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  1. 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 would like to thank the editor for the opportunity to submit our revised manuscript “Neuron- derived Thioredoxin-80: a novel regulator of type-I interferon response in microglia”. We thank the reviewers for their thorough analysis and thoughtful insights, we have considered all thequestions and issues aroused and modified the manuscript where appropriate and all changes in the manuscript are highlighted in yellow. We hope that this new improved version will be suitable for publication in an affiliated journal of Review Commons. Please find below a point-to-point description of the changes and the experiments that we plan to carry out.

    Reviewer #1:

    Major Comments:

    1.This work is potentially interesting, but the results are very preliminary. There is not a clear demonstration of the signaling pathways triggered by oxidative stress and leading to Trx80 production in neurons. The authors claim the role of Nrf2, but did not silence Nrf2, nor demonstrated the cascade downstream Nrf2 that is responsible for Trx80 production.

    This is a very valuable point raised by the reviewer. To better characterize the role of Nrf2 and the downstream cascade responsible for Trx80 production, we are currently running the following experiments:

    • We are silencing Nrf2 in neuronal primary cultures prior to 27-hydroxycholesterol (27-OHC) or Rotenone treatment. We will then measure Trx1 and Trx80 protein levels. We expect to see significant decrease in the protein levels of both Trx1 and Trx80 in control conditions, and a lack of effect of 27-OHC and rotenone in inducing an increase in their levels.

    Additionally, to confirm in our model that, as previously reported, ADAM10/17 α-secretases are responsible for the cleavage of Trx1 into Trx80, neuronal primary cultures will be treated with GW280264X, an inhibitor of ADAM10 and ADAM17 prior to 27-OHC or rotenone treatment. We expect to observe a decrease in the amount of Trx80 produced, whereas Trx1 protein levels should still increase in presence of 27-OHC and rotenone. We believe that these experiments will help to confirm the pathway responsible for the increased production of Trx80 downstream Nrf2 activation by 27-OHC or rotenone.

    Additionally, we will be more specific in the description of the oxidative-stress related signaling pathways that we describe from our RNAseq data, and determine whether know downstream targets of Nrf2 are indeed changing their expression levels upon 27OH treatment in neurons.

    1. In addition, Cyp27Tg mice show higher Trx80 levels only at a very old age and it is not at all shown that oxidative stress is responsible for Trx80 enhanced production in this mice model.

    We would like to point out that most of the studies regarding the oxidative effects caused by 27-OHC have been carried out in vitro, where it promotes the activation of cell survival pathways that appear to be modulated by Reactive Oxygen Species (ROS) (Vurusaner et al., 2018). Moreover, in vitro treatments with this oxysterol induce the expression of Nrf2 through extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt pathways (Vurusaner et al., 2016). Nrf2 is a transcription factors for many antioxidant proteins including heme oxygenase-1 (HO-1) that has also been found to be elevated upon 27-OHC treatment (Dasari et al., 2010). Despite this evidence in vitro, no study so far has evaluated 27-OHC-mediated oxidative stress in vivo.

    • To further clarify the pathways responsible for Trx80 production and its effects in Cyp27Tg in vivo we will perform fluorescence-activated-nuclei sorting (FANS) of neurons (Neun+), microglia (neun-, pu1+) and astrocytes (eaat1+, neun-) from 22 month old Cyp27 tg mouse cortex, followed by RNAseq analysis.
    1. The authors claim a role of Trx80 in promoting IRM phenotype in microglia. However, there is not any data showing its relevance in Alzheimer`s (AD progression).

    The role of IRMs in the brain is not yet completely understood. However, it has been reported that DNA damage (Hartlova and colleagues 2015) as well as amyloid plaques containing nucleic acids (Roy et al 2020) induce type-I interferon response in microglia. Dorman and colleagues have recently shown that type-I interferon responses in microglia can rapidly induce phagocytosis of damaged neurons (Dorman et al 2022). An increased phagocytic activity by microglia would also explain the decrease in amyloid-beta (Ab) previously reported in a Drosophila model overexpressing both human Trx80 and Ab42 (Gerenu et al., 2019).

    There are studies showing that type-I interferons and IRMs are present in human brain and actively play a role in aging and Alzheimer’s Disease (AD) (Roy et al 2020)(Olah et al., 2020). One potential model to explain the role of IRM and their relevance for AD is that exposure to damage associated molecular patterns (DAMPs) or secreted Trx80 from stressed neurons promote a type I-interferon response in microglia that subsequently triggers an autocrine loop that enhances phagocytic efficiency. Under physiological conditions, this mechanism might play an important role at dealing with bacterial and viral infections in the brain as well as removing debris and damaged and apoptotic neurons to keep a healthy brain homeostasis. However, these responses can become pathological if sustained high IFN-I levels trigger a exacerbated microglial response that leads to widespread cell death and neuroinflammation.

