C. Wright Mills' idea of "fringe-thoughts" is similar to Ahrens framing of "fleeting notes".

LER NOVAMENTE: Explicação de pq usar e( o que é pesquisa qualitativa), para responde a 3 sub-questão

Aqui é citado que em outra pesquisa foi descoberto que o desenvolvimento de sites acessíveis não é necessariamente custoso, mas pode vir a ser se a preocupação em cumprir os requisitos de a11y ser relizada no final do processo de desenvolvimento. (O que torna relevante a prática de testes de a11y durante todo o desenvolmento de sites)

O foco prático da pesquisa de forma resumida

O foco prático da pesquisa: "Dado os testes já aplicados por um conjunto de devs em projetos web financiados publicamentes, na Finlandia, quais técnicas de testes de acessibilidades podem ser aplicadas as atuais práticas de testes desses times de devs?"

Probably DHbox link should be https://dhbox.commons.gc.cuny.edu/

Objetivo

Reading this passage reminded me of the creation story in the Hawaiian culture. I have heard of a few other creation stories --- from the Bible, from my own culture, and from the academic culture. In all of them, I agree that it tells you a lot about how each of these groups of people understand and shape their own reality, how they choose to exist, and how they choose to move forward in life.

I think this is a very intelligent question that not even most people have the answer to. I think religion can tie into politics very easily, and it is. People do not want to admit that something like Christianity, which is suppose to be only good, can cause issues or have negative effects on the world. I still don't even fully have an answer for this question, which is why it makes it such a good one.

Stories are beautifully balanced in that there is both liight and dark within them. They can be told in beautiful metaphoprical ways but also used for deceit. It alligns directly with the good in bad in Adam and Eve. In the Native story, there is good and bad throughout the process.

I thought this as something important to highlight because as kids we think that we can go past the moon and back but as we get older we see that we can't go that far in reality but we can always imagine them, hence where the stories come in.

El texto se me hace muy ameno ya que explica muy en particular el cómo desarrollar un protocolo.

I agree that there is no education that is politically neutral because every class is taught by a human and every human is somewhat biased. However, it is teachers' and school's responsibility to work against curriculums that has anything hints of segregation.

I agree that there is no education that is politically neutral. Every class is taught by a human and every human is biased. It is the school's responsibility to keep curriculums as neutral as possible and to keep staff and workers work against segregation.

The text is saying that education is not neutral – it's political. For example, teaching only the works of "great white men" is a political choice. The challenge highlighted is that many people tend to ignore the political aspects of teaching, like racism or sexism. It emphasizes the ongoing effort needed to make people understand and acknowledge the political influences in education.

Racism is a difficult subject to talk about but there must be a conversation about it or else it's never going to go away. Even if it makes people uncomfortable. Many can go their entire life without thinking about racism because they are not affected by it and therefore don't consider it seriously.

o conciliador deve manter as partes informadas, sobre direitos e deveres, não havendo surpresa

Poor college students face a double challenge: not only do they have to deal with the stigmatization that comes with admission, but they also have to deal with a variety of dilemmas stemming from socioeconomic disparities. This includes negative comments, social isolation, economic challenges, and the difficulty of preparing for graduate or professional school without the support of an advanced academic background.

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

The manuscript describes the synergy among PI3Kbeta activators, providing compelling results concerning the mechanism of their activation. The particular strengths of the work arise to a great extent from the reconstitution system better mimicking the natural environment of the plasma membrane than previous setups have. The study will be a landmark contribution to the signaling field.

Public Reviews:

Reviewer #1 (Public Review):

The manuscript aims to provide mechanistic insight into the activation of PI3Kbeta by its known regulators tyrosine phosphorylated peptides, GTP-loaded Rac1 and G-protein beta-gamma subunits. To achieve this the authors have used supported lipid bilayers, engineered recombinant peptides and proteins (often tagged with fluorophores) and TIRF microscopy to enable bulk (averages of many molecules) and single molecule quantitation. The great strength of this approach is the precision and clarity of mechanistic insight. Although the study does not use "in transfecto" or in vivo models the experiments are performed using "physiologically-based" conditions and provide a powerful insight into core regulatory principles that will be relevant in vivo.

The results are beautiful, high quality, well controlled and internally consistent (and with other published work that overlaps on some points) and as a result are compelling. The primary conclusion is that the primary regulator of PI3Kbeta are tyrosine phosphorylated peptides (and by inference tyrosine phosphorylated receptors/adaptors) and that the other activators can synergise with that input but have relatively weak impacts on their own.

Although the methodology is not easily imported, for reasons of both cost and the experience needed to execute them well, the results have broad importance for the field and reverse an impression that had built in large parts of the broader signalling and PI3K communities that all of the inputs to PI3Kbeta were relatively equivalent, however, these conclusions were based on "in cell" or in vivo studies that were very difficult to interpret clearly.

Reviewer #2 (Public Review):

The manuscript of Duewell et al has made critical observations that help to understand the mechanisms of activation of the class IA PI3Ks. By using single-molecule kinetic measurements, the authors have made outstanding progress toward understanding how PI3Kbeta is uniquely activated by phosphorylated tyrosine kinase receptors, Gbeta/gamma heterodimers and the small G protein Rac1. While previous studies have defined these as activators of PI3Kbeta, the current manuscript makes clear the quantitative limitations of these previous observations. Most previous quantitative in vitro studies of PI3Kbeta activation have used soluble peptides derived from bis-phosphorylated receptors to stimulate the enzyme. These soluble peptides stimulate the enzyme, and even stimulate membrane interaction. Although these previous studies showed that the release of p85-mediated autoinhibition unmasks an intrinsic affinity of the enzyme for lipid membranes, they ignored what would be the consequence of these peptide sequences being present in the context of intrinsic membrane proteins. The current manuscript shows that the effect of membrane-conjugated peptides on the enzyme activity is profound, in terms of recruiting the enzyme to membranes. In this context, the authors show that G proteins associated with the membranes have an important contribution to membrane recruitment, but they also have a profound allosteric effect on the activity on the membrane, These are observations that would not have been possible with bulk measurements, and they do not simply recapitulate observations that were made for other class IA PI3Ks.

An important observation that the authors have made is that Gbeta/gamma heterodimers and RAc1 alone have almost no ability to recruit PI3Kbeta to the membranes that they are using, and this is central to one of the most profoundly novel activation mechanisms offered by the manuscript. The authors propose that the nSH2- and Gbeta/gamma binding sites partially overlap, so that Gbeta/gamma can only bind once the nSH2 domain releases the p110beta subunit. This mechanism would mean that once the nSH2 is engaged by membrane-conjugated pY, the Gbg heterodimer can bind and increase the association of the enzyme with membranes. Indeed, this increased membrane association is observed by the authors. However, the authors also show that this increased recruitment to membranes accounts for relatively little increase in activity, and that the far greater component of activation is due to an allosteric effect of the membrane association on the activity of the enzyme. The proposal for competition between Gbg binding and the nSH2 is consistent with the behavior of an nSH2 mutant that cannot bind to pY and which, consequently, does not vacate the Gbg-binding site. In addition to the outstanding contribution to understanding the kinetics of activation of PI3Kbeta, the authors have offered the first structural interpretation for the kinetics of Gbg activation in synergy with pY activation. The proposal for an overlapping nSH2/Gbg binding site is supported by predictions made by John Burke, using alphafold multimer. Although there is no experimental structure to support this structural model, it is consistent with HDX-MS analyses that were published previously.

Reviewer #1 (Recommendations For The Authors):

1. The approx relative concentrations (surface densities ) of Rac1-GTP, GBetagammas and PY-peptides used in experiments in Fig 1 are not easy to understand and useful to give an intuitive feel for the relative sensitivity of the PI3Kbeta reporter to those inputs.

In our revised manuscript, we provide densities of the individual signaling inputs used to reconstitute Dy647-PI3Kβ membrane recruitment (see Figure legend 1). We provide a more detailed explanation about our quantification method in subsequent figures where the membrane surface density of signaling inputs is varied to modulate the strength of PI3Kβ membrane localization and activity.

Building off the quantification of Rac1-GTP and pY membrane density measurements presented in our initial manuscript submission, we now include an estimate of the GβGγ membrane density. For these new measurements, we recombinantly expressed and purified additional SNAP-GβGγ protein, which we fluorescently labeled with AlexaFluor 555. The membrane surface density of GβGγ was quantified at equilibrium using a combination of AF488-SNAP-GβGγ (bulk signal) and dilute AF555-SNAP-GβGγ (0.0025%), which allowed us to resolve and count the single molecule density (Figure 3A). We calculate the total surface density of GβGγ based on the AF555-SNAP-GβGγ dilution factor. In the methods section titled, “surface density calibration,” we describe our protocol.

