LAG3 is not expressed in human and murine neurons and does not modulate α‐synucleinopathies

Abstract While the initial pathology of Parkinson’s disease and other α‐synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α‐synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α‐synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α‐synuclein pathology ex vivo. The overall survival of A53T α‐synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α‐synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α‐synucleinopathies is not universally valid.

1. "Histologically, PD is characterized by α-synuclein aggregates known as Lewy Bodies in neurons of the substantia nigra," That is not a good description of PD neuropathology. Lewy pathology is present in numerous areas of the CNS and PNS, and is not restricted to the substantia nigra. 2. "Growing evidence suggests that α-synuclein fibrils spread from cell to cell". While alpha-synuclein pathology can spread from cell to cell, it is not known if the fibrils are This study is of extremely high significance -we need mechanisms to deal with spectacular results in the literature that should not have been published because they are were uncompelling to begin with, but were published for various sociological/political reasons. Science won't progress if we don't find correction mechanisms for wrong conclusions. **Referee Cross-commenting** I agree with reviewers 1 and 3, especially with the suggestions made by reviewer 1, which should be instituted. I think we all concur that the paper should be published without new experiments. I believe testing a-synuclein propagation in vivo in LAG3 KO mice would be useful, but given the complete lack of replication of LAG3 expression in brain and of a-synuclein binding to LAG3, this is not necessary.

Review #3
1. How much time do you estimate the authors will need to complete the suggested revisions:

Estimated time to Complete Revisions (Required) (Decision Recommendation)
Less than 1 month 2. Evidence, reproducibility and clarity:

Evidence, reproducibility and clarity (Required)
It was proposed that LAG3 is important in the treatment of PD and related disorders, because it functions as a receptor of pathogenic α-synuclein and the treatment with anti-LAG3 antibodies attenuated the spread of pathological α-synuclein and drastically lowered the aggregation in vitro (Mao et al, Science 2016).
In this study, authors characterized 8 antibodies to LAG3 and investigated the presence of LAG3 in cultured cell lines, NSC-derived neural cultures, or organ homogenates for the presence of human or murine LAG3. But it was not detected in any of the neuronal samples tested. In addition, single cell (sc) RNAseq yielded only minimal counts for the LAG3 transcript in neurons, astrocytes, and mixed glial cells, and single-nucleus (sn) RNAseq human brain dataset for LAG3 expression across different cell types confirmed no LAG3 signals for any of 34 identified cell clusters, including 13 clusters of excitatory and 11 subtypes of inhibitory neurons, oligodendrocytes, oligodendrocyte precursor cells, microglia, astrocytes, and endothelial cells.
Authors also analyzed the binding of LAG3 with α-synuclein in mouse and human model systems, and concluded that the affinity of LAG3 for α-synuclein fibrils, if any, is micromolar or less.
Furthermore, authors studied the propagation of pre-formed fibrils (PFFs) of αsynuclein in neural stem cell (NSC)-derived neural cultures in the presence or absence of LAG3, and the impact of LAG3 on survival in ASYNA53T transgenic mice expressing wild-type LAG3 as well as hemizygous or homozygous deletions thereof. However, they were unable to see any significant role for LAG3 in these in vitro and in vivo models of α-synucleinopathies.
In this connection, the reviewer would like to ask one question: Have you conducted any experiments of the propagation of PFFs of α-synuclein in LAG3-KO mice ? If they did, what were the results ? **Minor point** In Page 10, I think it's a typo: ASYYN mice must be ASYN mice.

