PGENETICS-D-25-01100

Glial cell-intrinsic and non-cell autonomous toxicity in a Drosophila C9orf72 neurodegeneration model

PLOS Genetics

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PLOS Genetics

Anne O'Donnell-Luria

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PLOS Genetics

Aimée Dudley

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PLOS Genetics

Anne Goriely

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PLOS Genetics

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Isabel Hubbard, and Josh Dubnau. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Dubnau identified an interesting role of glia in neurodegeneration in fly models of C9ALS/FTD. Interestingly, blocking apoptosis promotes, rather than suppresses neural defects in these models. Overall, this is an interesting paper with solid data. My major concerns are about the relevance to disease and translation. Given the huge differences between human and fly glial system, it's unclear how these findings can be implicated in diseases. For example, the spg glia are very different in shape compared to oligodendrocytes, even though both construct the blood brain barrier. The spgs do not have the myolin sheath, which is disrupted in ALS/FTD. Furthermore, even though I agree with the cell non-autonomous role, I don't think their p35 data support such a role, as it can be simply explained as other cell-death pathways contribute to neuronal loss in their flies (e.g., necrosis, ferroptosis, etc.?). In addition, I don't quite understand the mdg4-TDP43 part. Can the authors explain its role in ALS more clearly.

Nevertheless, I think this is a good fly paper with solid science.

Reviewer #2: This is a systematic study of toxicity arising from expression of several C9orf72 models (expanded G4C2 repeats, RNA-only models, and alternative codon “DPR only” models) in Drosophila glia. This complements existing research in C9orf72 toxicity and TDP43- associated toxicity including several studies investigating toxicity emanating from astrocyte expression in cell model systems.

The authors demonstrate:

• Reduced lifespan with pan-glial expression

• Reduced lifespan when expression is restricted to sub-pial glia

• De-repression of the mdg4 endogenous retrovirus

• Differences in severity not explained by co-expressing RNA-only and pure DPR models suggesting some other mechanism is at play.

• Anti-apoptotic manipulations (overexpression of p35) exacerbated non-cell autonomous effects of C9 glia while pro-apoptotic manipulations (overexpression of reaper) improved these effects.

Overall, the experiments in this study are well done. While it is largely descriptive and no new mechanisms are provided, it still adds some useful data to the field. There is prior published work of non-cell autonomous effects from glia with pathologic C9orf72 G4C2 expansions and this group did previously do similar studies with TDP-43 overexpression. However, this study is unique in the systematic use of several models (a “full” producing RNA and DPRs, a “no RNA”, and a “pure DPR” for poly GA, GR, PR, and PA.

The manuscript does have some shortcomings that should be addressed.

1. The abstract would benefit from a final sentence which summarizes the central conclusion(s) of the manuscript.

2. The study is limited to male flies. Given that sexual dimorphism causes female flies to often exhibit different survival patterns compared to males, at least some comparison between female and male flies for key experiment al findings would strengthen the manuscript.

3. The tools used are a bit heavy handed, which limits interpretation. For example, killing the cells in which you are trying to express a toxic factor and then having that prevent the toxicity is a bit of a circular argument. It is impressive that getting rid of glia alone (via reaper expression) is so well tolerated, but that doesn’t make the results any less tautological. The opposite studies (suppressing apoptosis with p35) are confounded because cells dying by a means other than apoptosis may just release factors that are problematic for the neighboring neurons in a fashion that is non-specific. All these issues could be addressed by expressing or treating with something that kills the glia by a fashion other than apoptosis or ALS. If not, some rewording of their interpretation in the context of these limitations is warranted

4. I struggle with their over-interpretation of their findings. For example, while they imply/suggest that there is a connection between the non-cell autonomous toxicity observed in C9orf72 models and TDP-43/TBPH, they do not show this despite feasible experiments that could be done to delineate the relationship (e.g. assess for glial TBPH cytoplasmic localization, as was done here for fly neurons: Lee et al 2024, https://doi.org/10.1016/j.mocell.2023.12.003; Overexpress a form of TDP43 lacking a nuclear export signal in these same glia to see if you can suppress the repeat phenotype). Thus, they should either demonstrate a direct connection (something that would have real value to the field and would significantly enhance your manuscript) or they should not speculate based on the limited data they present.