1. The estimates of the PIP3 concentrations/densities measured using the BTK reporter seem good but its unclear (to me) how they were derived.

The density of PI(3,4,5)P3 lipids in our supported lipid bilayers was calculated based on the incorporation of a define molar ratio of PI(3,4,5)P3 in our small unilamellar vesicles. Based on the average footprint of 0.72 nm2 for a single lipid, we calculated the density of lipids per µm2. In the methods section titled, “kinetic measurements of PI(3,4,5)P3 lipid production,” we include the following description:

“Assuming an average footprint of 0.72 nm2 for phosphatidylcholine (Carnie et al., 1979; Hansen et al., 2019), we calculated a density of 2.8 × 104 PI(3,4,5)P3 lipids/μm2 for supported membranes that contain an initial concentrations of 2% PI(4,5)P2. We assume that the plateau fluorescence intensity of the AF488-SNAP-Btk sensor following reaction completion in the presence of PI3Kβ represents the production of 2% PI(3,4,5)P3. The bulk membrane intensity of AF488-SNAP-Btk was normalized from 0 to 1, and then multiplied times the total density of PI(3,4,5)P3 lipids to generate kinetic traces that report the kinetics of PI(3,4,5)P3 production.”

Minor points

l164; Rac1(GTP) AND GBeta gammas. In this context it should be OR. Or have I misunderstood?

l1093; kineticS measurementS.

Thank you for pointing out these typos. We made the appropriate edits.

The paper of Suire etal (Suire, S., Lécureuil, C., Anderson, K. E., Damoulakis, G., Niewczas, I., Davidson, K., Guillou, H., Pan, D., Jonathan Clark, Phillip T Hawkins, & Stephens, L. (2012). GPCR activation of Ras and PI3Kc in neutrophils depends on PLCb2/b3 and the RasGEF RasGRP4. The EMBO journal, 31(14), 3118-3129. https://doi.org/10.1038/emboj.2012.167) make the point that in vivo it appears that although Ras-activation is required for full activation of PI3Kgamma (and can activate PI3Kgamma in vitro directly) if you use tools to activate Ras in the absence of receptor and Gbetagamma signalling, it has no affect on PIP3 . This directly supports the authors conclusions.

Thank you for sharing this citation. We incorporated the reviewer’s insight into our discussion section to broaden the significance of our work.

Reviewer #2 (Recommendations For The Authors):

There are only a few relatively minor points that could be addressed to improve the paper:

1. Why is the density still going up after 10 minutes in Figure 1 Figure supplement 2? Doesn't this seem like a very long time? Are we seeing fast on/off combined with fast on/slow off? Are the particles eventually becoming stuck in odd places or are they slowly denaturing?

Our movies do not indicate a slow accumulation of immobilized or stuck Dy647-PI3Kβ particles on the membrane surface. On the long timescale, we believe that a small fraction of Dy647-PI3Kβ molecular do exhibit longer dwell times on membranes containing a high density of pY (>6,000 molecules/µm2). This is likely due to membrane hopping of Dy647-PI3Kβ. In other words, rather than Dy647-PI3Kβ dissociating from the membrane surface directly into the solution, the Dy647-PI3Kβ molecule immediately rebinds to another membrane conjugated pY peptide. This type of behavior of a peripheral membrane binding protein is generally correlated with there being a higher surface density of the binding partner (Yasui et al., 2014). Characterization of potential Dy647-PI3Kβ membrane hopping will require additional experimentation (e.g. PI3Kβ mutants) and quantitative analysis that goes beyond the scope of this study.

1. Lines 188-189. "By quantifying the average number of Alexa488-pY particles per unit area of supported membrane we calculated the absolute density of pY per μm2 (Figure 2D). I think this should be Figure 2C, right hand y-axis.

Thank you for identifying our typo. We’ve corrected the text for clarity.

1. Lines 102-193. "When Dy647-PI3Kβ was flowed over a membrane containing a low density of {less than or equal to} 500 pY/μm2, we observed rapid equilibration kinetics consistent with a 1:1 binding stoichiometry (Figure 2E).” There is no density shown in Fig. 2E. There is only "membrane intensity." Perhaps it was their intent to include a right-hand axis with density (number of particles/area), as they did in Figure 2C. However, they did not, so Figure 2E does not support the text. The value of Intensity/#py/um**2 does not appear to be the same for Figure 2C as for Figure 2E, assuming that the statement in the text is correct. The authors should include the density as a right-hand axis in 2E.

We have reworded this portion of the results section for clarity. In reading the reviewers comment, we recognize that a more convincing way to support our claim of a 1:1 binding stoichiometry would be to show that there are ~500 Dy647-PI3Kβ/μm2 membrane bound complexes when the pY surface density equals ~500 pY/μm2. For us to make this connection, we would need to perform experiments using a Dy647-PI3Kβ concentration that fully saturates all the binding pY binding sites. However, at this elevated Dy647-PI3Kβ solution concentration, individual Dy647-PI3Kβ complexes can start to bind to a single phosphotyrosine of the dually phosphorylated peptide due to competition for pY binding sites. As an alternative to performing the experiment described above, we can infer binding stoichiometry from the shape of the membrane absorption kinetic traces. For example, a simple bimolecular interaction exhibits rapid equilibration kinetics with a hyperbolic shaped kinetic trace. Systems that have more complex binding equilibria, however, generally take longer to equilibrate (due to the change in KOFF) and can often be broken down into 2 or 3 distinct dissociation constants (KD). This type of kinetic analysis has previously been used to describe multivalent membrane binding interactions for the Btk-PI(3,4,5)P3 (Chung et al., 2019) and PI3Kγ-GβGγ (Rathinaswamy et al., 2021) complexes. Considering that there are multiple interpretations of the Dy647-PI3Kβ membrane absorption traces show in Figure 2E, we refrain from saying that our results explicitly reveal a 1:1 binding stoichiometry. Instead, we provide several possible explanations for the results. Ultimately, additional experiments and kinetic modeling of wild type and mutant PI3Kβ is necessary to define the binding stoichiometry under different conditions.

1. Table 1. The authors have analysed the data to extract two dwell times and two diffusion coefficients. The legend should make this clear, referring to D1 as the slow diffusion component and D2 as fast diffusion, similarly, there are short and long dell times. This should be stated in the legend. There are two columns labelled "alpha". This presumably should be alpha1 and alpha2, the fractions of particles with short and long dwell times. The table legend should clarify this.

In our revision, additional text has been added to the figure legends and Table 1.

Text from Table 1: “Alpha (α) equals the fraction of molecules with the characteristic dwell time, τ1 (DT = dwell time). The fraction of molecules with the characteristic dwell time, τ2, equals 1-α. Alpha (αD) equals the fraction of molecules with the characteristic diffusion coefficient, D1. The fraction of molecules with diffusion coefficient, D2, equals 1-αD.”

1. In the legend for Figure 5 figure supplement 1, for part D, the "Cumulative membrane of binding events..." The "of" should be deleted.

Thank you for identifying this typo.

1. Lines 423-426: "We found that PI3Kβ kinase activity is also relatively insensitive to either Rac1(GTP) or GβGγ alone. This is in contrast to previous reports that showed Rho-GTPases (Fritsch et al. 2013) and GβGγ (Katada et al. 1999; Hashem A. Dbouk et al. 2012; Maier, Babich, and Nürnberg 1999) can activate PI3Kβ, albeit modest, compared to synergistic activation with pY peptides plus Rac1(GTP) or GβGγ." It is not clear what this statement means. On the surface, it might be interpreted as saying that these previous studies had some flaw that led the authors to conclude that there is some activation caused by Rac1 or Gbeta/gamma on their own. The current manuscript is an important contribution to understanding the mechanism of synergistic activation, but it is also true that the Hansen and his colleagues have not used the same membranes as were used previously. The authors state that they have used a wide range of membrane compositions, but the only ones that have appeared in the manuscript are nearly pure PC (with 2% PIP2) or PC with 20% PS. Extensive studies with varying membrane compositions are beyond the scope of the current study, since the current manuscript concisely makes important observations regarding mechanism. However, it would be helpful for readers if the authors at least mention the differences in membrane compositions among the studies.