Significance (Required)
These negative findings about the LAG in α-synucleinopathies shown in this manuscript do not provide any new insight into the mechanisms of α-synuclein propagation. However, it is clear that LAG3 is not expressed in neuronal cells and the binding of LAG3 to α-synuclein fibrils appears limited. Overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data shown in this manuscript are convincing and the information is very important in terms of correcting the direction of disease treatment and research. **Referee Cross-commenting** I agree with reviewers 1 and 2. This paper should be published as soon as possible.
1. "Histologically, PD is characterized by α-synuclein aggregates known as Lewy Bodies in neurons of the substantia nigra," That is not a good description of PD neuropathology. Lewy pathology is present in numerous areas of the CNS and PNS and is not restricted to the substantia nigra.
We have added a more detailed account: "Histologically, PD is characterized by α-synuclein inclusions known as Lewy Bodies whose accumulation is associated with neurodegeneration (Dickson, 2012; Mullin and Schapira, 2015; Corbillé et al., 2016). These inclusions affect the Substantia nigra and other mesencephalic regions as well as, in some cases, the amygdala and neocortex (Dickson, 2018)." 2. "Growing evidence suggests that α-synuclein fibrils spread from cell to cell". While alpha-synuclein pathology can spread from cell to cell, it is not known if the fibrils are the species (alone or combined with other conformers) that cause the spreading of the pathology in a seeding fashion, or if smaller alpha-synuclein assemblies play that role.
We have reformulated the sentence to credit the fact that we do not know which synuclein species is the one that is transmitted: ." This sentence gives the impression that the corresponding author has led the field when it comes to alpha-synuclein's prionid properties. That is not really the case, and it would be appropriate to cite the literature in a more scholarly fashion that reflects how this part of the alphasynuclein research field developed. I cannot disagree, and in fact I suspect that the present paper may be my second and possibly last experimental contribution to the synuclein field! However, I do claim intellectual parenthood of the prionoid (not "prionid") concept, which I first expounded in a 2009 Nature paper. Anyway, we now provide a more balanced citation: 4. "Interrupting transmission of a-synuclein may slow down or abrogate the disease course." This is a bold statement and far from certain. While one might propose that this is the case, it is still just a hypothesis and the Introduction should reflect that.
We have rewritten the sentence in a more subdued manner: "It is thought that interrupting transmission of a-synuclein may slow down or abrogate the disease course." 18th Jun 2021 Authors' response to Reviewer Comments (transferred files) Reviewer #2 (Evidence, reproducibility and clarity (Required)): This study conclusively shows that LAG3 is not the receptor for a-synuclein that underlies the spread of synucleinopathic damage in various PD-related conditions. The paper is done extremely carefully and comprehensively. My only suggestion is to indicate the significance level in Figure 5a, as it may turn out that LAG3 is actually protective. We have added the significance level in Fig. 5A, in the legend: "The survivals of ASYN A53T LAG3 -/-, LAG3 +/and LAG3 +/+ mice were similar (Mantel-Cox log-rank test, p-value = 0.165)." **Referee Cross-commenting** I agree with reviewers 1 and 3, especially with the suggestions made by reviewer 1, which should be instituted. I think we all concur that the paper should be published without new experiments. I believe testing a-synuclein propagation in vivo in LAG3 KO mice would be useful, but given the complete lack of replication of LAG3 expression in brain and of a-synuclein binding to LAG3, this is not necessary.
We considered running experiments in addition to those performed in vivo in ASYN A53T transgenic mice (including LAG3 KO) and ex vivo in organotypic slices, the latter using pre-formed fibrils. However, the outcome of these experiments, along with the absence of LAG3 expression in neurons and its unclear binding, convinced us that the usage of further animals and reagents would be unwarranted.
Reviewer #3 (Evidence, reproducibility and clarity (Required)): Have you conducted any experiments of the propagation of PFFs of α-synuclein in LAG3-KO mice ? If they did, what were the results ?
We did consider the possibility of replicating the experiments using PFFs in LAG3 KO mice. However, as stated above, we felt that our experiments -including the survival study in vivo in ASYN A53T transgenic mice -were unambiguous. After critical consideration, we remained unconvinced that this additional experiment would change the weight of our evidence in a substantial manner that would justify the inoculation of other animals and the utilisation of more resources. Thank you for the submission of your manuscript to EMBO Molecular Medicine. I have now had a chance to carefully read your manuscript and the point-by-point response to the concerns raised by Review Commons referees. I also discussed your work and your response to the referees' comments with the other members of our editorial team. We are satisfied with the modifications made and would like to invite a minor revision of your manuscript.
We noticed that in your point-by-point response, some of the referees' comments are missing. Please make sure that you include all the comments from referees, including the *Referee Crosscommenting*.
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The findings are important and reported clearly. The experiments are conducted in a rigorous way by numerous participating laboratories.
Reviewer #1 (Significance (Required)): Very high significance, both from a molecular biology and clinical standpoints. This is an important manuscript that challenges the findings and conclusions of a prior high-profile paper in Science by Ma et al 2016, claiming that LAG3 is a receptor for aggregation-prone species of alpha-synuclein and that deletion of LAG3 results in reduced cell to cell propagation of alphasynuclein aggregates.