5. Similarly, I struggle with interpretation of the mdg4 story. It appears to be de-repressed more in some contexts but not others but not always in perfect correlation with phenotypes. They interpret this as evidence that it is not a cause of the phenotype. The right experiment to assess for a direct role for mdg4 would be expression of mdg4 in isolation in glial cells and see if that recapitulates the phenotype. To test the opposite, you could express an siRNA against mdg4 or knock it out and see if the repeat/DPR effects are mitigated. If mdg4 is really a marker of an ongoing process rather than a driver, then you could target those processes more directly. Without these more focused studies, I think their discussion needs to be amended.

Reviewer #3: C9ORF72 G4C2 repeat expansion is the most common genetic cause of both ALS and FTD. The repeat expansion is bidirectionally transcribed into sense and anti-sense RNA foci, and also produces dipeptide repeats (DPRs) via a non-canonical translation mechanism. In this manuscript, the authors used glial cell type-specific expression of G4C2 repeats, or individual DPRs, or RNA repeat-only to systematically investigate both the glial cell-intrinsic and non-cell autonomous toxicity. They show that GR and G4C2 repeats produce the highest toxicity in glia. Only G4C2 repeat transgene produces non-cell autonomous toxicity that result in loss of nearby neurons. It was hypothesized that inhibiting glial cell death would increase neuronal toxicity. However, blocking apoptosis in GR- or G4C2-expressing glia using the p35 caspase inhibitor neither prevented glial death nor increased neuronal loss, yet still exacerbated the reduction in lifespan. Although this observation is intriguing, the underlying mechanism remains unclear.

1. Translation in the GA frame is most efficient among the GP, GA, and GR frames on G4C2 repeats, due to the presence of a near-cognate start codon. However, the data in this manuscript show substantially higher levels of GR, which is inconsistent with prior studies. The sequence of the G4C2 transgene therefore needs to be clarified, particularly the region upstream of the repeats. If this sequence does not reflect the authentic C9orf72 context, the relative toxicity observed from the G4C2 transgene compared with DPR-only or repeat-only constructs may not accurately represent endogenous mechanisms. The conclusions should be interpreted more cautiously, and a more careful discussion is needed.

2. In Figure 2B, the overall signal in the G4C2 and GR groups appears markedly dimmer, which is inconsistent with the quantification shown in Figure 2C. If there is an approximately 50% reduction in SPG cell number, one would expect a loss of signal in roughly half of the cells, with the remaining cells exhibiting comparable intensity to controls, rather than a uniform decrease in signal intensity across all cells.

3. In Figure 3E, the overall image quality is low and shows issues similar to those noted in Figure 2B. Both GA and GR conditions also exhibit reduced signal intensity, and the staining in the G4C2 condition seems not work. These technical concerns make it difficult to interpret the data.

4. Figure 4, transgene expression levels should be measured to confirm the expression is comparable in each group.

5. Apoptosis markers should be stained for validate the apoptosis is inhibited for enhanced by the modifiers.

6. Blocking apoptosis in GR- or G4C2-expressing glia using the p35 caspase inhibitor neither prevented glial death nor increased neuronal loss, yet nevertheless exacerbated the reduction in lifespan. No mechanistic explanation is provided to account for this discrepancy. The manuscript claims that “these findings support the idea that in response to either (G4C2)36 or GR36 expression in SPG, cell-intrinsic effects that lead to apoptotic cell death have a protective role to the surrounding tissue and to the animal’s lifespan”. However, no evidence is presented to support an effect on surrounding tissues, and the basis for changes in lifespan remains unclear.

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No:

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Reviewer #1: Yes:  Ke Zhang

Reviewer #2: No

Reviewer #3: No

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Dear Dr Dubnau,

We are pleased to inform you that your manuscript entitled "Glial cell toxicity in a Drosophila C9orf72 neurodegeneration model" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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PLOS Genetics

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors addressed my comments. I have two more suggestions:

1) Define G4C2

2) Put, in the Discussion, the authors' response on my concern about the relevance to disease and translation.

Reviewer #2: This is an interesting story regarding the role of SPG glia expressing GR DPR producing C9-relevant transgenes leading to cell non-autonomous toxicity independent of neuronal loss. This adds value to the C9 field and potentially to the intersection of neurodegeneration and aging. While the manuscript does not provide a clear mechanism by which this occurs, we do not feel that such is requisite to justify publication.

Reviewer #3: The manuscript has been strengthened by the addition of new data and careful revisions to improve clarity and avoid overstating the conclusions. Although the underlying molecular mechanism remains unclear, the findings are of sufficient interest to warrant publication, provided that the conclusions are presented cautiously and do not overstate the implications of the results.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: None

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Reviewer #1: Yes:  Ke Zhang

Reviewer #2: Yes:  Peter Todd

Reviewer #3: No

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