A monocarboxylate transporter rescues frontotemporal dementia and Alzheimer’s disease models

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   Review Commons

   Jun 28, 2023

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   Reply to the reviewers

   We thank the reviewers for their comments and insights, we feel the manuscript is now greatly improved. Please find below our answers to the reviewer’s queries

   Reviewer #1 (Evidence, reproducibility and clarity):

   The manuscript by Niccoli et al. describes the identification of a novel modifier of C9orf72-derived toxicity based on the manipulation of the brain metabolic pathways. The premise for this work is supported by strong literature describing the aberrant glucose metabolism in FTD, AD and other degenerative disorders. The idea tested here is whether increasing the import of pyruvate produced in glia into neurons. They test three different types of …

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

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   Reply to the reviewers

   We thank the reviewers for their comments and insights, we feel the manuscript is now greatly improved. Please find below our answers to the reviewer’s queries

   Reviewer #1 (Evidence, reproducibility and clarity):

   The manuscript by Niccoli et al. describes the identification of a novel modifier of C9orf72-derived toxicity based on the manipulation of the brain metabolic pathways. The premise for this work is supported by strong literature describing the aberrant glucose metabolism in FTD, AD and other degenerative disorders. The idea tested here is whether increasing the import of pyruvate produced in glia into neurons. They test three different types of importers and find that one of them, Bumpel, the orthologue of human SLC5A12, suppresses toxicity and reduces the accumulation of arginine-containing repeats, GP and PR. The authors investigate several potential mechanisms mediating this reduction of toxic DPRs, but do not find strong evidence linking pyruvate import and increase autophagy or mitochondria metabolism.

   Overall, this is an interesting discovery based on a candidate approach that shows the power of Drosophila to efficiently identify novel mediators of neurodegeneration. The article is well written, although more detailed explanations of some experiments would be helpful. The weaknesses of the manuscript are the lack of a clear mechanism mediating the protective activity of pyruvate, the incomplete experiments lacking relevant controls, and the presentation of western blots.

   Specific comments:

   1. The reduced levels of DPRs require that the expression of C9 mRNA or the GR and PR constructs is examined by qPCR. In figure 3E, GP is not even detectable_

   We agree with the reviewer, ideally we would have measured the RNA by qPCR. However, the C9 repeats and the DPR constructs are highly repetitive, it is therefore impossible to do a qPCR for them. The upstream and downstream sequence is identical for the C9 and the bumpel constructs, there isn’t, to our knowledge any unique sequence we can use to measure levels of expression in the presence of bumpel.

   We did run a GFP control (Fig 2D) and did not see any difference and we have now carried out a qPCR for Gal4-GeneSwitch (Fig S3) to show that the levels of the driver do not change.

   1. I wonder if there are constructs available to silence Bumpel or overexpress the human orthologues of bumpel. These would be nice controls for the effects observed with the Bumpel overexpression

   This would be an extremely interesting experiment, however bumpel is normally only expressed in glia, therefore we can’t down-regulated it in glia whilst upregulating 36R in neurons, as we are limited to one driver (since everything is driven by the Gal4/UAS system). Expression of C9 in glia does not have a clear phenotype (our observation), so we can’t drive both in glia. We tried over-expressing the human homologue SLC5A12 , but it did not rescue the C9 phenotype (data not shown), possibly because it requires (like other human SLC5A type transporters) PDZK1 as extra co-factor (Srivastava S. et al, 2019), and this is not present in flies.

   1. The argument about bumpel modulating autophagy downstream of Atg1 is not supported by the experimental data

   We now have imaging data showing that bumpel modulates the formation of lysosomes, downstream of Atg1 (Fig 5). We also show that bumpel and Atg1 can act synergistically, leading to a much stronger rescue of C9 expression (See Fig 5I.), which also suggests that the two are acting at different points in the same pathway. We also show that bumpel rescues the downregulation of TFEB targets (Fig 5J)

   1. Western blots throughout show no control lanes and in several occasions are created with cutout bands. The standard for this type of experiments should be more stringent, with entire gels showing all experimental conditions, which requires consistent methods and results vs selecting the best bands from different gels.

   We apologise if this was mis-understood, the lanes shows are all from the same blot, where other samples were run too, and it would be confusing for the reader to include them. We have re-run samples where we had remaining sample from our quantifications, so that the lanes are now contiguous and we provide original blot images in the supplemental information for those we could not re-run. The control for all experiments are the C9 expressing line without bumpel, and this is always present, if the reviewer means we are missing -RU controls, these do not produce any DPRs so are not included in western blot or ELISA quantifications as the signal is not above back-ground.