    Trx80 has been previously reported to be depleted in AD brains (Gil-Bea et al., 2012) and to decrease in APPNL-G-F mice, a mouse model of amyloid pathology as amyloid accumulation worsens. This pathology is characterized by a generalized loss of neurons, which as we show in our study, are the main producers of Trx80. Moreover, an increasing accumulation of amyloid pathology might as well explain the decrease in Trx80 and its effects on microglia. Evidence supporting this possibility come from studies showing that the presence of amyloid-plaques promote the generation of disease-associated microglia, which are transcriptionally different from IRMs (Sala-Frigerio et al., 2019)(Keren-Shaul et al., 2017). This shift in microglia phenotype might be preceded by a shift in the signaling mechanism that governs microglial functions, from a reduction in the production and secretion of neuronal Trx80 to the generation of a different type of signaling molecules that promote disease-associated microglial functions.

    Nevertheless, we agree with the reviewers that the main current limitation of this work is that it ha s been mainly performed in vitro. To better understand the involvement of Trx80 in regulating microglia function in vivo and its relevance in an AD context, we will:

    • Induce Trx80 production in neurons in vivo by performing stereotaxic injections in the prefrontal cortex of adeno-associated virus (AAVs) carrying the Trx80 sequence under the neuronal promoter synapsin, that will allow for Trx80 overexpression exclusively in neurons. We will analyze the effects of neuronal Trx80 overexpression on surrounding microglia by determining the expression of IRM signatures both by immunofluorescence and RT-qPCR.

    • We will also use this system to analyze the effects of Trx80 in APPNL-G-F mice, where we will determine the effects of Trx80 overproduction in neurons on amyloid pathology in the Trx80-transduced hemisphere compared to the opposite hemisphere of the same mouse that will be transduced with control virus. These mice develop plaques at 3 months, we will therefore perform injections in 2 month-old mice and determine the effects of Trx80 at 1, 2 and 4 weeks post-transduction.

    1. They show that Trem2 silencing in vitro prevents Trx80-dependent expression of genes characterizing IRM phenotype in microglia. Notably, they did not show any data about the expression of these genes in 3 and 10 months old APPNL-G-F mice, not in young and 22 months old Cyp27Tg. Enhanced Trx80 levels in 22 months old Cyp27Tg do parallel enhanced expression of IRM markers?

    We did not further look at the interferon response genes in the APPNL-G-F mice because their presence in this mouse model was previously reported at a single cell resolution in microglia (Sala-Frigerio et al., 2019). We will change the text in our manuscript to a more detailed explanation about this previously reported data on IRM gene expression at different ages of the APPNL-G-F mouse.

    Regarding IRM markers in Cyp27Tg mice, we did look at microglia expressing ISG15, an interferon response protein, in 22 months old Cyp27Tg mice and their age matched controls by immunofluorescence. As we report in Figure 4D, we found that 22 months old Cyp27Tg mice had a higher proportion of microglia ISG15 positive. Looking at other markers was limited due to antibody availability and specificity.

    As we mention in point 2, we will further determine the presence of IRMs by performing FANS of neurons, microglia and astrocytes from 22 month old Cyp27Tg mice. We expect that, even in theIRM population is small, by performing RNAseq analysis in a cell-specific manner, we will be able to find an increase in IRM molecular signatures in microglia.

    1. Is there brain inflammation in 22 months old 22 months old Cyp27Tg?

    We appreciate the point raised by the reviewer. 27-hydroxycholesterol (27-OHC) has been previously described to induce inflammation in the periphery (Umetani et al., 2014) as well as in the brain in the form of S100A8-RAGE signaling pathway (Loera-Valencia et al., 2021). However, to confirm this in our experimental model, we will run an V-PLEX Plus Mouse Cytokine 19-Plex Kit of inflammatory markers by ELISA (MSD). This panel will allow us to accurately measure the levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-15, IL-17A/F, IL-27p28/IL-30, IL-33, IP-10, KC/GRO, MCP-1, MIP-1α, MIP-2 and TNF-α in 22 months old Cyp27Tg and age-matched control brain homogenates from the prefrontal cortex.

    This point will further be clarified by the FANS-RNAseq experiment described above.

    1. Decreased levels of Trx80 in 10 months old APPNL-G-F mice do parallel decreased levels of IRM markers compared to age matched control mice?