The experiments in this paper are numerous and employ a variety of techniques. The overall conclusions are that LAG3 is not expressed by the relevant neurons and that LAG3 is not a receptor for alpha-synuclein fibrils (of different sizes). Therefore, the authors conclude that LAG3 is unlikely to play a role in the spread of alpha-synuclein pathology in Parkinson's disease and related disorders.
There are, however, some weaknesses. For example, the Introduction contains passages that are not written in a stringent way: 1. "Histologically, PD is characterized by α-synuclein aggregates known as Lewy Bodies in neurons of the substantia nigra," That is not a good description of PD neuropathology. Lewy pathology is present in numerous areas of the CNS and PNS, and is not restricted to the substantia nigra.
We have added a more detailed account: "Histologically, PD is characterized by α-synuclein inclusions known as Lewy Bodies whose accumulation is associated with neurodegeneration (Dickson, 2012; Mullin and Schapira, 2015; Corbillé et al., 2016). These inclusions affect the Substantia nigra and other mesencephalic regions as well as, in some cases, the amygdala and neocortex (Dickson, 2018)." 2. "Growing evidence suggests that α-synuclein fibrils spread from cell to cell". While alpha-synuclein pathology can spread from cell to cell, it is not known if the fibrils are the species (alone or combined with other conformers) that cause the spreading of the pathology in a seeding fashion, or if smaller alpha-synuclein assemblies play that role.
We have reformulated the sentence to credit the fact that we do not know which synuclein species is the one that is transmitted: ." This sentence gives the impression that the corresponding author has led the field when it comes to alpha-synuclein's prionid properties. That is not really the case, and it would be 25th Jun 2021 1st Authors' Response to Reviewers appropriate to cite the literature in a more scholarly fashion that reflects how this part of the alphasynuclein research field developed.
I cannot disagree, and in fact I suspect that the present paper may be my second and possibly last experimental contribution to the synuclein field! However, I do claim intellectual parenthood of the prionoid (not "prionid") concept, which I first expounded in a 2009 Nature paper. Anyway, we now provide a more balanced citation: 4. "Interrupting transmission of a-synuclein may slow down or abrogate the disease course." This is a bold statement and far from certain. While one might propose that this is the case, it is still just a hypothesis and the Introduction should reflect that.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This study conclusively shows that LAG3 is not the receptor for a-synuclein that underlies the spread of synucleinopathic damage in various PD-related conditions. The paper is done extremely carefully and comprehensively. My only suggestion is to indicate the significance level in Figure 5a, as it may turn out that LAG3 is actually protective. We have added the significance level in Fig. 5A, in the legend: "The survivals of ASYN A53T LAG3 -/-, LAG3 +/and LAG3 +/+ mice were similar (Mantel-Cox log-rank test, p-value = 0.165)." Reviewer #2 (Significance (Required)): This study is of extremely high significance -we need mechanisms to deal with spectacular results in the literature that should not have been published because they are were uncompelling to begin with, but were published for various sociological/political reasons. Science won't progress if we don't find correction mechanisms for wrong conclusions. **Referee Cross-commenting** I agree with reviewers 1 and 3, especially with the suggestions made by reviewer 1, which should be instituted. I think we all concur that the paper should be published without new experiments. I believe testing a-synuclein propagation in vivo in LAG3 KO mice would be useful, but given the complete lack of replication of LAG3 expression in brain and of a-synuclein binding to LAG3, this is not necessary.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):
It was proposed that LAG3 is important in the treatment of PD and related disorders, because it functions as a receptor of pathogenic α-synuclein and the treatment with anti-LAG3 antibodies attenuated the spread of pathological α-synuclein and drastically lowered the aggregation in vitro (Mao et al, Science 2016).
In this study, authors characterized 8 antibodies to LAG3 and investigated the presence of LAG3 in cultured cell lines, NSC-derived neural cultures, or organ homogenates for the presence of human or murine LAG3. But it was not detected in any of the neuronal samples tested. In addition, single cell (sc) RNAseq yielded only minimal counts for the LAG3 transcript in neurons, astrocytes, and mixed glial cells, and single-nucleus (sn) RNAseq human brain dataset for LAG3 expression across different cell types confirmed no LAG3 signals for any of 34 identified cell clusters, including 13 clusters of excitatory and 11 subtypes of inhibitory neurons, oligodendrocytes, oligodendrocyte precursor cells, microglia, astrocytes, and endothelial cells.
Authors also analyzed the binding of LAG3 with α-synuclein in mouse and human model systems, and concluded that the affinity of LAG3 for α-synuclein fibrils, if any, is micromolar or less.
Furthermore, authors studied the propagation of pre-formed fibrils (PFFs) of α-synuclein in neural stem cell (NSC)-derived neural cultures in the presence or absence of LAG3, and the impact of LAG3 on survival in ASYNA53T transgenic mice expressing wild-type LAG3 as well as hemizygous or homozygous deletions thereof. However, they were unable to see any significant role for LAG3 in these in vitro and in vivo models of α-synucleinopathies.
In this connection, the reviewer would like to ask one question: Have you conducted any experiments of the propagation of PFFs of α-synuclein in LAG3-KO mice ? If they did, what were the results ?
We did consider the possibility of replicating the experiments using PFFs in LAG3 KO mice. However, as stated above, we felt that our experiments -including the survival study in vivo in ASYN A53T transgenic mice -were unambiguous. After critical consideration, we remained unconvinced that this additional experiment would change the weight of our evidence in a substantial manner that would justify the inoculation of other animals and the utilisation of more resources. **Minor point** In Page 10, I think it's a typo: ASYYN mice must be ASYN mice.
Thank you for pointing this out. We corrected it.