   1. For figures 2B and 5C, please, show representative WBs

   These are ELISA quantifications, not western blots, we choose to run these when possible, as they are more quantitative.

   1. Figure 5D describes the survival curve as significantly rescued. Statistical tests can indicate differences, but that is in no way convincing. The test may show the curves are different, but the abeta Atg1 flies also seem to start falling early, so an argument could be made in both directions, as a suppressor or an enhancer.

   We agree the rescue is not strong enough, we have now removed this lifespan.

   1. It is unclear why several results are placed in the supplemental materials. In general, all this material seems highly relevant and related to what is shown in the main figures

   We are happy to include them in the main manuscript if this would help the reader, and we have now placed all mitochondrial data in Fig 4.

   Minor comments:

   Please, define several abbreviations throughout

   We apologise for this over-sight, we have now does this.

   A couple of sections could be improved by carefully sequencing human vs Drosophila background to advance the argument rather than going in circles. There is also a section on mitophagy in between two sections related to autophagy that could be sequenced better.

   We have re-structured the sections, we think this has improved the flow.

   There is a sentence at the end of page 6 that seems misplaced

   We apologise for the over-sight, and we have removed this

   Reviewer #1 (Significance):

   Overall, this is an interesting discovery based on a candidate approach that shows the power of Drosophila to efficiently identify novel mediators of neurodegeneration. The article is well written, although more detailed explanations of some experiments would be helpful. The weaknesses of the manuscript are the lack of a clear mechanism mediating the protective activity of pyruvate, the incomplete experiments lacking relevant controls, and the presentation of western blots.

   We thank the reviewer for the helpful comments, we have added some details in the methods section, we apologise for not having made it clear that the westerns were all derived from the same blot (we have now placed the originals in the supplemental materials). Regarding mechanism, we now show that bumpel over-expression increases clearance of late stage autolysosomes, possibly by increasing transcription of TFEB target lysosomal genes.

   Reviewer #2 (Evidence, reproducibility and clarity):

   Summary:
   Project investigates the role in dementias of glial glucose uptake, conversion to lactate and shuttling via transporters to neurons to produce pyruvate to fuel TCA cycle production of ATG. The experiments are conducted in Drosophila melanogaster, which have become a powerful model system for understanding neurodegeneration mechanisms associated with ALS/FTD associated C9orf72 pathology. Bumple misexpression is shown to rescue early death phenotype in flies expressing a C9orf72 expansion and flies expressing arginine containing di-peptide repeat proteins. The report describes novel insight into the function of bumpel, demonstrating that this conserved orthologue of human SLC14A functions as a sodium exchange transporter for monocarboxylates pyruvate and lactate. These findings conclude that increased neuronal pyruvate, but not its metabolites, rescues C9orf72 associated pathology.
   The authors next set out to describe the mechanism by which increase pyruvate rescues survival in C9orf72 expressing flies. Levels of autolysosomes were increased in C9orf72 expressing flies, and stimulation of autophagy by overexpression of atg1 shown to decrease levels of DPRs (though not to same extent as bumple expression). Expression of bumple in C9orf72 flies led to a modest increase in LC3-II, indicating increased autophagy. Co-overexpression of bumple and atg1 did not have an additive effect, suggesting bumple activates autophagy downstream or independent of atg1 activity. Finally the author extend their findings to amyloid models, suggest a common protective mechanism for elevating neuronal pyruvate levels in neurodegenerative disease.

   Major comments

   Prior data suggests that bumpel is expressed in glia (for example Yildirim et al 2022). In their study the authors do not present any data to demonstrate that the transporter is normally expressed in neurons in flies. This calls into questions the physiological relevance of their findings, that neuronal upregulation of bumpel is protective against C9orf72 associated pathology in neurons, from which it is reasonable for a reader to conclude that bumpel may be a neuronal target for therapeutic intervention. However, the report well demonstrates that regardless of whether the transporter in native to neurons, the increase in monocarboxylates it facilitates is projective against C9orf72 pathology and thus the overall conclusion of the project is supported by experimental evidence. The point of upregulation of a natively expressed gene versus misexpression of a glial enriched transporter should be considered in a bit more detail in the discussion text. The authors may consider speculating the identify of members of the sodium coupled monocarboxylate transporters that are enriched in neurons. Are any of the bumple human orthologues expressed in neurons?_

   We thank the reviewer for this comment and suggestion. The reviewer correctly points out that we do not show whether there is a defect in pyruvate import in C9 expressing flies. We could not identify a validated sodium coupled pyruvate transporter in flies with a strong neuronal expression, we have added a comment in the discussion about this. There are a number of human homologues, some, such as SLC5A8, are expressed in neurons, thus providing a possible therapeutic target. We have added a sentence to this regard in the discussion.

