that alterations in the synaptic vesicle release/recycling cycle could indeed contribute to the locomotion defect, in addition to the acidification impairment observed in VhaAC45L knockdown larvae. As suggested by the reviewer, we plan to carry out FM-dye assays to measure endocytosis and exocytosis at the neuromuscular junction of control versus VhaAC45L-KD animals. If successful, a new figure will be added to the final version of the paper.
What compartments are affected? (markers for synaptic vesicles versus lysosomal compartments!).
Finding out whether VhaAC45L is specifically involved in the acidification of synaptic vesicles, or if it also plays a similar role in other synaptic organelles, in particular lysosomes, would be very interesting indeed. However, we found that it was technically difficult to address this issue in the Drosophila nervous system. A good way would be to check whether the lysosomal pH is affected by VhaAC45L knockdown, as it is the case for synaptic vesicles.
Unfortunately, because lysosomes are not abundant in neurons, lysosome-specific pH-sensitive probes such as Lysotracker do not yield detectable signals at Drosophila larval synapses. So, whether VhaAC45L is specific for synaptic vesicles or involved in the regulation of V-ATPase activity in all neuronal compartments reminas an open question for now.
- molecular function: is vhaAC45L required for v-ATPase assembly? (IP/Pull-downs of v- ATPase complexes in the presence or absence of vhaAC45L with other subunits!).
In accordance with the reviewer, we are also very much eager to learn more about the precise molecular function of VhaAC45L, and in particular whether it is required or not for assembly of the V-ATPase complex. Pull-downs of V-ATPase proteins in controls versus VhaAC45L-KD could be used to address this question, but this would require a large quantity of antibodies directed against subunits of the V0 and V1 domains, respectively. Unfortunately, there are no such antibodies commercially available against Drosophila V-ATPase proteins. We have tried several antibodies that recognize V-ATPase subunits from other species and were predicted to react against Drosophila homologs, but with no success. The only V-ATPase antibodies currently at our disposal were samples generously sent to us by other laboratories in insufficient quantities for carrying out such experiments. To our regret, therefore, we were not able to answer this question until now because of the lack of appropriate tools.
- vha100 was proposed in Drosophila to function on synaptic vesicles and the lysosomal pathway, but, if I remember correctly, here quantal size was normal. I am missing a comparison between the two.
We thank the reviewer for this comment. A comparison with previously published results on subunit Vha100-1 has now been added (lines 458-469) in the discussion related to this topic in the revised manuscript.
- The V5 knock-in is used both as a mutant as well as a tool to analyze protein localization. This is likely okay, but a little concern of course has to be that by creating a mutant protein through stop codon deletion its subcellular localization, turnover, etc. are not normal. Similarly, anti-V5 co-IPs will isolate proteins bound to the mutant variant of vhaAC45L. Minimally, IPs or pull- downs using other members of the V0 complex should be done to understand the role of vhaAC45L in direct comparison with vhaAC45 on complex assembly and possibly targeting to the synapse (or ideally targeting to specific compartments).
It is indeed a legitimate concern to question the physiological relevance of results obtained by studying V5-tagged VhaAC45L. However, the V5 tag is very small (14 amino acids) and we fused it in place of the stop codon to keep intact the whole sequence of the protein. In addition, we found that the V5 knock-in flies are viable and fertile as homozygous. Given that the null mutants, as well as strong RNAi knockdowns, are lethal at early developmental stage, this suggests that the V5 knock-in has limited negative effects, if any, on VhaAC45L function. This led us to believe that at least a good portion of the V5-tagged protein might be targeted to the right subcellular compartment, and associate to its physiological partners.
Significance:
There is significance to the reporting of an accessory v-ATPase subunit required for SOME function of the v-ATPase in neurons. There is some lack of significance in the absence of basic mechanistic insight as to what vhaAC45L does to the v-ATPase in neurons.
We agree that we did not elucidate here the precise molecular mechanisms by which VhaAC45L contributes to synaptic vesicle acidification. It is rather an initial description of a novel neuronal protein that appears to be essential for proper synaptic functioning, and we provide consistent evidence that its function requires specific interaction with the V-ATPase complex, and in particular with three subunits that reproducibly co-immunoprecipitated with VhaAC45L (namely Vha1C39-1, Vha100-1 and ATP6AP2). Please note that it took many years and many papers before the molecular mechanisms of action of comparable accessory subunits, such as ATP6AP1/AC45 or ATP6AP2, was better understood, and it is still nowadays a matter of investigation. It is therefore very demanding to expect that we describe the exact function of the previously uncharacterized VhaAC45L at all levels in a single first paper.