The reviewer raises an important point concerning our interpretation of PI3Kβ activation data in relationship to existing literature. In our original submission, we made conclusions concerning how individual signaling inputs modulate PI3Kβ activity, without showing all our data or providing sufficient explanation. In our revised manuscript, we include PI3Kβ kinase activity measurements performed in the presence of either pY, Rac1(GTP), or GβGγ alone (Figure 5B-5C). These experiments were reconstituted on supported membranes in the absence or presence of 20% PS lipids. We found that increasing the density of anionic lipids increased the overall activity of PI3Kβ in the presence of pY or GβGγ alone. This is consistent with a subtle increase in PI3Kβ membrane affinity due to the negatively charged PS lipids. Mutations that disrupt the direct interaction between PI3Kβ and GβGγ eliminated the observed lipid kinase activity. We were unable to detect PI3Kβ activity in the presence of Rac1(GTP) alone. In conclusion, we’re able to detect some PI3Kβ activity in the presence of GβGγ alone, which is consistent with previous reports (Dbouk et al., 2010; Katada et al., 1999; Maier et al., 2000). In the future, a more comprehensive analysis will be required to map the relationship between PI3Kβ activity, membrane localization, and lipid composition. For example, previous reconstitutions have revealed differential activation of PI3Kα that depends on the most abundant lipid being phosphatidylethanolamine (PE) rather than phosphatidylcholine (PC) (Hon et al., 2012; Ziemba et al., 2016). PE lipids comprise 25-30% of the cellular plasma membrane (Yang et al., 2018) and have been used in previous studies to measure PI3K lipid kinase activity on small unilamellar vesicles (Dbouk et al., 2010; Hon et al., 2012).

In this study, we elected to use a simplified membrane composition that minimized non-specific membrane localization of fluorescently labeled PI3Kβ. This allowed us to more clearly define the strength of individual and combinations of protein-protein interactions that regulate PI3Kβ localization and kinase activity. When reconstituting amphiphilic molecules (i.e. lipids) in aqueous solution a variety of structures, including micelles, inverted micelles, and planar bilayers can form based on the lipid composition (Kulkarni, 2019). The organization of these membrane structures is related to the molecular packing parameter of the individual phospholipids (Israelachvili et al., 1976). The packing parameter (P=v⁄((a•l_c))) depends on the volume of the hydrocarbon (v), area of the lipid head group (a), and the lipid tail length (l_c). When generating supported lipid bilayers on a flat two-dimensional glass surface, we aim to create a fluid lamellar membrane. We find that phosphatidylcholine (PC) lipids are ideal for making supported lipid bilayers because they have a packing parameter of ~1 (Costigan et al., 2000). In other words, PC lipids are cylindrical like a paper towel roll. In contrast, cholesterol and phosphatidylethanolamine (PE) lipids have packing parameters of 1.22 and 1.11, respectively (Angelov et al., 1999; Carnie et al., 1979). This gives cholesterol and PE lipids an inverted truncated cone shape, which prefers to adopt a non-lamellar phase structure. Due to the intrinsic negative curvature of PE lipids, they can spontaneously form inverted micelles (i.e. hexagonal II phase) in aqueous solution when they are the predominant lipid species (Israelachvili et al., 1980; Kobierski et al., 2022; Wnętrzak et al., 2013). In the methods section of our manuscript, we note that from our experience incorporation of PE lipids dramatically reduces the protein-maleimide coupling efficiency, displayed more membrane defects, and resulted in a larger fraction of surface immobilized Dy647-PI3Kβ. This could be related to the intrinsic negative curvature of PE membranes. However, further investigation is needed to decipher these issues.

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This point confuses me, does this mean the text is better defined by the average reader or just scholars?

DOI: 10.1016/j.molcel.2023.12.007

Resource: AB_2934034

Curator: @scibot

SciCrunch record: RRID:AB_2934034

What is this?

DOI: 10.1016/j.celrep.2023.113658

Resource: (Thermo Fisher Scientific Cat# 12-7321-82, RRID:AB_466199)

Curator: @scibot

SciCrunch record: RRID:AB_466199

What is this?

I understand what Viramontes is trying to say here because of how descriptive she is. Her word choice of describing the mother helps readers and myself to create our own thoughts and images about hear. When I read this, I felt heartbroken. The way Viramontes relates her veins to a grape vine made my mind think about the women and men. They are working so harvesting and producing these grape vines that they don't even have time to take care of themselves.

Very interesting how the first line went straight into setting the idea of this make believe future.

Claiming the holy site in Mecca as their own, they have no further raised themselves over the other faiths. They do not want those under other religions to share this holy site they wish to maintain it for themselves, and they succded in their conquest. They also use this as a way to conquer mecca within the boundaries of their faith.

This breaks the 2nd amendment which states that free men are allowed to carry with them ammunition of some sort. However, the "free men" in this situation were not really free, to be considered eligible for this rule.

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

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Reply to the reviewers

1. General Statements We appreciate the insightful reviewer comments. Both reviewers alluded to the logical lack of connection between two themes in the original paper. Specifically, we showed that N-cad differentially regulates migration in different environments, and that leader and follower cells differ phenotypically, but did not connect the two themes. In this revised version, we've performed additional experiments and undertaken a comprehensive reorganization of both the manuscript and figures. The major changes are outlined below:

2. Figure 4 A-C has been moved to Figure 6 F-H.

3. Figure 5 has been moved to Figure S3 F-H.
4. Figure 6 F has been moved to Figure 7 A.
5. Figure 6 G-H have been moved to Figure 7 D-E.
6. Figure 6 I-J have been moved to Figure S5 A-B.
7. Figure 7 C-F have been moved to Figure S5 C-F.
8. Added transcriptome profiling of control and N-cad-depleted cells and of leader and follower cells (Figures 6 E, S1 H and S4 C-D, Tables S2 and S3). We have incorporated additional figures (Figure 4 and 5 in the revised manuscript) to support the idea that the amount of N-cad at the cell surface is regulated by endocytic recycling, thereby stimulating glioma migration in the different local environments. Furthermore, our new findings showed that YAP1/TAZ regulates the surface level of N-cad during glioma migration (Figure 8). We trust that these additions contribute to the clarity and robust justification of our paper.

Similar to other types of tumors, our findings revealed that pediatric high-grade gliomas migrate collectively, possibly contributing to a more aggressive invasion than single cells. In this study, we found that N-cad mediates this collective glioma migration. Interestingly, within these migrating groups, leader and follower cells dynamically interchange positions during migration, accompanied by changes their phenotypic characteristics. This suggests that differences in phenotypes, including N-cad recycling, proliferation and YAP activation, may be predominantly regulated by cell-extrinsic factors rather than being predetermined by genetic or epigenetic factors. Moreover, our new RNA-sequencing results indicate minimal difference between leader and follower cells, except for the upregulation of YAP response and wound healing migration genes in leader cells. Although genomic alterations still possibly encode the leader-follower exchange, our findings strongly suggest that the activation of YAP1 and glioma migration are regulated by the cellular context, specifically where cells are located within the group.

Contrary to our initial findings suggesting a positive feedback loop between N-cad endocytosis and nuclear YAP1, our revised data indicates that nuclear YAP appears to be independent of N-cad. We observed that homotypic interactions with N-cad present in the surrounding environment, such as neurons (Figure 6 C-D) or N-cad extracellular domain-coated surface (Figure 7 B-C), did not affect nuclear YAP1. However, YAP1/TAZ depletion decreased N-cad expression and altered its localization at the surface (Figure 8). This leads us to propose a revised model where nuclear YAP1 stimulates surface N-cad, thereby facilitating the distinct modes of migration on ECM and neurons (Figure 8 I).

1. Point-by-point description of the revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

In this manuscript, Kim and colleagues describe the role of N-Cadherin during pediatric glioma migration. They compare cell lines that have similar transcripts but different levels of N-Cadherin protein and find that N-Cadherin levels influence the route of migration - whether it be on ECM or other tissues. They also describe molecular feedback between N-Cadherin and YAP in leader vs follow cells of their systems. The data are clear, well presented, and convincing; and the conclusions described by the manuscript are mostly justified. My major criticism of the manuscript is that the line of questioning undertaken does not appear well justified. At many points, I was left asking "but why are they doing this?" and I could not understand the rationale for some of the experiments that were performed (even if they were performed well). The manuscript opens by validly describing how gliomas are highly invasive, poorly understood and that N-Cadherin was highly expressed in comparison to other adhesion proteins. This opened the path for the questions and experiments performed that contributed to Figures 1-3, which I thought were interesting. From there on, I found the logic of the story unclear and poorly justified. For example, I do not know why leader and follower cells were justified - when it had nothing to do with N-Cadherin which was the focus of the work prior. And then, having rightly concluded in Figure 4 that the data suggested that leader and follower cells dynamically exchange positions rather than being pre-determined, they went onto further figures focusing on differences between leader and follower cells, which left my quite confused. I am likewise confused by the model proposed in that, they authors describe that the difference between leader and follower cells contributes to a nuclear YAP/N-Cad endocytosis feedback loop that feeds into the speed of migration. Yet, the authors describe earlier that leader and follower cells frequently exchange positions, with no evidence that they are pre-determined. How do the authors square these seemingly conflicting points? And further, what is the relevance of this to understanding the differing modes of migration (on ECM or other tissues)? On this issue, I suggest authors re-consider whether the order of figures or logic of the story is appropriate (perhaps consider moving some figures to supplement?), and to clearly justify in the text the elements that are being addressed. Overall, I think the messaging, logic and justification could be use significant improvement; the experiments however are well performed, and the figures are very clear and nicely presented, and I don't have any qualms about them.