   [_OPTIONAL] cDNA overexpression of neuron specific sodium coupled monocarboxylate transporters in C9orf72 fly models would strengthen the conclusion their physiological relevance for ALS/FTD. Fly lines for these are not available in repositories, but could be generated and tested at reasonable cost (<£700, ~3 month duration).

   This would be an ideal experiment, however, we could not find a neuronal sodium coupled transporter which is known to import monocarboxylates. There are a number of sodium coupled neuronal transporters, but they are mostly homologous to SLC5A6, which is a glucose coupled transporter. Going forward, we will screen a number of transporters to identify if there are any which import pyruvate.

   The role of bumple expression in survival (Figure 1) could be a technical artifact due to dilution of Gal4 between C9orf72 and bumple-ORF transgenes. No expression control is shown (for example GFP, LacZ etc). This theory is unlikely as no improvement in survival was seen for the SLC14A class of transporters which have a matching site directed transgene insertion. For clarity this point relating to controls should be commented on in the text.

   The reviewer is correct, there could be a dilution of the Gal4. We don’t like using GFP as a control as we have often seen a worsening when expressing other highly stable proteins at high levels. We have generated an “empty” flyORF line (generated by injecting the empty plasmid into the identical attP site), and used it as a control to check for dilution effects, bumpel still rescued relative to this control, we now include this is the supplementary (Fig S1B).

   Reduced Mito-GFP levels are used to support a role for bumple in increasing mitophagy. As mito-GFP is a marker for mitochondria but not specifically mitophagy, an alternative explanation for decreased levels could be reduced mitochondria biogenesis. The text should be amended to clarify this point.
   The role of Pink1 RNAi in modifying mitophagy is a bit overstated. Whilst Pink1 is involved in stress associated mitophagy, its role in basal mitochondria turnover is less well defined. Text should be adapted.

   We have added qualifying statements regarding the possibility of reduced mitochondrial biogenesis, and the fact that Pink1’s role in basal mitophagy is not very clear. The use of the mitophagy inducer drug, Kaempferol, however, suggests that mitophagy is unlikely to be a cause of the DRP reduction.

   Minor comments

   Introduction well describes current state of C9orf72 fly models. Introduction would benefit from a few comparable lines for AD models. The first paragraph of reports may also be better placed in the introduction._

   We thank the reviewer for the suggestion, and have added a more in depth introduction to Aß and have moved the first paragraph of the results section to the introduction

   Figure 1 presents survival for three SLC16A transporters and bumple. The C9 control curve appears to be consistent between charts, likely indicating the same control used across experiments, rather than independent controls for each chart. The authors should considered showing either all SLC16A and bumple data on a single chart, or clarify in the figure legend that a common control dataset is used. GFP control is used in later experiments (Figure 2).

   We have now indicated that the SLC16A transporters were run together in the figure legend.

   Choice of amyloid model needs a line of explanation, particularly with regard to extra/intracellular deposition of amyloid in this model.

   We have now added a few sentences describing this when the model is introduced

   Fruit Fly Injection method section needs a bit more detail to describe site of injection (head, body etc). This is not clear in the result section either.

   We have now added this, the injection was done in the abdomen.

   How were bumple orthologues identified? What degree of conservation (sequence homology etc?)

   The bumpel orthologues are those identified as most similar by flybase. We have now added the degree of conservation in the text

   The speculative mechanism for C9 pathology modification involves interaction of neurons and glia, monocarboxylate transporters and changes in autophagy activity. For clarity a diagram showing the model may be a helpful addition.

   We have now added a diagram explaining how we think the rescue is achieved

   Typos:
   Figure 1 Legend - "p values of ona way ANOVA "

   We apologise for the error, and have now corrected it

   Figure S2 Legend - Atg1 RNAi genotypes from S2 legend are mentioned erroneously

   We apologise for the error, and have now corrected it

   Repetition of text in results: "Bumpel, together with its paralogues kumpel and rumpel, is expressed in glia in flies, where it is thought to promote transport of substrates across the brain (31)."