Reviewer #2
In this study and using Drosophila melanogaster as a model system, Dulac et al report the very interesting discovery of a previously uncharacterized neuronal specific regulator of the V-ATPase called VhaAC45L. They combine genetics, IHC, Mass spec and ephys to unravel the expression pattern and function of this protein. They find that it is required to acidify synaptic vesicles in glutamatergic neurons of the Drosophila larval neuromuscular junction, for appropriate synaptic transmission and for larval locomotion. The experiments are very well performed, the data presented very convincing and the paper is well written. Nonetheless, a few additional pieces of evidence and some level of expanded analysis would strengthen the conclusions and increase the depth of the work.
Major comments:
- Figure 1F: the while the localization to the presynaptic terminal is convincing, where exactly the protein is localized to is not studied. The imaging in these experiments could use increased resolution and concomitantly colocalization studies with more specific synaptic vesicle markers.
We agree that it would be very good to show this additional result. However, confocal microscopy does not provide sufficient resolution to localize the protein at the membrane of individual synaptic vesicles. Another way would be to see if VhaAC45L immunostaining co- localizes with domains enriched in synaptic vesicle markers, but these organelles are rather ubiquitously distributed in the synaptic boutons at the Drosophila neuromuscular junction. To correctly perform this experiment, we would have to do immuno-electron microscopy, a technique we do not master in our laboratory and that we did not plan to implement for the present work.
- Figure 3B-G: these experiments should be complemented by a rescue experiment, ideally of the null mutant using a UAS construct and a pan neuronal driver, or - if such animals are viable to the third larval instar stage - a glutamatergic driver. If possible, it would also be good to study the NMJ phenotype of the null mutant rescued to viability using a neuronal driver that does not express in motor neurons (e.g. Chat-G4).
Although a rescue experiment could potentially add a further evidence that Vha45ACL deficiency is responsible for the synaptic vesicle acidification defect described in Figure 3, we don’t think that it is a requisite here because we obtained similar results by knocking down the gene using two different RNAis. As described in the manuscript, the pan-neuronal expression of Vha45ACL could rescue the embryonic lethality of the null mutant, so it would be theoretically possible to check the acidity level of synaptic vesicles at the neuromuscular junction of the recued larvae. However, this would involve making rather complex genetic constructions to express VMAT-pHluorin in motor neurons in rescued mutant background. In addition, the conclusions we could draw from such experiment would be limited by the lack of comparison. Indeed, in Figure 3 the defect was observed in knockdown context, and the same experiment could not be performed in knockout larvae due to the early lethality. If we could measure the acidity level of rescued null mutants, we would not have any comparison point besides the knockdown experiments. As knockout and knockdown are not likely to produce identical phenotype (especially in terms of magnitude of effect), the ideal would be to compare the rescued phenotype to the null mutant expressing VhaAC45L in all neurons except motoneurons, as suggested by the reviewer. However, such genotype would certainly not be viable, since we observed that expression of VhaAC45L RNAis with a stronger motoneurons driver (D42-Gal4) was sufficient to induce lethality at early developmental stage.
- Figure 5: the authors focus on quantal size which measures the postsynaptic response to spontaneous release from the presynaptic terminal. However, it is unclear how this directly relates to the locomotor deficit beyond signaling potential deficits in vesicle loading or fusion. It would be more convincing to also study evoked release, and expand the analysis of presynaptic properties (number of events, amplitude, frequency).
We fully agree with this comment shared by Reviewers 2 and 3 related to the electrophysiology experiments. Note that these experiments have been carried out in collaboration with another laboratory located in another city. The Covid-19 situation during the past year has prevented, and is still complicating, movements between labs, preventing us from going further with the electrophysiology analyses of VhaAC45L KD. If the situation in the near future allows it, we would very much like to add a more extensive electrophysiological analysis, including in particular the study of evoked release. In the revised manuscript, we have nevertheless completed Figure 5 by adding representative distributions of spontaneous mEPSP amplitudes in control and VhaAC45L knockdown larvae, as well as the results of new analyses showing lack of effects the KD on the mean EPSP frequency.