We appreciate your insightful comments, recognizing the need for logical and justifiable improvements in certain sections of our previous manuscript. Please see Section 1, General Statements, for an explanation of changes made.

Minor Comments

1. Not required, but the authors may wish to consider putting t=0 pictures of the experiments in the supplement as supportive evidence for the circles of the initial seeding location they show in Fig 1.

We provide the t=0 images in Figure S1 N and O.

1. I assume the title of the second results section should say "migration speed" rather than "speed migration"

The new title of the second results section is “N-cad stimulates and inhibits migration through intercellular homotypic interaction”.

1. Fig. 4D - Are both example cell pictures leaders? If so, I'm not sure why two have been provided; I'm guessing the bottom set are supposed to be follower cells. If so, please label as appropriate. (And if not, a representative set of pictures from a follower cell should be provided).

We have enhanced the clarity of the labels. We provide representative high magnification images of leader and follower cells. The updated figure can be found in Figure 5 A.

1. Figure 5 Legend - the title of this figure is too definitive, and exaggerates further than the main text does, which was correct in saying that the experiments only suggest that N-Cadherin endocytosis might regulate the localisation of b-catenin and p120-catenin. Probably I would go further and say that there is no experimental evidence provided that even suggests that in the first place, and that this is a hypothesis that remains to be tested. The authors should inhibit endocytosis specifically (rather than just depleting N-Cad) and see the effect, to justify their conclusion.

We appreciated your points and concerns. Following your earlier suggestion, we have moved the figure to the supplementary section (Figure S3 F-H). Moreover, we have addressed the reciprocal regulation of N-cad and catenins by knocking down p120-, β- or α-catenin. Our new findings showed that p120-, β- or α-catenin depletion decrease the amount of N-cad at the cell surface, not steady-state protein level, resulting in decreased migration on astrocytes but increased migration on ECM (see Figure 4). These findings support the idea that catenins play a role in glioma migration according to the environment by altering surface N-cad level. With that, we updated the figure title to “Catenins regulate N-cad surface levels to stimulate or inhibit migration.”

Reviewer #1 (Significance (Required)):

The manuscript provides a characterised of invasive glioma migration that was previously lacking. It also provides interesting observations related to the role of N-Cadherin for different modes of migration (on ECM or on tissues) that will be of interest for further exploration. It makes a good advance in terms of addressing a highly invasive cell type that has poor prognosis. I anticipate that now this initial characterisation has been performed, authors and others will be interested in gaining a deeper understanding as to how these two modes of migration are controlled, how there might be interplay between them and how such findings contribute to its highly invasive nature. I have expertise in collective cell migration and directed cell migration.

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

Summary In the submitted manuscript, Kim et al. describe various aspects of N-cadherin function in the collective migration of PBT-05 cells, a pediatric high-grade glioma line, on laminin, 3D-matrigel, neurons or astrocytes. N-cadherin promotes the collective migration on neurons or astrocytes, whereas it suppresses the migration on laminin or 3D-matrigel. The authors also show that N-cadherin is actively internalized and recycled in the leader, but not follower, cells of the collective, which induce the nuclear accumulation of YAP/TAZ proteins. YAP/TAZ proteins are shown to regulate the collective migration.

Thank you for the comments. Please see Section 1, General Statements, for a summary of changes made. Please also note that our new experiments failed to show that N-cad levels or traffic regulate YAP/TAZ nuclear accumulation. Rather, YAP/TAZ are regulated by cell density independent of N-cad, and YAP/TAZ regulate N-cad protein levels and traffic independent of changes in N-cad RNA levels

Major comments

1. In Fig. 1G, N-cadherin knockdown seems to affect the distribution of astrocytes. The authors should stain a marker for astrocytes, instead of actin, and the red alone images should be provided.

Astrocytes were cultured for 4 days to generate 3D scaffolds before adding the glioma spheroid, essentially as described (Gritsenko et al., Histochem Cell Biol, 2017). Co-cultures were stained for human-specific vimentin (glioma) or actin (glioma and astrocytes) (see Figure 1 G and separate channels in new Figure S1 P). There do not appear to be major changes in astrocyte organization outside the migration front, but we lack a way to stain for astrocytes specifically and cannot visualize astrocytes under the glioma cells. It remains possible that astrocytes may be affected differently by contact with control and N-cad-deficient glioma cells. However, we added a new experiment, assaying migration on decellularized astrocyte ECM. While N-cad stimulated migration on astrocytes it inhibited migration on astrocyte ECM (Figures 1 I and J and S1 Q). Thus N-cad stimulates glioma migration on astrocyte cells and not their ECM.

1. The colocalization between N-cadherin and Rab11 may not be high in Figs. 4F and S2B. It is unclear whether the majority of the internalized N-cadherin is recycled to the plasma membrane. In Fig. S2B, the internalized N-cadherin may be located mainly at the early endosomes before transported to the recycling endosomes (Is it 20 min after the N-cadherin antibody internalization?). First, the authors should analyze the colocalization between the N-cadherin and Rab11 at 30-40 min after the internalization. If the colocalization with Rab11 would be still low at that time point, some of the internalized N-cadherin might be degraded in the lysosomes. To test this possibility, the authors should analyze the colocalization between N-cadherin and LAMP1 under the treatment with a lysosome inhibitor.

At steady state, N-cad co-localized better with Rab5 than with Rab11 or LAMP1 (Figure 5 C-D). In kinetics experiments, N-cad antibodies were internalized for 40 min. They colocalized better with Rab5 or EEA1 than with Rab11 or LAMP1. When we allowed recycling for an additional 20 min, the surface level of N-cad antibodies partially recovered in leader cells more than follower cells (see Figures 5 G and S3 D). We tested whether treatment with lysosomal inhibitors would increase co-localization of N-cad with Rab11 in recycling endosomes. Surprisingly, however, Chloroquine or Bafilomycin A1 decreased the amount of internalized N-cad antibody in leader and follower cells, and long-term treatment did not increase total N-cad levels. Therefore, the fate of internalized N-cad in follower cells remains unclear.

1. When N-cadherin is depleted, dissociated single cells are increased, but these cells are not well characterized. A high magnification image of the dissociated single cells is required. In addition, the migration speed of the dissociated single cells should be measured.

We didn’t quantify single cell migration because only a minority of cells separate from the collective even when N-cad is depleted. Therefore, we quantified migration directionality and speed for cells at or near the front of collective migration (Figure 2 D-I). We have updated the image of single cells, providing representative high-magnification images in Figure S1 N and O.

1. In Fig. S2D, treatment with Pitstop-2 alone or Dyngo-4a alone is required. Dynamin is also involved in clathrin-independent endocytosis and N-cadherin is reported to be internalized via caveolin-1-mediated endocytosis as well as clathrin-mediated during neuronal migration. It would be better to clarify which type of endocytosis occurs in the leader cells.

We have removed results showing inhibition of cell migration and N-cad endocytosis by Pitstop-2 and Dyngo-4a from the paper. Treatment with either chemical alone had much less effect on internalization or migration than adding both together (see figure below). This is hard to explain. Pitstop-2 should inhibit clathrin-coated pit formation and Dyngo-4a should inhibit clathrin and caveolin-mediated endocytosis. Caveolin-1 and 2 transcripts were not detected in our cells (Table S2). There may be some other form of clathin-independent endocytosis. Interpretation is also challenging since these inhibitors will inhibit endocytosis of many receptors, not just N-cad. Accordingly, we have removed these results in the revised manuscript.

1. In Fig. 2, N-cadherin depletion disturbs the migration directionality. Is this a result from disruption of cell polarity? To test this, the position of centrosome or Golgi or lamellipodia in the leader cells should be analyzed. (OPTIONAL)

We elected not to perform this analysis.

1. I cannot understand the significance of Fig. 5F and 5G. If the authors would speculate that alpha- and beta-Catenins may transduce the intracellular signaling from the internalized N-cadherin, the authors should perform the knockdown experiments of the Catenins and analyze whether it may affect the nuclear accumulation of YAP/TAZ. (OPTIONAL)

We agree. In the initial manuscript, we showed that N-cad depletion altered the localization of p120-, β-, and α-catenin (previously shown in Figure 5 F-G). For better clarity and logic, these figures have been moved to Figure S2 H in the revised manuscript. Additionally, to test whether catenins regulate N-cad and YAP1, we depleted p120-, β-, or α-catenin using shRNA. We found that downregulation of p120-, β-, or α-catenin decreased N-cad surface levels, consequently slowing migration on astrocytes and stimulating migration on laminin (Figure 4). In other words, depleting catenins altered migration in parallel with the changes in N-cad surface level. Catenin depletion also increased single-cell dissociation, reduced the crowding of leader and follower cells, and increased nuclear YAP1 (see figure below). These findings suggest that the main role of p120-, β-, or α-catenin is to regulate surface N-cad. Since this result does not support a role for catenins transducing an N-cad signal to YAP1, we have not included it in the paper.