    According to Sala-Frigerio and colleagues that evaluated the gene expression profile of microglia isolated from APPNL-G-F mice at single-cell resolution, IRM population progressively increases with age (Sala-Frigerio et al., 2019). This suggests that several factors other than exposure to Trx80, including DNA-damage accumulation, that has been previously reported to be associated with an IRM-like response in human brains (Mathys et al., 2017) might as well promote and sustain the IRM phenotype. Further research will be therefore necessary to fully understand the finely-tuned mechanisms that regulate microglia states, both with temporal and spatial resolution.

    alterations. The authors need to clarify the functional relevance of their data, so several experiments are necessary.

    We thank the reviewer for her/his comment, and we agree that studying the functional relevance of this system will help to greatly improve the quality of this work.

    As described in point 4., we will determine the functional relevance of Trx80 in vivo and whether it influences amyloid-beta-induced alterations by performing stereotaxic injections of AAVs carrying Trx80 before the first plaques appear in the mouse and at different time-points post transduction(1,2 and 4 weeks) to determine how Trx80-overexpression induced reactive microglia in vivo alters amyloid pathology-derived alterations.

    Reviewer #1:

    1. Minor comments: Figure 3f: in the text is written "neurons", while in the figure legends is written microglia.

    We apologize for this mistake and we have now changed the text accordingly (p.11, l. 258).

    CROSS-CONSULTATION COMMENTS

    Microglia are quite resistant to viral transduction so the 50% knockdown by siRNA further raises the question of their identity in culture

    We would like to apologize for the misunderstanding regarding the Trem2 silencing transduction since it is missing in the methodology part. We did not use viral transduction, we used siRNA mediated transduction (Horizon SMARTpool siRNA) as it has been previously and successfully used in primary microglia cultures (Ruan et al., 2022). We have now added this information to the methods part of the manuscript (p.6, l.137-142).

    The subject of study is potentially very interesting because of investigating the role of Tx80 in microgliaand showing that Trx80 acts through Trem2, which is implicated in AD. Microglia and oxidative stressare considered playing a key role in AD progression. However, data are very preliminary and thismanuscript does not present data showing the functional relevance of Trx80 in AD.

    • We agree and thank the reviewer for her/his comments. We believe that the newly planned experiments describe above will help to address the function of Trx80 in vivo and its relevance in an AD context (by determining its effect in the APPNL-G-F mouse model of amyloid pathology) will help to greatly improve this study.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Goikolea and colleagues aim to describe how neuronal thioredoxin-80 (Trx80)influences microglial reactivity. Because Trx80 levels track with age and amyloid pathology, understanding this signaling axis provides insight into the pathogenesis of AD. The authors show that pyramidal neuron expression of Trx80, rather than its precursor, is upregulated in aging and the APP mouse model of AD. They show that Trx80 induces interferon response gene induction in microglia cultures and that knockdown of Trem2 prevents this. This work has the potential to be very impactful.

    Minor points:

    One wonders if the discrepancy between gene, precursors, and Trx80 is due to lack of degradationof Trx80 with age. If anything is known about this, the authors might comment on its regulation inthe discussion.

    We apologize for the misunderstanding. We will improve our explanation on Trx80 regulation in thediscussion.

    Methods for siRNA knockdown appear to be missing.

    We apologize for this mistake. We have now included it in the text p.6, l.137-142).

    Reviewer #2 (Significance (Required)):

    This work has the potential to advance our understanding of how a known anti-oxidant Trx80 contributes to microglial states. Given the major limitation of being an in vitro study, the extrapolation of these findings into AD pathogenesis are not possible.

    We agree and thank the reviewer for her/his comments. We believe that the newly planned experiments describe above will help to address the function of Trx80 in vivo and its relevance in an AD context (by determining its effect in the APPNL-G-F mouse model of amyloid pathology)will help to greatly improve this study in this regard.

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  2. 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

    In this manuscript, Goikolea and colleagues aim to describe how neuronal thioredoxin-80 (Trx80) influences microglial reactivity. Because Trx80 levels track with age and amyloid pathology, understanding this signaling axis provides insight into the pathogenesis of AD. The authors show that pyramidal neuron expression of Trx80, rather than its precursor, is upregulated in aging and the APP mouse model of AD. They show that Trx80 induces interferon response gene induction in microglia cultures and that knockdown of Trem2 prevents this. This work has the potential to be very impactful.

    The major limitation of this work is that it is conducted strictly in vitro or ex vivo - without return to the invivo state with cell-specific knockout of Trx80 and subsequent analyses of microglial phenotypes. This is of particular importance given that microglia are exquisitely sensitive to manipulation and there is increasing evidence that in vitro states (especially not those derived from mixed glial cultures) are not representative of true in vivo production. Microglia are quite resistant to viral transduction so the 50% knockdown by siRNA further raises the question of their identity in culture. If the authors cannot manipulate Trx80 in vivo in a cell specific way, they might consider using more highly purified more invivo like cultures such as those championed by Bohlen et al, Neuron, 2018.