- General: showing some level of genetic interaction with V-ATPase subunits in at least some of the assays would be welcome.
We are definitely in accordance with the reviewer on that point, but we think that this would involve a lot of work and be beyond the scope of the present initial description. Here we show by proteomic analyses that at least 12 proteins co-precipitate and so potentially interact with VhaAC45L, three of them being previously identified constitutive or accessory V-ATPase subunits. In our opinion, studying the interactions between VhaAC45L and these proteins through genetic and molecular studies will be the subject of future works. As stated by Reviewer 2 in the Referees cross commenting below: “further biochemical analysis is interesting but probably beyond the scope of this initial description and would take too much time”. We fully agree with this statement.
Minor comments:
Some of the images, especially those in Figure 3, should be larger for ease of visualization.
As requested, the images of Figure 3 have been enlarged.
Significance
The discovery of a neuronal specific regulator of the V-ATPase is very interesting. To my knowledge it is the first description of a neuronal specific V-ATPase related protein since the description of Vha100-1 by Hiesinger and colleagues in 2005. The work is therefore of great interest to researchers working on synaptic function in general and on synaptic vesicle biology in particular.
We are grateful to the reviewer for his very positive assessment of our work.
I note that I do not have in depth expertise in electrophysiology, although I am sufficiently familiar with basic NMJ physiology experiments to render the opinions stated above.
Reviewer #3
In this study, Dulac and colleagues investigated roles of VhaAC45-like gene, which codes one of the V-ATPase accessory proteins in Drosophila, in synaptic transmission. First, they demonstrated that VhaZC45L transcripts are expressed selectively in neurons and that the gene products are addressed to synaptic areas. Second, they showed that VhaAC45L is co- immunoprecipitated with some subunits of V-ATPases, which is consistent with bio-informatics predictions. They further demonstrated that VhaAC45L-knockdown (KD) resulted in defects in synaptic vesicle acidification as well as a reduction in quantal size of glutamate, indicating that VhaAC45L play a key role in regulating neurotransmitter release by modulating the driving force for transmitter uptake. Last, not least, they demonstrated that VhaAC45L-KD in motoneurons attenuated larvae locomotor performance, indicating its physiological relevance. Overall, this study is rigorously executed and nicely presented, and adds one more component of the V- ATPase that is responsible for neurotransmitter uptake into synaptic vesicles. However, since this study simply confirmed an established notion from other species such as yeast and mammals that AC45 is one of the accessory proteins of the V-ATPase complex, a conceptual novelty beyond the previous knowledge is relatively poor in its present form. Thus, this reviewer would suggest several issues as following to improve the comprehensiveness as well as novelty of the current manuscript.
- The reason why the authors focused on VhaAC45-'like' is somewhat obscure, and therefore should be explained. How different VhaAC45 and VhaAC45L are in terms of amino acid sequences, tissue distributions, and KO phenotypes. It seems more comprehensive if the authors provide some experimental evidence on VhaAC45; e.g. whether it is also expressed in neurons or not (Fig. 1), and, if VhaAC45 is neuronal, whether it can rescue the phenotypes of VhaAC45L- KD to certain degree (Figs 4 & 5).
Following the reviewer’s request, we have added a sequence alignment of VhaAC45 and VhaAC45L, as well as a graph showing tissue distributions of both genes in Supplementary Figure 1 of the revised manuscript. To our knowledge, there is no published functional study of VhaAC45 in Drosophila, so we can only make assumptions derived from studies on predicted homologs in evolutionarily distant species. For that reason, it is difficult to compare VhaAC45 to VhaAC45L, as it would first require an entire new study of VhaAC45 function in flies. Since our interest is to study neuronal physiology, we focused on VhaAC45L because compelling evidence indicates that this subunit is specific to the nervous system, as described in our manuscript, rather than on VhaAC45 which seems to be expressed in all tissues. In addition, homologs of VhaAC45L have never been functionally characterized to date in any species, making it very interesting to study this new protein in a genetically tractable organism.
- What is the mechanism of Ac45 in regulating V-ATPase activity? In mammals, it has been suggested that Ac45 is essential for proper sorting of the V-ATPase to the destined organelles (e.g. Jansen et al., Mol. Biol. Cell., 2010; Jansen et al., BBA, 2008). In this context, it should be examined whether VhaAC45L-KD would affect the synaptic localization of other V-ATPase subunits.