   We apologise and have rectified this

   "Modulation of Atg1 when bumpel was co-overexpressed, however, did not affect GP
   levels (Fig 4E, F)" - Should be refering to Fig 4D, E)

   We apologise and have rectified this

   Reviewer #2 (Significance):

   The study will be of broadly of interest to researcher working in the fields of neurodegeneration and metabolism, providing evidence for a protective role of elevated pyruvate in neuron that provide new understand relating to pathology in C9orf72 associated motor neuron disease and frontotemporal dementia.

   Strengths:
   The study presents novel data to demonstrate that overexpression of fly monocarboxylate transporter bumple rescues an early death phenotype associate with ALS/FTD gene C9orf72. Any novel therapeutic strategies of ALS are of interest to the field, and the strategy demonstrated here may be readily translated to human cell culture systems for proof of principle translational studies to a more physiologically relevant system. This study further demonstrates the utility of invertebrate models to generate novel understanding of C9orf72 pathology.

   Limitations:
   The study speculates that there is a link between pyruvate levels and increased autophagy, however the mechanisms by which this occurs is not defined in present study. This is a limitation of the experiment, though opens up an interesting question for future studies._

   We thank the reviewer for their comments, and we have now added experiments characterising the role of bumpel in autophagy, particularly showing its rescue of a late autolysosomal block.

   Reviewer expertise: The reviewer researches ALS and dementia associated neurodegeneration, utilising Drosophila, rodent and stem cell derived model systems.

   Reviewer #3 (Evidence, reproducibility and clarity):

   This is an interesting manuscript in which the authors provide evidence that elevated neuronal expression of the pyruvate transporter bumpel can partially rescue shortened lifespan in fly models of frontotemporal dementia and Alzheimer's disease. In addition, elevated neuronal bumpel expression can reduce accumulation of arginine containing FTD-linked dipeptide repeat proteins. Some evidence is presented that elevated neuronal bumpel expression may activate autophagy. These findings are novel and may have implications for therapeutic interventions based on pyruvate import/metabolism to treat neurodegenerative disorders. However, I have several concerns as follows:

   Major Comments:

   1. The authors provide no explanation as to why they targeted bumpel overexpression in neurons. Endogenous bumpel appears to be predominately expressed in glia cells so why not target these cells instead?

   We wanted to increase pyruvate import in neurons, so we over-expressed a number of pyruvate transporter that were available in the fly ORF stock centre (so that they would all be inserted into the same site and therefore directly comparable), we were mainly interested in cell autonomous effects of importing glycolytic metabolites. Over-expressing bumpel in glia would be indeed an extremely interesting experiment, unfortunately we do not have the ability to express C9 in neurons while over-expressing bumpel in glia as we only have one over-expression system that works. We are working towards generating a new C9 model so we can then use the Gal 4 system to over-express bumpel in glia, but this is currently not available yet. Over-expression of C9 in glia is not toxic and not a good model of disease.

   1. Data is shown that overexpressed bumpel can suppress GR and PR dipeptide repeat toxicity when these peptides are translated using an ATG start codon (Fig 2D,E). Does bumpel mediated neuroprotection also correlate with a reduction in DPR levels driven with an ATG start codon?

   This would be a very interesting question, unfortunately, whist the Isaacs lab kindly made available the GR antibody for the initial ELISA experiment, we no longer have that antibody available and we do not have a working PR antibody. GR and PR westerns are not possible to carry out as the proteins are too positively charged to run. We do show that bumpel can down-regulate Aß from a UAS promoter, so its effect is not specific to RAN translation.

   1. The authors provide some evidence suggesting that overexpression of bumpel increases autophagy in the fly brain. However, knockdown of Atg1 while co-expressing bumpel (Fig 4E) did not result in increased GP protein levels. In addition, Atg1 knockdown did not attenuate the protective effects of bumpel overexpression (Fig 4I), suggesting that bumpel is working through a pathway independent of autophagy to promote DPR clearance and protection against toxic peptide accumulation. The authors need to modify the interpretation of their data and temper their claim that autophagy contributes to bumpel-mediated protective effects in the CNS.

   We apologise the data was not strong enough. We have now added evidence that bumpel acts downstream of Atg1, on late stage autolysosomal clearance. We also show that bumpel and Atg1 can act synergistically to improve the C9 phenotype when over-expressed, this is now described in Fig 5.