    Minor points:

    One wonders if the discrepancy between gene, precursors, and Trx80 is due to lack of degradation of Trx80 with age. If anything is known about this, the authors might comment on its regulation in the discussion Methods for siRNA knockdown appear to be missing.

    Significance

    This work has the potential to advance our understanding of how a known anti-oxidant Trx80 contributes to microglial states.

    Given the major limitation of being an in vitro study, the extrapolation of these findings into AD pathogenesis are not possible.

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  3. 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

    Summary:

    The manuscript entitled "Neuron-derived Thioredoxin-80: a novel regulator of type-I interferon response in microglia" demonstrates that neurons are the major source of Trx80, derived from the cleavage of Trx, into the brain. Trx80 production increases during normal aging. On the contrary, APPNL-G-F mice shows an opposite trend compared to wild type mice: Trx80 levels are significantly higher in 3 months old mice compared to age matched littermate, while significantly decreased in 10 months old APPNL-G-F mice compared to controls. The authors revealed that oxidative stress induced Trx80 production in neurons and this effect is Nrf2-dependent. Indeed, rotenone - an oxidative stress inducer- enhanced both Nrf2 expression and Trx80 levels. In agreement, 27-hydroxycholesterol (27-OHC), which promotes oxidative stress, promoted Trx80 production and Trx2 expression in neurons. In addition, 22 months old 27-OHC over-producing (Cyp27Tg) mice showed enhanced levels of Trx80. By gene expression analysis, the authors reported that Trx80 treated microglia showed a IRM gene expression profile, because of an enhanced expression of genes considered as IRN markers. They demonstrated in vitro that Trx80 promotes the IRN phenotype in microglia through Trem2. Knock down of Trem2 in microglia prevented Trx80-mediated enhanced expression of IRM markers.

    Major Comments:

    This work is potentially interesting, but the results are very preliminary. There is not a clear demonstration of the signaling pathways triggered by oxidative stress and leading to Trx80 production in neurons. The authors claim the role of Nrf2, but did not silence Nrf2, nor demonstrated the cascade downstream Nrf2 that is responsible for Trx80 production. In addition, Cyp27Tg mice show higher Trx80 levels only at a very old age and it is not at all shown that oxidative stress is responsible for Trx80 enhanced production in this mice model.

    The authors claim a role of Trx80 in promoting IRM phenotype in microglia. However, there is not any data showing its relevance in Alzheimer`s (AD progression). They show that Trem2 silencing in vitro prevents Trx80-dependent expression of genes characterizing IRM phenotype in microglia. Notably, they did not show any data about the expression of these genes in 3 and 10 months old APPNL-G-F mice, not in young and 22 months old Cyp27Tg. Enhanced Trx80 levels in 22 months old Cyp27Tg do parallel enhanced expression of IRM markers? There is brain inflammation in 22 months old 22 months old Cyp27Tg? Decreased levels of Trx80 in 10 months old APPNL-G-F mice do parallel decreased levels of IRM markers compared to age matched control mice? Which is the significance of this pathway in AD?

    It may be interesting to analyze whether microglia pre-treatment with Trx80 alters Abeta-induced alterations. The authors need to clarify the functional relevance of their data, so several experiments are necessary. Methods and data are sufficiently described.

    Minor comments:

    Figure 3f: in the text is written "neurons", while in the figure legends is written microglia

    Referees cross-commenting

    Comments to reviewer 2 opinion: "The major limitation of this work is that it is conducted strictly in vitro or ex vivo - without return to the invivo state with cell-specific knockout of Trx80 and subsequent analyses of microglial phenotypes. This is of particular importance given that microglia are exquisitely sensitive to manipulation and there is increasing evidence that in vitro states (especially not those derived from mixed glial cultures) are not representative of true in vivo production. Microglia are quite resistant to viral transduction so the 50% knockdown by siRNA further raises the question of their identity in culture. If the authors cannot manipulate Trx80 in vivo in a cell specific way, they might consider using more highly purified more invivo like cultures such as those championed by Bohlen et al, Neuron, 2018." I agree that in vivo experiments are necessary. Moreover, also the in vitro studies presented are preliminary.

    Significance

    The subject of study is potentially very interesting because of investigating the role of Tx80 in microglia and showing that Trx80 acts through Trem2, which is implicated in AD. Microglia and oxidative stress are considered playing a key role in AD progression. However, data are very preliminary and this manuscript does not present data showing the functional relevance of Trx80 in AD. The audience that maybe interested in the subject proposed by the authors are scientist working on: AD, aging, oxidative stress, microglia activation.

    My fields of expertise: Alzheimer, oxidative stress, TXNIP function, inflammation, neurodegeneration, microglia, gene expression

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