Minor comments

1. The quantitative data is required in Fig. 5E.

Quantitative data from three independent experiment are now presented in Figure S2 G.

1. Vinculin is associated with the cadherin-catenin complex and it may not be a good loading control (Fig. 3C and 3L).

The Western blot data has been updated and is now presented in new Figure 3 B and 3 F, with β-tubulin as a loading control.

**Referees cross-commenting**

I totally agree with the other Reviewers' comments and evaluation. As the reviewer-1 pointed out, I also think the experiments are well performed, but it would lack logic at least in part (see my comment-6). In addition, as the reviewer-3 pointed out, the linking mechanism of N-cadherin homophilic interaction with YAP/TAZ signaling is important to improve this manuscript

We hope the revisions have improved the logical flow. We have also added new results showing that YAP/TAZ regulate N-cad protein levels and localization but not N-cad RNA. N-cad is not needed for cell density-dependent regulation of YAP1 localization. The model is shown in Figure 8 I.

Reviewer #2 (Significance (Required)):

Strength N-cadherin has multiple function in cancer and neuronal migration, and both positive and negative effects of N-cadherin on cancer cell migration have been reported. In this regard, different behaviors of N-cadherin in the leader and follower cells of the collective are interesting and may explain the controversial previous results.

Limitation This study reveals various aspects of N-cadherin function in the collective migration of the glioma cell line, but it is unclear whether these findings are applied to pediatric high-grade gliomas in vivo.

Thus, this study is a potentially important and informative to cell biologists and researchers in cancer biology, although this reviewer also found several weak points that should be improved.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this manuscript, the authors explore the role of N-cadherin in the migratory/infiltrative behavior of human pediatric brain tumor cells, in light of their surrounding microenvironment. Their in-depth phenotype analysis allows to document the behavior of migrating cells and revisit the concept of leading/follower migratory cells (somehow more commonly applied to endothelial cells). They suspected that the YAP/TAZ pathway might modulate N-cadherin endocytosis and vice versa, using imagery-based cell tracking.

Major comments

1. To control for co-culture models, migration should be evaluated on decellularized matrices from astrocyte and neuron cultures.

We thank for your suggestion. We tested glioma migration on astrocyte-derived decellularized matrices. The mouse astrocytes we used are known to produce various extracellular matrices with a composition similar to Matrigel, except for laminin α5. (Gritsenko et al., J Cell Sci, 2018). N-cad shRNA cells migrated faster on decellularized ECM than control (Figure 1 I-J and S1 Q). This result agrees with N-cad depletion increasing migration on ECM but is opposite to migration on astrocytes.

1. N-cadherin was stably knocked down with shRNA, which raises the question of adaptative/compensatory mechanisms. First, one could ask what happen in knockout conditions. Similarly, transient siRNA-mediated silencing might help to strengthen the findings. Second, is there any impact of Ncad knock down on alternate adhesive receptors (either cell-cell or cell-ECM). This should be verified with bulk RNAseq.

Transient knockdown with N-cad siRNA also increased migration on laminin-coated surface (Figure S1 L-M). Unfortunately, N-cad depletion with siRNA was short-lived, precluding its use for long-term assays, like coculture with neurons or astrocytes. To test whether there is any impact of N-cad knockdown on alternative adhesion receptors, we performed RNA-Seq (Figure S1 H, Table S2). We found N-cad depletion did not alter expression of other cell-cell and cell-ECM adhesive receptors except CDH3 (2.8-fold increase compared with 7-fold decrease in CDH2). Integrin expression was unchanged.

1. It would be interesting to evaluate the impact of N-cadherin/N-cadherin homotypic interactions on YAP/TAZ signaling, using for instance N-cad-coated surface.

We observed that the homotypic interaction of N-cad with surrounding neurons and astrocytes did not hinder the accumulation of nuclear YAP1 in leader cells (Figure 6 C-D). To further support the idea that N-cad does not directly regulate YAP1 signaling, we have now measured YAP1 localization in cells migrating over N-cad ECD. The new data confirms that N-cad does not directly regulate YAP1 localization (Figure 7 B-C).

1. along this line, the impact of mechanical cues (stiffness, 2D vs 3D) is not explored.

We appreciate your suggestion. It is possible that different mechanical and cytoskeletal cues between leader and follower cells affect YAP1 signaling. In this study, we would like to focus more on the role of N-cad-mediated cell adhesions in YAP signaling.

Minor comments

1. Levels of N-cadherin expression in normal Astro and Neurons to compare with pediatric brain cancer cells (S1C)

A new western blot analysis to show N-cad levels in DMG, PHGG and mouse cerebellar neurons and astrocytes has been added to Figure S1 F.

1. Low versus high density culture conditions should be controlled and its further impact on the YAP/Ncad endocytosis route should be supported experimentally, or to be omitted from their proposed model.

We previously used different size of micropattern discs to control low or high cell density. Smaller cell clusters, with more edge cells and hence fewer cell-cell interactions, had higher nuclear YAP1 (Figure 7 D-E). We have repeated this experiment, including N-cad ECD antibodies to measure N-cad endocytosis. Smaller cell clusters had higher N-cad antibody internalization (Figure 7 F). Together with our evidence that leader cells have higher YAP1 and more N-cad internalization than followers, and that YAP/TAZ knockdown inhibits N-cad internalization, these results high YAP/TAZ in leader cells regulates N-cad internalization.

Reviewer #3 (Significance (Required)):

This paper presents robust image analysis of human pediatric brain tumor migration in the context of the different microenvironment that they might encounter (matrices, neurons, astrocytes). This study brings new concepts on the way N-cadherin might contribute to tumor cell migratory behavior based on the nature of the interactions in which N-cadherin is involved. As a limitation, it remains unclear the mechanism by which N-cadherin endocytosis is driven.

We now discuss the limitations of the study as follows:

“The mechanisms by which YAP1 regulates N-cad levels and trafficking remain to be explored. YAP1 is widely expressed in human brain tumors and strongly associated poor survival. Leader cells expressed higher levels of YAP1-response and wound-healing gene transcripts, but transcript levels of N-cad and proteins known to regulate cadherin traffic, such as p120-catenin, Rab5/11 and Rac1, were similar. Therefore, N-cad is likely regulated at the level of protein synthesis or turnover. More endosomal N-cad recycled to the surface of leader than follower cells, implying that follower cells might divert more N-cad for lysosomal degradation, but our attempts to interfere with N-cad endocytosis or degradation specifically were unsuccessful. Further understanding of the mechanism and function of N-cad recycling for glioma cell migration will require cargo-specific ways to selectively regulate endocytosis and recycling”.

Overall, this article had a lot of interesting and bold points. The incorrect usage of Hawaiian diacritics, however made the paper a little harder to read as it did not flow as well as it could if it was spelt correctly. Moreover, the italicization of Hawaiian words made words and phrases in Hawaiian seem like a foreign language. That is to say, that when the author uses quotes from Hawaiian sources, they did not use quotation marks around the Hawaiian, only the English, such as this example. This was present throughout the paper.

Etapas essenciais para a construção de uma pesquisa.

Segundo o autor GIL (2004), uma pesquisa deve ter alguns critérios para estabelecer um tema, são eles: espacial, temporal e populacional.

É um tipo em que o problema da pesquisa será resolvido com algum tipo de intervenção.

É um tipo de pesquisa em que o pesquisador participa ativamente, diferente de só observar de longe.

É um tipo de pesquisa, principalmente feita pelas humanas, em que o pesquisador consulta referências bibliográficas, o que já é imprescindível para qualquer pesquisa. Porém, existem pesquisas feitas inteiremante só de bibliográfia, para isso da-se o nome de revisão bibliográfica.

Pesquisa que foca em um objeto de estudo bem delimitado e específico.

Um tipo de pesquisa onde se faz uma análise de causa e efeito, pode ser de campo ou de laboratório.

I do not think we need o include some.

The discussion section of a paper is an opinion

definitions of silence

summary of Rappaport's arguments

Quizas aquí tocaría un punto y aparte para pasar a la interpretación espacial. Si no quieres punto y a parte, puedes empezar la frase con un "On the other hand," "From the spatial perspective," o algo así, como para indicar que sigues discutiendo los mismos resultados pero desde otra perspectiva.