   1. Although the authors present evidence that increased bumpel expression can activate autophagy, the data is not convincing that the neuroprotective effects associated with bumpel are mediated through autophagy. Pyruvate, in some circumstances, can non-enzymatically scavenge hydrogen peroxide or in other cases trigger oxidative stress resistance through hormetic ROS signaling. The authors should consider these alternative possibilities.

   These are indeed possibilities, we have added a sentence to that effect in the discussion, we have now also showed that bumpel is affecting late clearance of autolysosomes, and is leading to an increase in TFEB targets.

   1. The authors rely on overexpressing bumpel to attenuate C9 toxicity in flies. They should perform the opposite experiment and knockdown bumpel to demonstrate that reduced bumpel expression results in potentiation of C9 and amyloid beta neurotoxicity. In addition, then should show that knockdown of bumpel expression has some effect on autophagy.

   This would be a very interesting experiment, unfortunately bumpel is expressed only in a few glia subtypes in a wild type fly, and we can’t downregulate it in glia while over-expressing toxic proteins in neurons, because of limitations of our expression system, both genes need to be over-expressed in the same cell type. We have tried downregulating bumpel in neurons, and don’t get an effect on phenotype, and no effect on DPR levels, but bumpel expression in neurons is extremely low. Moreover, bumpel has 2 paralogs, rumpel and kumpel,(also only present in glia) and all three need to be knocked out for phenotypes to become visible in glia (Yildirim et al, 2022). These experiments would be interesting but outside out scope.

   We are in the process of generating new C9 models to be able to do these experiments, but these are currently outside the scope of this work.

   Minor Comments:

   1. Neuronal overexpression of bumpel appears to shorten lifespan of wild type flies (Fig 2A). It is possible that neuronal import of pyruvate may drive mitochondrial oxidative phosphorylation and ROS formation. The authors should comment on this possibility in the discussion._

   This is a very good point, we have added a point to that effect.

   1. In Fig 3 the authors used a mixture of sodium pyruvate and ethyl pyruvate to demonstrate the import properties of bumpel. The rationale for using ethyl pyruvate is unclear as this membrane-permeable metabolite can by-pass any transporters.

   The ethyl pyruvate was only used in the injection of flies, not for the FRET experiments looking at the import properties of bumpel. Since we were not over-expressing bumpel, we needed the pyruvate to by-pass the requirement for a transporter. We were showing that delivery of pyruvate by another methods (other than by a transporter) was able to phenocopy the over-expression of bumpel, thus showing the effect is mediated by pyruvate entrance into the cell.

   1. In the introduction several acronyms are used (i.e. GRN, MAPT, TREM2) that are not defined.

   We apologise and have now rectified this.

   Reviewer #3 (Significance):

   To my knowledge, this is the first study to identify that bumpel can permit the import of pyruvate and lactate into neurons when ectopically expressed in the fly brain. The fact that increased neuronal pyruvate import can partially protect against toxic peptide accumulation is unexpected and quite novel. Although some evidence is presented that bumpel can trigger autophagy, it is not clear if autophagy is mediating bumpel neuroprotective effects. Alternative mechanisms related to pyruvate effects on ROS and oxidative stress resistance should be considered.

   We thank the reviewer for their comments, and have added clarifying statements regarding the potential role of ROS.

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   Review Commons

   Jun 28, 2023

   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

   This is an interesting manuscript in which the authors provide evidence that elevated neuronal expression of the pyruvate transporter bumpel can partially rescue shortened lifespan in fly models of frontotemporal dementia and Alzheimer's disease. In addition, elevated neuronal bumpel expression can reduce accumulation of arginine containing FTD-linked dipeptide repeat proteins. Some evidence is presented that elevated neuronal bumpel expression may activate autophagy. These findings are novel and may have implications for therapeutic interventions based on pyruvate import/metabolism to treat neurodegenerative disorders. However, I … More

   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

   This is an interesting manuscript in which the authors provide evidence that elevated neuronal expression of the pyruvate transporter bumpel can partially rescue shortened lifespan in fly models of frontotemporal dementia and Alzheimer's disease. In addition, elevated neuronal bumpel expression can reduce accumulation of arginine containing FTD-linked dipeptide repeat proteins. Some evidence is presented that elevated neuronal bumpel expression may activate autophagy. These findings are novel and may have implications for therapeutic interventions based on pyruvate import/metabolism to treat neurodegenerative disorders. However, I have several concerns as follows:

   Major Comments:

   1. The authors provide no explanation as to why they targeted bumpel overexpression in neurons. Endogenous bumpel appears to be predominately expressed in glia cells so why not target these cells instead?
   2. Data is shown that overexpressed bumpel can suppress GR and PR dipeptide repeat toxicity when these peptides are translated using an ATG start codon (Fig 2D,E). Does bumpel mediated neuroprotection also correlate with a reduction in DPR levels driven with an ATG start codon?
   3. The authors provide some evidence suggesting that overexpression of bumpel increases autophagy in the fly brain. However, knockdown of Atg1 while co-expressing bumpel (Fig 4E) did not result in increased GP protein levels. In addition, Atg1 knockdown did not attenuate the protective effects of bumpel overexpression (Fig 4I), suggesting that bumpel is working through a pathway independent of autophagy to promote DPR clearance and protection against toxic peptide accumulation. The authors need to modify the interpretation of their data and temper their claim that autophagy contributes to bumpel-mediated protective effects in the CNS.
   4. Although the authors present evidence that increased bumpel expression can activate autophagy, the data is not convincing that the neuroprotective effects associated with bumpel are mediated through autophagy. Pyruvate, in some circumstances, can non-enzymatically scavenge hydrogen peroxide or in other cases trigger oxidative stress resistance through hormetic ROS signaling. The authors should consider these alternative possibilities.
   5. The authors rely on overexpressing bumpel to attenuate C9 toxicity in flies. They should perform the opposite experiment and knockdown bumpel to demonstrate that reduced bumpel expression results in potentiation of C9 and amyloid beta neurotoxicity. In addition, then should show that knockdown of bumpel expression has some effect on autophagy.

   Minor Comments:

   1. Neuronal overexpression of bumpel appears to shorten lifespan of wild type flies (Fig 2A). It is possible that neuronal import of pyruvate may drive mitochondrial oxidative phosphorylation and ROS formation. The authors should comment on this possibility in the discussion.
   2. In Fig 3 the authors used a mixture of sodium pyruvate and ethyl pyruvate to demonstrate the import properties of bumpel. The rationale for using ethyl pyruvate is unclear as this membrane-permeable metabolite can by-pass any transporters.
   3. In the introduction several acronyms are used (i.e. GRN, MAPT, TREM2) that are not defined.

   Significance

   To my knowledge, this is the first study to identify that bumpel can permit the import of pyruvate and lactate into neurons when ectopically expressed in the fly brain. The fact that increased neuronal pyruvate import can partially protect against toxic peptide accumulation is unexpected and quite novel. Although some evidence is presented that bumpel can trigger autophagy, it is not clear if autophagy is mediating bumpel neuroprotective effects. Alternative mechanisms related to pyruvate effects on ROS and oxidative stress resistance should be considered.

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3. 

   Review Commons

   Jun 28, 2023

   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

   Summary:

   Project investigates the role in dementias of glial glucose uptake, conversion to lactate and shuttling via transporters to neurons to produce pyruvate to fuel TCA cycle production of ATG. The experiments are conducted in Drosophila melanogaster, which have become a powerful model system for understanding neurodegeneration mechanisms associated with ALS/FTD associated C9orf72 pathology. Bumple misexpression is shown to rescue early death phenotype in flies expressing a C9orf72 expansion and flies expressing arginine containing di-peptide repeat proteins. The report describes novel insight into the function of bumpel, … More

   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

   Summary:

   Project investigates the role in dementias of glial glucose uptake, conversion to lactate and shuttling via transporters to neurons to produce pyruvate to fuel TCA cycle production of ATG. The experiments are conducted in Drosophila melanogaster, which have become a powerful model system for understanding neurodegeneration mechanisms associated with ALS/FTD associated C9orf72 pathology. Bumple misexpression is shown to rescue early death phenotype in flies expressing a C9orf72 expansion and flies expressing arginine containing di-peptide repeat proteins. The report describes novel insight into the function of bumpel, demonstrating that this conserved orthologue of human SLC14A functions as a sodium exchange transporter for monocarboxylates pyruvate and lactate. These findings conclude that increased neuronal pyruvate, but not its metabolites, rescues C9orf72 associated pathology.