Reviewer #3 (Significance (Required)):
These negative findings about the LAG in α-synucleinopathies shown in this manuscript do not provide any new insight into the mechanisms of α-synuclein propagation. However, it is clear that LAG3 is not expressed in neuronal cells and the binding of LAG3 to α-synuclein fibrils appears limited. Overexpression of LAG3 in cultured human neural cells did not cause any worsening of αsynuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data shown in this manuscript are convincing and the information is very important in terms of correcting the direction of disease treatment and research. **Referee Cross-commenting** 30th Jun 2021 1st Revision -Editorial Decision 30t h Jun 2021 , Thank you for submitting your revised manuscript to EMBO Molecular Medicine, and thank you for addressing the editorial issues. I am pleased to inform you that we will be able to accept your manuscript pending the following amendments: 1. in the main manuscript file: -please provide up to 5 keywords and incorporate them in the main text.
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Can you please let us know whether you are okay with the modified text and resized image, or if you would like to introduce future modifications?
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-Since this study does not generate large-scale datasets, please only include the following sentence in this section-"This study includes no data deposited in external repositories". Done.
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6. We would also encourage you to include the source data for figure panels that show essential data. Numerical data should be provided as individual .xls or .csv files (including a 5th Jul 2021 2nd Authors' Response to Reviewers tab describing the data). For blots or microscopy, uncropped images should be submitted (using a zip archive if multiple images need to be supplied for one panel). Additional information on source data and instruction on how to label the files are available at https://www.embopress.org/page/journal/17574684/authorguide#sourcedata We are eager to provide everything helpful to the community. We have now put together a zip file that includes all source files. 7. I only made a minor change in the synopsis text (see attached). I have also adjusted your synopsis image to the requested resolution (see attached).
Can you please let us know whether you are okay with the modified text and resized image, or if you would like to introduce future modifications?
We are fine with the modified text.
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All further communications concerning your paper proofs should quote reference number EMM-2021-14745-V3 and be directed to the production office at embopressproduction@wiley.com. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
Is there an estimate of variation within each group of data?

Reporting Checklist For Life Sciences Articles (Rev. June 2017)
This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript.

Journal Submitted to: EMBO Molecular Medicine
Corresponding Author Name: Adriano Aguzzi YOU MUST COMPLETE ALL CELLS WITH A PINK BACKGROUND ê B-Statistics and general methods the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.

definitions of statistical methods and measures:
a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).

The data shown in figures should satisfy the following conditions:
Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
We adhered to best practice guidelines established in our field of science and chose sample sizes accordingly.
graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
We used standard statistical methods for analyses in all animal experiments. N were estimaed based on our previous work We have not excluded samples or animals from our analyses. The criteria would have been preestablished as is standard in the field.
In all analysis includng animal experimentation, the experimenter was blinded.
In all analysis includng animal experimentation, the experimenter was blinded.
In all analysis includng animal experimentation, the experimenter was blinded.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.