Uma pesquisa ainda pode ser descrita como básica e aplicada. A básica é uma pesquisa mais ampla, sem nenhuma aplicação prática, já a aplica utiliza os conhecimentos da pesquisa básica para trazer aplicabilidade no mundo real e solucionar um problema específico.

Para a pesquisa quantitativa, as variavéis, números e dados se sobressaem das questões sociais espeficíficas que possam ter na pesquisa.

Existe a abordagem qualitativa e a quantitativa. A qualitativa foca nas informações sociais, o problema em questão não são os números e varíaveis, ao contrário da quantitativa.

Tipos de metódos de pesquisa.

Author Response

The following is the authors’ response to the original reviews.

Reviewer 1 (Public Review):

Weakness: Although the cross-links stimulate ATP hydrolysis, further controls are needed to convince me that the TM1 conformations observed in the structures are physiologically relevant, since they have been trapped by "large" substrates covalently-tethered by crosslinks.

Our response: Reviewer 1 raised concerns about the relatively large size of our covalently attached AAC substrate that would potentially distort TM1 in Pgp. We would like to clarify that AAC has a molecular weight of 462 Da, which, in comparison to many known Pgp substrates ranging from 250 to over 1,000 Da, is not a large compound. For instance, the few other Pgp substrates mentioned in our manuscript all have a comparable or larger size: verapamil, 455 Da; doxorubicin, 544 Da; FK506, 804 Da; valinomycin, 1,111 Da; cyclosporin A, 1,203 Da.

Furthermore, AAC was strategically attached to a site distant from TM1 in the inwardfacing Pgp conformation. After it was exported to the outward-facing state, several TM helices accommodate the compound. The observation that only TM1 exhibited significant conformational changes suggests its potential role in the transport mechanism. This hypothesis is supported by our findings, where a conservative substitution (G72A) in TM1 resulted in a dramatic loss of transport function for various drug substrates and impaired verapamil-stimulated ATPase activity.

Reviewer 1 (Recommendations for the Authors):

I understand the need for an unconventional approach to understanding the translocation pathway. What would help to support this model is to cross-link a much smaller substrate, as the one used is quite large and could potentially distort TM1 in the outward-state when cross-linked.

Our response: We thank the reviewer for this recommendation, and we have outlined plans for future experiments involving other substrates, including smaller ones, to further investigate our proposed model. However, it is important to acknowledge that conducting these studies will require a significant amount of effort and resources, which we believe extend beyond the scope of our current manuscript.

In unbiased MD simulations starting from the IF state are there any simulations where the substrate follows the same path as proposed here?

Our response: All our MD simulations were performed in the outward-facing state to focus on potential substrate release pathways. Starting MD simulations from the inwardfacing state would introduce complexities in capturing the necessary domain motions and nucleotide binding and hydrolysis required for substrate translocations. Therefore, we opted not to perform MD studies starting from the inward-facing state.

Reviewer 2 (Public Review):

Weakness: There is much to like about the experimental work here but I am less sanguine on the interpretation. The main idea is to covalently link via disulfide bonds a model tripeptide substrate under different conditions that mimic transport and then image the resulting conformations. The choice of the Pgp cysteine mutants here is critical but also poses questions regarding the interpretation. What seems to be missing, or not reported, is a series of control experiments for further cysteine mutations.

Our response: Reviewer 2 raised concerns about the interpretation of our results and suggested the need for additional mutant designs to validate our proposed TM1 mechanism. Firstly, we believe that the observed TM1 conformational changes are valid in our cryoEM structures, despite the use of different conditions and several mutants to capture Pgp in the outward-facing state.

Regarding the G72A mutant, we consider it conclusive that this single point mutation in the TM1 has a profound effect. Importantly, the G72A mutant was readily expressed and purifiable as a stable protein. We were able to resolve a high-resolution structure of the G72A mutant (without the substrate), confirming that the protein is not generally destabilized but properly folded.

Above all, we appreciate the Reviewer’s suggestion to explore additional mutations and intend to do so in future studies.

Reviewer 2 (Recommendations for the Authors):

I am sold on the results regarding TM1 conformational changes as they are evident in the cryoEM structures. However, the set of states compared between mutants are not biochemically equivalent: for 335 and 978 they used an ATP-impaired Pgp whereas for 971 they used what appears to be WT, and the conformation was imaged presumably subsequent to ATP hydrolysis and Vanadate trapping. This is significant if the authors were unable to trap the OF in the impaired mutant background and should be highlighted. I have to believe that they tried that condition but I could be wrong.

Our response: We acknowledge the point made by the Reviewer about the biochemical equivalence of mutant states and the potential significance of using an ATP-impaired mutant for trapping the outward-facing conformation of 971. We have not yet attempted to use the ATPase-deficient 971C mutant for crosslinking and intend to address this question in future studies.

In our current approach, we used the ATPase-active 971C for two specific reasons:

1) Our biochemistry data, as shown in Fig 1C, indicates that 971C only crosslinks in the presence of ATP hydrolysis conditions. Vanadate trapping was employed to stabilize the outward-facing conformation.

2) Based on our experience, we have observed that the conformations of ATP-bound (mutant) and vanadate-trapped states of an ABC transporter are structurally equivalent at this resolution level of our study (see ref. 21: Hoffmann et al. NATURE 2019).

The authors propose a new model for substrate translocation. It is based on three mutants and a number of structures. If the authors were not challenging the current dogma I would not have written the next comment. Considering the impact of the findings, I would have designed a couple more cysteine mutants based on their model. For instance, this pathway has a number of stabilizing interactions, can't they make a mutant that preserves conformational switching but eliminates substrate translocation? I like the G97A mutant result but I am worried that the effect could just be a general destabilization or misfolding as part of the cryoEM particles seem to suggest. The authors advance one interpretation of the disorder observed in this mutant but it could easily be my interpretation.

Our response: We thank the reviewer for the suggestion to design additional mutants to further validate our proposed model for substrate translocation. We agree that this would be highly valuable, considering the potential impact of our findings. However, given the time-intensive nature of our approach, we believe that presenting these additional designs in a future study is a reasonable course of action.

Regarding the G72A mutation, we believe that our current data fully supports our model and the role of TM1 in regulating the Pgp activity. Importantly, we would like to emphasize that the G72A mutant was readily expressed and purifiable as a stable protein. Additionally, our cryoEM structural determination of the G72A mutant at high resolution confirmed that the protein is not generally destabilized but properly folded.

There are a couple of troubling methodological questions that I want the authors to address or clarify:

1. In the methods they report that the final sample for cryoEM was prepared on a SEC devoid of detergent. It is obvious that the sample was folded but I was wondering why the detergent was removed? Was that critical for observing these structures with multiple ligands? Did they observe any lipids in their cryoEM?

Our response: We avoid detergent in the buffer on final SEC purification. This step is to remove free detergent from the background which helps during cryoEM imaging. Of course, this cannot be done with every detergent but due to the very low CMC of LMNG it is possible. By now, we have verified this method for several other transporters with the same success. While this procedure helps us to obtain better images it is not necessary to obtain specific conformations or ligand bound states, nor does it affect these states or conformations.

In our cryoEM structures , we did observe multiple cholesterol hemisuccinate (CHS) molecules on the outer transmembrane surface of Pgp.

1. Can the authors comment on why labeling was carried out in the presence of ATP? Does it matter if the substrate was added prior to ATP and incubated for a few minutes?

Our response: For every dataset, we first added the substrate to be cross-linked and afterwards added the ATP. In the cases of 335C and 978C, labeling was successful before ATP was added, as evidenced by the inward-facing structures with cross-linked substrate. However, for 971C, cross-linking only occurred after the addition of ATP. We interpret this data to suggest that the 971 site is inaccessible to the substrate in the inward-facing state, and cross-linking can only occur after the transporter transitions to outward-facing state. This is in line with our inward-facing structure which does not show a cross-linked substrate, and our biochemical data shown in Fig 1C, where 971C only crosslinked in the presence of ATP.

1. I am not an expert on MD simulations and I understand that carrying out simulations at higher temperatures used to be a trick to accelerate the process. Is this still necessary? Why didn't the author use approaches such as WESTPA?

Our response: Most so-called enhanced sampling methods, including WESTPA, explicitly define a reaction coordinate for the process of interest, usually based on intuition or prior studies. If this coordinate is chosen poorly, enhanced sampling usually fails, either because the sampling becomes inefficient or because the sampling biases the transition pathway (or both). Lacking reliable intuition or prior knowledge on which motions would result in substrate release, we chose temperature to speed up the process. High temperature largely avoids the introduction of an any bias through the definition of a progress coordinate. By contrast, the weighted ensemble method underlying WESTPA is a great method to simulate unbiased dynamics of a process with a known progress coordinate, but unfortunately requires to choose a progress coordinate prior to the simulation and will then mostly sample the process along this progress coordinate, because this is the only direction in which sampling is improved. High temperature MD on the other hand accelerates all processes in the system under study. Indeed, we have now confirmed that the pathway found at high temperature is also feasible at near-ambient conditions.