   The authors next set out to describe the mechanism by which increase pyruvate rescues survival in C9orf72 expressing flies. Levels of autolysosomes were increased in C9orf72 expressing flies, and stimulation of autophagy by overexpression of atg1 shown to decrease levels of DPRs (though not to same extent as bumple expression). Expression of bumple in C9orf72 flies led to a modest increase in LC3-II, indicating increased autophagy. Co-overexpression of bumple and atg1 did not have an additive effect, suggesting bumple activates autophagy downstream or independent of atg1 activity. Finally the author extend their findings to amyloid models, suggest a common protective mechanism for elevating neuronal pyruvate levels in neurodegenerative disease.

   Major comments

   Prior data suggests that bumpel is expressed in glia (for example Yildirim et al 2022). In their study the authors do not present any data to demonstrate that the transporter is normally expressed in neurons in flies. This calls into questions the physiological relevance of their findings, that neuronal upregulation of bumpel is protective against C9orf72 associated pathology in neurons, from which it is reasonable for a reader to conclude that bumpel may be a neuronal target for therapeutic intervention. However, the report well demonstrates that regardless of whether the transporter in native to neurons, the increase in monocarboxylates it facilitates is projective against C9orf72 pathology and thus the overall conclusion of the project is supported by experimental evidence. The point of upregulation of a natively expressed gene versus misexpression of a glial enriched transporter should be considered in a bit more detail in the discussion text. The authors may consider speculating the identify of members of the sodium coupled monocarboxylate transporters that are enriched in neurons. Are any of the bumple human orthologues expressed in neurons?
   [OPTIONAL] cDNA overexpression of neuron specific sodium coupled monocarboxylate transporters in C9orf72 fly models would strengthen the conclusion their physiological relevance for ALS/FTD. Fly lines for these are not available in repositories, but could be generated and tested at reasonable cost (<£700, ~3 month duration).
   The role of bumple expression in survival (Figure 1) could be a technical artifact due to dilution of Gal4 between C9orf72 and bumple-ORF transgenes. No expression control is shown (for example GFP, LacZ etc). This theory is unlikely as no improvement in survival was seen for the SLC14A class of transporters which have a matching site directed transgene insertion. For clarity this point relating to controls should be commented on in the text.
   Reduced Mito-GFP levels are used to support a role for bumple in increasing mitophagy. As mito-GFP is a marker for mitochondria but not specifically mitophagy, an alternative explanation for decreased levels could be reduced mitochondria biogenesis. The text should be amended to clarify this point.
   The role of Pink1 RNAi in modifying mitophagy is a bit overstated. Whilst Pink1 is involved in stress associated mitophagy, its role in basal mitochondria turnover is less well defined. Text should be adapted.

   Minor comments

   Introduction well describes current state of C9orf72 fly models. Introduction would benefit from a few comparable lines for AD models. The first paragraph of reports may also be better placed in the introduction.

   Figure 1 presents survival for three SLC16A transporters and bumple. The C9 control curve appears to be consistent between charts, likely indicating the same control used across experiments, rather than independent controls for each chart. The authors should considered showing either all SLC16A and bumple data on a single chart, or clarify in the figure legend that a common control dataset is used. GFP control is used in later experiments (Figure 2).

   Choice of amyloid model needs a line of explanation, particularly with regard to extra/intracellular deposition of amyloid in this model.

   Fruit Fly Injection method section needs a bit more detail to describe site of injection (head, body etc). This is not clear in the result section either.

   How were bumple orthologues identified? What degree of conservation (sequence homology etc?)

   The speculative mechanism for C9 pathology modification involves interaction of neurons and glia, monocarboxylate transporters and changes in autophagy activity. For clarity a diagram showing the model may be a helpful addition.

   Typos:

   Figure 1 Legend - "p values of ona way ANOVA "

   Figure S2 Legend - Atg1 RNAi genotypes from S2 legend are mentioned erroneously

   Repetition of text in results: "Bumpel, together with its paralogues kumpel and rumpel, is expressed in glia in flies, where it is thought to promote transport of substrates across the brain (31)."

   "Modulation of Atg1 when bumpel was co-overexpressed, however, did not affect GP
   levels (Fig 4E, F)" - Should be refering to Fig 4D, E)

   Significance

   The study will be of broadly of interest to researcher working in the fields of neurodegeneration and metabolism, providing evidence for a protective role of elevated pyruvate in neuron that provide new understand relating to pathology in C9orf72 associated motor neuron disease and frontotemporal dementia.

