PGENETICS-D-25-00724

LINE-1 replication in a mouse TDP-43 model of neurodegeneration marks motor cortex neurons for cell-intrinsic and non-cell autonomous programmed cell death

PLOS Genetics

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PLOS Genetics

Hua Tang

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PLOS Genetics

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PLOS Genetics

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note that one review is uploaded as an attachment.

Reviewer #1: The review is uploaded as an attachment.

Reviewer #2: Korada and colleagues investigate the role of TDP-43 in repressing retrotranspon elements (RTEs) using an established two mouse models of TDP-43 pathology, the mild overexpression of wildtype or Q331K human TDP-43. They show that the Q331K mouse has early motor deficits compared to a later deficit in the WT. They cross the mice to a LINE1 GFP reporter line to observe LINE1 re-integration in the motor cortex. They show that this LINE1 integration is enhanced at particular time points in the TDP-43 mutant lines, and that this is accompanied by markers of cell death in LINE1-positive neurons, along with nearby cells.

This is a very interesting paper for the field, showing that TDP-43 is associated with LINE de-repression and suggesting that this de-repression is killing both neurons and neighboring cells. Overall, the paper is well written and the methods are clearly described. I have few major comments, but would suggest that the figures are improved to be more legible and consistent across panels.

Major comment: are the authors planning to look at TDP-43 dependent splicing changes in their RNA-seq data? It would be useful to show the accumulation of both loss-of-function (cryptic exons) and gain of function (skiptic exons) at different time points to see whether these splicing changes co-occur with the RTE changes observed. There is a database of mouse TDP-43 splicing events in Fratta et al, 2018 (PMID: 29764981) that could be queried.

Reviewer #3: This is a very nice, compact paper which makes some important discoveries that advance the field as a whole. The transient upregulation of RTEs is a significant finding. The correlation of the upregulation with onset of neurological phenotypes in the two models (WT and mutant TDP) is a significant finding. The death of neurons by apoptosis, and the “action at a distance” (non-cell autonomous death of neighboring cells) are also significant findings, and help to explain previous findings on RTE expression in human post-mortem samples. As a whole, the data are of sufficient quality to support these conclusions, and this warrants the publication of this study.

Major issues

While the apparent increase of L1 retrotransposition, as evidenced by the L1-EGFP reporter, is a very nice touch, given the results obtained, it also injects a certain degree of uncertainty on what’s actually going on.

1) GFP-positive “cell clusters” are seen at 3 and 6 months but not later. This correlates well with the wave of endogenous RTE expression seen in the mutant-TDP mice but not at all in the WT-TDP mice. I’m struggling to find an explanation for this. The authors should provide some speculations on this.

2) Relevant to the above, the L1-EGFP line is poorly defined (I looked up the Gage paper). It was made by pronuclear injection, so likely contains many copies of the reporter (at one or several genomic loci). Is anything known about this? These factors probably affect its expression and regulation. Also, how many times has the line been backcrossed to C57? The TDP models from JAX are on a C57BN background. Are the experiments in this paper done on a mixed background?

3) The GFP signal tracks successful retrotransposition but not expression of the reporter itself. The L1-EGFP is human, and thus would be expressed either from its own (quite weak) promoter (inside the 5’ UTR) or from a cellular promoter at the site of its insertion. I see no reason why the human L1 5’UTR would be regulated in concert with endogenous mouse L1s. Given that the authors note that the upregulation of endogenous RTEs was “similar across both evolutionarily recent and more ancient transposable elements”, one possibility is that the upregulation is rather nonspecific and reflects a large-scale chromatin relaxation. It is possible that the transgene integrated in a heterochromatic region and thus could also ride this wave. In any case, it’s easy to monitor the expression of the L1-EGFP reporter (it’s human) by qRT-PCR, and this should be done. An alternative explanation could be that the reporter is on all the time, but that the cellular environment becomes supportive of productive retrotransposition at the observed times. It is important to address these issues because of the discrepancy noted in 1) above.

4) Currently, endogenous RTE expression is monitored by RNA-seq. This is limiting for two reasons: 1) well known issues of deconvoluting expression of repetitive sequences (and assigning functional vs. non-functional L1s), 2) does not provide single-cell resolution, while much of the other data in this paper do. There is a good antibody to mouse L1 ORF1 (validated by Alex Bortvin, Abcam AB216324), and it would be easy to perform and include those experiments on the specimens already in hand.

Minor issues

1) The authors keep referring to “clusters”, defined as “large, spatially defined groups of L1-EGFP-labeled cells”. The manner in which they are scored is described verbally (which is clear), but it would be very helpful to have some visuals on this. For example, in some of the images presented, draw a dashed line around what is being referred to as a “cluster”.

2) Row designations in Fig. 2a, b are missing.

3) Is the lifespan of mutant-TDP Tg mice known? They are missing from the 17 month timepoint.

4) Fig. 3: not clear what is being plotted here. Each dot corresponds to one mouse, and the position on the Y axis is the average number of GFP positive cells in that mouse? How were the violin outlines computed?

5) Sentence line 709-711, meaning is not clear. What is meant by any upregulated RTE? Both significantly and non-significantly upregulated RTEs? Was there no significant overlap between significantly upregulated in WT hTDP and significantly upregulated in Q331K hTDP?

6) Sentence on line 713-715: “We then compared . . . “ Where is the result of this comparison? Something seems to be missing.

7) The authors keep referring to “replication” of L1, as evidenced by the L1-EGFP reporter. I would prefer to use “retrotransposition” – I believe it is a more descriptive and accurate term.

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Reviewer #1: None

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Comments_July 2025.pdf

Dear Dr Sher,

We are pleased to inform you that your manuscript entitled "LINE-1 retrotransposition in a mouse TDP-43 model of neurodegeneration marks motor cortex neurons for cell-intrinsic and cell non-autonomous programmed cell death" has been editorially accepted in principle for publication in PLOS Genetics. Congratulations! Please address the final minor points by reviewer 3 along with requested editorial formatting (see below).

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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 have addressed the reviewers' questions and concerns. Each point has been clearly responded to, and the corresponding revisions have been appropriately incorporated into the manuscript. The overall of the manuscript is clearer after the corrections and revisions.

Reviewer #2: I am satisfied that the authors have addressed my comments.

Reviewer #3: Rev. 3, point 1): Please include your thoughts and and interpretations in the Discussion. Speculative is fine, this is what discussions are for.

Point 3): please include your response (or a shortened version of it) in the discussion.

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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: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:  John Sedivy

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