We thank the reviewer for pointing out these very interesting references. We have indeed tried to determine the relative abundance of two other V-ATPase subunits at the larval neuromuscular junction in control and VhaAC45L knockdown contexts. However, because the tested subunits are not specific to neurons, and are expressed at relatively low levels in synapses, it was not possible for us to properly separate the synaptic signal from the background immunostaining in surrounding muscles. This unfortunately prevented us from performing an accurate and reliable quantification.
- Given that a rodent brain SV contains a few copies of the V-ATPase on average (Takamori et al., 2006, and some newer papers by others), it is interesting that >80% reduction of Ac45 showed moderate effects on quantal size. If SVs under study also contains 1 or 2 V-ATPase per SV, there must be some SVs lacking VhAC45L upon KD. In this context, it is interesting to see how VhaAC-KD (RNAi1~3) affect the frequencies of minis.
The reviewer’s valuable comment prompted us to undertake new analyses on our electrophysiological recordings. We have now added in Figure 5E graphs showing the mean EPSP frequency for larvae expressing VhAC45L RNAi1 and RNAi2, which are the ones that were used in the quantal analysis. Both of these RNAi apparently decreased the frequency compared to controls, but this difference was not statistically significant. As detailed in the Discussion (line 458-469), this may suggest that VhaAC45L does not influence the abundance of the V-ATPase complex at nerve terminals, but rather its efficiency.
- In general, decrease in mini amplitudes is accounted for by changes in postsynaptic sensitivity for neurotransmitters. Although acidification deficits would support that decrease in quantal size is due to the decrease in the driving force for glutamate uptake, it should be examined whether the postsynaptic receptor fields are not affected by VhaAC45L-KD by recording postsynaptic response upon application of non-saturable concentrations of glutamate.
Testing for potential postsynaptic receptor field alteration by glutamate application would be an interesting experiment indeed, but, as we believe, not a critical control for the present manuscript. Because we expressed RNAis presynaptically, any modification in the postsynaptic receptor field would have to be an indirect consequence of VhaAC45L downregulation in motoneurons, and so, likely to be related to the synaptic vesicle acidification defect. It would not change, therefore, our conclusion that VhaAC45L deficiency in motoneurons induces a decrease in quantal size. Because electrophysiology experiments were carried out in collaboration with another laboratory located in another city, the current sanitary context has so far prevented us from performing this test (please refer to our answer to comment 3 of Reviewer 2 for more details).
- Related to 4, it is also interesting to see if evoked responses are also attenuated as a result of VhaAC45L-KD, which is more physiologically relevant for locomotor activity phenotype than minis.
We also agree with this comment, shared by Reviewer 2, to which we already responded above in our answer to comment 3 of Reviewer 2.
Minor points:
- Quantal size of glutamate is not affected by reduced expression of DVGLUT (Daniels et al., Neuron, 2006), which highly contrasts with VhaAC45L, expression of which defines quantal size. Distinct regulation of quantal size by the transporter and the V-ATPase subunit should be discussed.
As suggested by the reviewer, a discussion of this point has been added (lines 458-469). and Daniels et al. 2006 is now cited in the revised manuscript.
- For electrophysiological experiments, respective sample traces should be shown in Figure 5.
Quantal size is not directly visible in sample traces, so we added instead representative distributions of spontaneous mEPSP amplitudes in control and VhaAC45L knockdown larvae in the new Figure 5C.
- <![endif]>Only RNAi1 and RNAi2 lines were examined for SV pH estimation and mini analysis. The results from RNAi3 should be presented, or at least mentioned in the text.
These experiments were performed using two different RNAi constructs to ensure that similar effects were observed and to exclude the possibility of potential off-targets. Knocking down VhaAC45L in neurons with RNAi1 and 2 was lethal at pupal stages, suggesting that they give similar levels of inactivation. RNAi3 systematically induced lighter phenotypes, producing viable adults, which led us to believe it had a lower efficiency. Because the results on synaptic vesicle acidification and electrophysiology were very consistent with RNAi1 and RNAi2, we considered that it was not necessary to repeat the experiment with RNAi3.