In new simulations, we have now observed a similar release pathway at T=330 K. As the only difference, the substrate has not fully dissociated from the protein after 2.5 us, with weak interactions persisting at the top part of TM1 from the extracellular side. Importantly, this is a configuration observed also in higher temperature simulations but with much shorter lifetime.

In response, we now included these new findings and a new Extended Data Fig. 15 in the revised manuscript.

1. One way to show that the two substrates binding mode is biochemically relevant is to measure Vmax at different substrate concentrations. One would expect a cooperative transition if that interaction is mechanistically important.<br /> Our response: We have measured Vmax as a function of QZ-Ala concentration in a previous report (ref. 24), supporting positive cooperativity for binding to two sites.

Reviewer 3 (Public Review):

We thank Reviewer 3 for recommending the acceptance of our manuscript as is.

Reviewer 3 (Recommendations for the Authors):

Page 4, last line: Pgp302 should be Pgp1302. In addition, I can only encourage the authors to add an additional table to the manuscript. Here, the mutation, the obtained structure(s), IF or OF, the resolution, and the main message should be summarized.

Our response: Following the reviewer’s suggestion, we have added Extended Data Table 2 summarizing the Pgp mutants and respective structural data in the revised manuscript.<br /> We verified that Pgp302 is the correct term on Page 4, last line.

Pg. 5, section 'Covalent ligand design for Pgp labeling', it is mentioned that even in the presence of Mg2+ATP, Pgp302 could not react with AAC-DNPT. Maybe it would be worthwhile to add the data either in Supplementary Information or state 'data not shown'.

Our response: We stated ‘data not shown’ in the text.

Pg. 47, last line : A space is missing between M68, and M74.

Our response: Space was added.

Pg. 7, line 2: The authors mention that a single dataset of ATP-bound Pgp335 revealed three different OF conformations: ligand-free, single-ligand-bound, and double-ligandbound. However, the percentage fraction of each dataset sums up to be more than 100%. Would request the authors to recalculate the fraction size of each conformation.

Our response: We have corrected the error in our calculation, based on the particle distribution in our dataset (OF335-nolig: 1,437,110 particles, 40.4%; OF335-1lig: 1,184,253 particles, 33.3%; and OF335-2lig: 939,924 particles, 26.4%).

Pg 53, Figure legend of Extended Data Fig. 11: Please include the color coding for the helix TM1 and also the residues colored plum.

Our response: We added the color coding for TM1 and other residues in the figure legend.

Pg. 8, line 3: While referring to the structure of OF971-1lig, the authors nicely point towards the conserved residues M74 and F78 which coordinate the ligand. However, in Fig. 3b, residues M74 and F78 should also be indicated.

Our response: We updated Fig. 3b by adding arrows pointing towards the residues M74 and F78.

Pg. 54, Extended data Fig. 12: The authors should adopt a single writing style. In some places, Pgp is referred to as P-gp while in others as Pgp.

Our response: We updated the protein labels in Extended Data Fig. 12.

Pg. 54, Extended data Fig. 12: The authors should clearly mention which OF335 structure (1st panel) was used for visualizing the interactions.

Our response: To clarify, we added the following sentences in the figure legend: “Pgp335 OF in the top panel refers to OF335-1lig. In the bottom panel describing OF335-2lig, the left and right diagrams refer to the binding positions of non-covalent and covalent ligand, respectively”.

Pg. 18, section 'synthesis of dipeptide 8': In the text it is mentioned that for the synthesis of thiazole acid 6, compound 3 was dissolved in a mixture of THF/MeOH/H2O (3:1:1), while in the corresponding figure (Extended Data Fig. 1), the ratio is stated as 5:1:2.

Our response: 3:1:1 ratio is correct. We made the correction in Extended Data Fig. 1.

Pg. 19, section 'synthesis of linear tripeptide 10': Same as above for compounds 10 and 4, respectively.

Our response: We corrected the conditions in the Extended Data Fig. 1 accordingly.

Pg. 20, section 'Synthesis of cyclic peptide 11': There seems to be a discrepancy in the synthesis protocol between the text and the extended figure 1, especially regarding the use of THF/MeOH/H20, followed by NaOH and TFA or only NaOH and TFA.

Our response: we further clarified the conditions of using NaOH in THF/MeOH/H2O (3:1:1) and TFA in DCM in the text for synthesis and Extend Data Fig. 1.

Pg. 40, Extended Data Fig. 1: In the bottom last panel showing the synthesis of peptide 11, the authors have missed showing peptide 10 as the starting material for the reaction.

Our response: Label for the peptide 10 was added following the suggestion.

Pg. 26, third last line: 'o' is missing from the last word cry'o'

Our response: We corrected the typo.

Pg. 63 and 64, Extended Data Table 1: The Cryo-EM data collection, refinement, and validation statistics for OF971-1lig, IF971-1lig, OF978-1lig, and IF978-2lig are mentioned twice in the table.

Our response: This was now corrected in the revision.

Waarom? Het kan uiteraard wel, maar een koppeltabel is eigenlijk alleen noodzakelijk bij een n:n relatie. Bij een 1:n relatie volstaat het om in een database de relatie in de tabel bij het object of gegevensgroep aan de n kant op te slaan ook al ligt de relatie van de 1 naar de n kant. Worden 1:n relaties in de data hub ook gerealiseerd met een koppeltabel of komt daar de relatie ook in de tabel aan de n kant te staan?

I tabel

Misschien ook even uitleggen hoe dat werkt bij 1:n relaties tussen gegevensgroepen onderling of tussen gegevensgroep en object. Bij 1:n relaties wordt er voor de gegevensgroep aan de n kant in de data hub bv. een aparte tabel gemaakt. In dat geval leg je wel een relatie tussen een gegevensgroep (record) en een versie (voorkomen) van een object.

I- tabel

I- tabel

O conhecimento filosófico é fruto das indagações que vem a partir do conhecimento vulgar e mítico-religioso.

Conhecimento vulgar ou senso-comum: É um conhecimento que não precisa de comprovação científica, apesar de ter uma base real de alguma forma.

Outro desmembramento do conhecimento, dessa vez em tácito e explicito. O tácito seria algo que não conseguimos explicar, subjetivo e análogo, exemplo: andar de bicicleta. O explicito é mais racional, possui uma metodologia, exemplo: uma receita de bolo.

Existem duas formas de conhecimento a sensível e a não sensível. A primera está relacionada a consciência de eventos em que despertam meus sentidos físicos. Já a segunda está ligada a percepção intelecto-racional que temos sobre algo.

Dude, I can totally relate!

¿Los ensayos en casa deben estar en doble espacio o en espacio único?

the production over the years of film and the origins of "movies'

O Princípio da unidade de tesouraria se refere à determinação de depósito, em uma única conta, dos recursos arrecadados, com o fito de facilitar a gestão de tais recursos.

No entanto, há a exceção prevista no art. 43, § 1º, LRF quanto à vedação da unificação dos recursos da Previdência Social com os demais entes.

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

The manuscript is very-well written. Although the study is well-conducted the authors should be more convincing on how bacteria residing in tissues do not induce death. The association with IL-10 cytokine production appears weak and more experiments are needed to make it more robust

Reviewer #2 (Public Review):

Iske et al. provide experimental data that NAD+ lessens disease severity in bacterial sepsis without impacting on the host pathogen load. They show that in macrophages, NAD+ prevents Il1b secretion potentially mediated by Caspase11.

While the in vivo and in vitro data is interesting and hints towards a crucial role of NAD+ to promote metabolic adaptation in sepsis, the manuscript has shortcomings and would profit from several changes and additional experiments that support the claims.

Conceptually, the definition of sepsis is outdated. Sepsis is not SIRS, as in sepsis-2. Sepsis-3 defines sepsis as infection-associated organ dysfunction. This concept needs to be taken into account for the introduction and when describing the potential effects of NAD+ in sepsis. Also, LPS application cannot be considered a sepsis model, since it only recapitulates the consequence of TLR-4 activation. It is a model of endotoxemia. Also, the LPS data does not allow to draw conclusions about bacterial clearance (L135).

The authors state that protective effects by NAD were independent of the host pathogen load. This clearly indicates that NAD confers protection via enhancing a disease tolerance mechanism, potentially via reducing immunopathology. This aspect is not considered by the authors. The authors should incorporate the concept of disease tolerance in their work, cite the relevant literature on the topic and discuss it their findings in light of the published evidence for metabolic alteration sand adaptations in sepsis.