   Strengths:

   The study presents novel data to demonstrate that overexpression of fly monocarboxylate transporter bumple rescues an early death phenotype associate with ALS/FTD gene C9orf72. Any novel therapeutic strategies of ALS are of interest to the field, and the strategy demonstrated here may be readily translated to human cell culture systems for proof of principle translational studies to a more physiologically relevant system. This study further demonstrates the utility of invertebrate models to generate novel understanding of C9orf72 pathology.

   Limitations:

   The study speculates that there is a link between pyruvate levels and increased autophagy, however the mechanisms by which this occurs is not defined in present study. This is a limitation of the experiment, though opens up an interesting question for future studies.

   Reviewer expertise: The reviewer researches ALS and dementia associated neurodegeneration, utilising Drosophila, rodent and stem cell derived model systems.

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4. 

   Review Commons

   Jun 28, 2023

   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 #1

   Evidence, reproducibility and clarity

   The manuscript by Niccoli et al. describes the identification of a novel modifier of C9orf72-derived toxicity based on the manipulation of the brain metabolic pathways. The premise for this work is supported by strong literature describing the aberrant glucose metabolism in FTD, AD and other degenerative disorders. The idea tested here is whether increasing the import of pyruvate produced in glia into neurons. They test three different types of importers and find that one of them, Bumpel, the orthologue of human SLC5A12, suppresses toxicity and reduces the accumulation of arginine-containing repeats, GP and PR. The authors … More

   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 by Niccoli et al. describes the identification of a novel modifier of C9orf72-derived toxicity based on the manipulation of the brain metabolic pathways. The premise for this work is supported by strong literature describing the aberrant glucose metabolism in FTD, AD and other degenerative disorders. The idea tested here is whether increasing the import of pyruvate produced in glia into neurons. They test three different types of importers and find that one of them, Bumpel, the orthologue of human SLC5A12, suppresses toxicity and reduces the accumulation of arginine-containing repeats, GP and PR. The authors investigate several potential mechanisms mediating this reduction of toxic DPRs, but do not find strong evidence linking pyruvate import and increase autophagy or mitochondria metabolism.

   Overall, this is an interesting discovery based on a candidate approach that shows the power of Drosophila to efficiently identify novel mediators of neurodegeneration. The article is well written, although more detailed explanations of some experiments would be helpful. The weaknesses of the manuscript are the lack of a clear mechanism mediating the protective activity of pyruvate, the incomplete experiments lacking relevant controls, and the presentation of western blots.

   Specific comments:

   1. The reduced levels of DPRs require that the expression of C9 mRNA or the GR and PR constructs is examined by qPCR. In figure 3E, GP is not even detectable
   2. I wonder if there are constructs available to silence Bumpel or overexpress the human orthologues of bumpel. These would be nice controls for the effects observed with the Bumpel overexpression
   3. The argument about bumpel modulating autophagy downstream of Atg1 is not supported by the experimental data
   4. Western blots throughout show no control lanes and in several occasions are created with cutout bands. The standard for this type of experiments should be more stringent, with entire gels showing all experimental conditions, which requires consistent methods and results vs selecting the best bands from different gels.
   5. For figures 2B and 5C, please, show representative WBs
   6. Figure 5D describes the survival curve as significantly rescued. Statistical tests can indicate differences, but that is in no way convincing. The test may show the curves are different, but the abeta Atg1 flies also seem to start falling early, so an argument could be made in both directions, as a suppressor or an enhancer.
   7. It is unclear why several results are placed in the supplemental materials. In general, all this material seems highly relevant and related to what is shown in the main figures

   Minor comments:

   Please, define several abbreviations throughout

   A couple of sections could be improved by carefully sequencing human vs Drosophila background to advance the argument rather than going in circles. There is also a section on mitophagy in between two sections related to autophagy that could be sequenced better.

   There is a sentence at the end of page 6 that seems misplaced

   Significance

   Overall, this is an interesting discovery based on a candidate approach that shows the power of Drosophila to efficiently identify novel mediators of neurodegeneration. The article is well written, although more detailed explanations of some experiments would be helpful. The weaknesses of the manuscript are the lack of a clear mechanism mediating the protective activity of pyruvate, the incomplete experiments lacking relevant controls, and the presentation of western blots.

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   Read the original source
5. 

   Version published to 10.1101/2023.06.25.546435v1 on bioRxiv

   Jun 26, 2023