Significance
As mentioned above, as it stands, the authors merely confirmed the pre-existing bioinformatic knowledge on one of the AC45 homologues in Drosophila. The audience of The EMBO Journal might be interested in how different/similar VhaAC45 and VhaAC45-like are, and their functional relevance. In particular, is VhaAC45 also mandatory for the V-ATPase functioning in neurons? Adding some basic information of VhaAC45, e.g. tissue distribution, KO phenotypes, and ability to rescue the VhaAC45-like-KD phenotypes, will certainly improve the comprehensiveness of this study, and capture audience's attention.
As mentioned in our response to point 1 of the reviewer above, we have added more data comparing the structure and distribution of VhaAC45 and VhaAC45L in the revised manuscript. VhaAC45 appears to be ubiquitously expressed whereas VhaAC45L is neuron-specific.
Comparing VhaAC45 to VhaAC45L would require a completely new study of VhaAC45 function, because it has never been done before in Drosophila to our knowledge. This would require repeating all the experiments with this other gene, probably involving two more years of work, and would make for a much longer and very different manuscript. It is understandable that this cannot be envisaged. Because homologs of VhaAC45L have never been functionally characterized to date in any species, we considered that it was worth studying this new protein on its own.
Reviewer #4
We have reviewed "A specific regulator of neuronal V-ATPase in Drosophila melanogaster." by Dulac et al. The authors have identified VhaAC45L as a regulator of neuronal V-ATPase in Drosophila melanogaster. The authors have utilized multiple techniques to determine the localization of VhaAC45L in neurons and specifically in the synapse. The use of multiple approaches including determining RNA levels in different regions of the fly, and using CRISPR- Cas9 technique to insert V5 tag, makes a very convincing argument about the synapse-specific expression of VhaAC45L.
The combined use of co-immunoprecipitation technique and LC/MS to show that VhaAC45L co- precipitated with V-ATPase complex subunits is convincing that VhaAC45L is a subunit of V- ATPase. To determine the role of VhaAC45L in acidification of synaptic vesicles the authors have utilized pHluorins in combination with multiple RNAi lines. The authors have used a well- designed experiment to prove that VhaAC45L regulates acidification of the synaptic vesicles.
Further, larval locomotion and quantal size determination using VhaAC45LRNAi which is known to be altered due to pH gradient of synaptic vesicles shows the functional role of VhaAC45L in synaptic vesicle acidification.
Minor comments:
- For all graphs, please remove gridlines to make data points more visible.
We found that gridlines can be helpful for the readers to assess approximate values on the graphs. So, we have not removed them but rather changed the colour to a light grey so it does not affect any more visibility. We have also placed the points over the error bars in all the graphs, so they become more apparent.
- Line 120-123: Authors indicate the VhaAC45LRNAi induced lethal phenotype when expressed in glutamatergic and cholinergic drivers but the figure is missing. Please indicate as "data not shown" if not included in Figure.
This mention has been added in the manuscript (line 125).
- A diagram summarizing the role of VhaAC45L in V-ATPase enzymatic complex and specific role is recommended.
We believe that it is too early in this first report to draw an accurate diagram summarizing the role of this new protein in the V-ATPase complex.
Significance
V-ATPase play a crucial role at the synapse by being responsible for acidification of the synaptic vesicles and identification of a synaptic vesicle specific regulator of V-ATPase is important to understand the complex regulation of synapse function. The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.
We thank the reviewer for his very positive appreciation of our work.
Referees cross commenting
(Written by Reviewer 2)
There seems to be overall consensus among the reviewers on 3 issues:
- A somewhat more precise understanding of the role of vhaAC45L in the synaptic vesicle cycle through better localization studies and some classic assays (like FM dye uptake).
—See our answers to comments 1 of Reviewer 1 and Reviewer 2.
- A little more characterization of the transmission defects (e.g. studying evoked responses) would be welcome.
—See our answers comment 3 of Reviewer 2.
- Ascertaining the validity of the alleles with rescue experiments, perhaps in the V5 mutant background to allow localization analysis in a rescued background.
—See our answers to comment 2 of Reviewer 2.
I think further biochemical analysis is interesting but probably beyond the scope of this initial description and would take too much time.
We fully agree with this statement.
The minor issues are easy to address
We have addressed all of them in the preliminary revised version of the manuscript.