For the in vitro data, the manuscript would benefit from additional experiments using in vitro infection models.

In the merge manuscript, the authors provide two different versions of the figures. In one, bar plots are shown without individual data and in the other with scatter blots. All bar plots need to be provided as scatter plots showing individual values.

The authors should show further serology data for kidney and liver failure etc. as well as further cytokine data such as IL-6 and TNF to better characterize their models.

Careful revision of the entire manuscript, the figure legends and figures is required. The figure legend should not repeat the methods and materials section. The nomenclature for mouse protein and genes needs to be thoroughly revised.

L350. The authors write that they dissect the capacity of NAD+ to dampen auto- and alloimmunity. In this work, no data that supports this statement is shown and experiments with autoantigens or alloantigens are not performed.

L163 The authors describe pyroptosis but in the figure legend call it apoptosis. Specific markers for each cell death should be measured and determined which cell death mechanisms is involved.

Animal data comes from an infection model and LPS application. The RNAseq data is obtained from cells primed with Pam3CSK4 and subsequently subjected to LPS. It is unclear how the cell culture model reflects the animal model. As such the link between IFN signaling and the bacterial infection/LPS model are not convincing and need to be further elaborated.

Figure 5: It is unclear how many independent survival experiments were done, how many mice per group were used and whether the difference between groups was statistical significant. This information should be added.

Further experiments with primary cells from Il10 k.o. and Caspase11 k.o. animals should be provided that support the findings in macrophages.

Author Response:

Reviewer #1 (Public Review):

“The manuscript is very-well written. Although the study is well-conducted the authors should be more convincing on how bacteria residing in tissues do not induce death. The association with IL-10 cytokine production appears weak and more experiments are needed to make it more robust.”

Thank you very much for your thoughtful and constructive feedback on our manuscript. We appreciate your positive assessment of the writing quality and the acknowledgment of the wel-lconducted nature of the study.

In regard to the reviewer's comment that "The association with IL-10 cytokine production appears weak," we would like to provide a comprehensive response based on the findings and insights presented in our study (Fig 5). We would like to emphasize several key points to further elucidate this association:

The established knowledge underscores IL-10's capacity to hinder the activation and proliferation of macrophages, thereby safeguarding against an overly aggressive immune-inflammatory reaction (as referenced). In our earlier investigations, we demonstrated that NAD+ orchestrates a systemic generation of IL-10, which assumes a pivotal function in curtailing proinflammatory responses across various conditions, such as autoimmune diseases (as referenced), alloimmunity (as referenced), and bacterial infections (as referenced). In our latest research, we divulge that the introduction of NAD+ leads to an elevated occurrence of IL-10-producing CD4+ T cells, CD8+ T cells, and macrophages, although not dendritic cells (depicted in Figure 5B and C). Furthermore, our comprehensive analyses have substantiated that NAD+ administration thwarts pyroptosis by specifically targeting the non-canonical inflammasome pathway. Intriguingly, our in vitro outcomes suggest that the neutralization of the autocrine IL-10 signaling pathway through a neutralizing antibody and an IL-10 receptor antagonist partially reverses the NAD+-mediated blockage of pyroptosis. These in vitro results imply that NAD+ induces the production of IL-10 cytokines by macrophages, contributing to the suppression of pyroptosis. To corroborate our in vitro conclusions, we employed IL-10 knockout mice and wild-type mice, both treated with either NAD+ or a placebo solution. The wild-type mice treated with NAD+ displayed a survival rate exceeding 80%, whereas the IL-10 knockout mice exhibited a survival rate of "only" 40%. These in vivo findings align with our in vitro discoveries, underscoring the crucial role of NAD+mediated IL-10 cytokine production in impeding pyroptosis through NAD+ and shielding against septic shock. Drawing from our prior and current investigations, we respectfully disagree with the reviewer's characterization of our work as "weak."

Recommendations for the authors

‘’I suggest that animals subject to E. coli infection need to be followed-up for longer and sacrificed at a later time points. It is too difficult to believe that mice are surviving with full resting bacteria in tissues. Do results suggest a full shut-down of the mechanism? What was the level of infiltration of the tissues by neutrophils?’’

‘’I have difficulty to agree with the survival results of the IL-10(-/-) mice of Figure 5E. Can the authors provide the p-values and follow-up for longer? Why the WT and the IL-10(-/-) mice survive the same?’’

Thank you for your thoughtful and constructive comments on our manuscript. We appreciate your valuable insights, and we have carefully considered your suggestions.

We thank the reviewers for this comment. We have indeed followed-up for a longer period of time mice subjected to E. Coli infection and LPS (54mg/kg). Mice infected and treated with NAD+ survived for several months and recovered fully after 10 days. Mice survived for at least a year following infection. We have now included a sentence regarding the long-term survival in the results section of Figure 1 entitled “NAD+ protects mice against septic shock not via bacterial clearance but via inflammasome blockade”. Figure illustrating the level of infiltration of the tissues by neutrophils was added in supplementary data as supplementary figure 4.

In contrast, WT and IL-10-/- mice failed to withstand E. Coli or LPS (54mg/kg) administration when treated with a placebo solution. To our knowledge, our investigation represents the pioneering instance of successfully conferring protection against the lethal doses of E. Coli and LPS administered to animals. Considering the potent immunosuppressive nature of IL-10, our anticipation was that IL-10-/- mice would manifest an exacerbated inflammatory response subsequent to LPS administration, in contrast to WT mice. Our in vivo findings indeed corroborate this assumption, revealing that IL-10-/- mice succumbed more swiftly to LPS administration, displaying statistically significant disparities in survival rates compared to WT mice (p value of 0.0154). The pertinent p-value has been thoughtfully included in Figure 5E of our study.

Reviewer #2 (Public Review):

“Iske et al. provide experimental data that NAD+ lessens disease severity in bacterial sepsis without impacting on the host pathogen load. They show that in macrophages, NAD+ prevents Il1b secretion potentially mediated by Caspase11.

While the in vivo and in vitro data is interesting and hints towards a crucial role of NAD+ to promote metabolic adaptation in sepsis, the manuscript has shortcomings and would profit from several changes and additional experiments that support the claims.

Conceptually, the definition of sepsis is outdated. Sepsis is not SIRS, as in sepsis-2. Sepsis-3 defines sepsis as infection-associated organ dysfunction. This concept needs to be taken into account for the introduction and when describing the potential effects of NAD+ in sepsis. Also, LPS application cannot be considered a sepsis model, since it only recapitulates the consequence of TLR-4 activation. It is a model of endotoxemia. Also, the LPS data does not allow to draw conclusions about bacterial clearance (L135).

The authors state that protective effects by NAD were independent of the host pathogen load. This clearly indicates that NAD confers protection via enhancing a disease tolerance mechanism, potentially via reducing immunopathology. This aspect is not considered by the authors. The authors should incorporate the concept of disease tolerance in their work, cite the relevant literature on the topic and discuss it their findings in light of the published evidence for metabolic alteration sand adaptations in sepsis.

For the in vitro data, the manuscript would benefit from additional experiments using in vitro infection models.

In the merge manuscript, the authors provide two different versions of the figures. In one, bar plots are shown without individual data and in the other with scatter blots. All bar plots need to be provided as scatter plots showing individual values.

The authors should show further serology data for kidney and liver failure etc. as well as further cytokine data such as IL-6 and TNF to better characterize their models.

Careful revision of the entire manuscript, the figure legends and figures is required. The figure legend should not repeat the methods and materials section. The nomenclature for mouse protein and genes needs to be thoroughly revised.

L350. The authors write that they dissect the capacity of NAD+ to dampen auto- and alloimmunity. In this work, no data that supports this statement is shown and experiments with autoantigens or alloantigens are not performed.

L163 The authors describe pyroptosis but in the figure legend call it apoptosis. Specific markers for each cell death should be measured and determined which cell death mechanisms is involved.

Animal data comes from an infection model and LPS application. The RNAseq data is obtained from cells primed with Pam3CSK4 and subsequently subjected to LPS. It is unclear how the cell culture model reflects the animal model. As such the link between IFN signaling and the bacterial infection/LPS model are not convincing and need to be further elaborated.

Figure 5: It is unclear how many independent survival experiments were done, how many mice per group were used and whether the difference between groups was statistical significant. This information should be added.

Further experiments with primary cells from Il10 k.o. and Caspase11 k.o. animals should be provided that support the findings in macrophages.”

Thank you for taking the time to review our manuscript. We appreciate your insightful comments and valuable feedback regarding our study on the role protective role and underlying mechanisms of NAD+ in septic shock.

“While the in vivo and in vitro data is interesting and hints towards a crucial role of NAD+ to promote metabolic adaptation in sepsis, the manuscript has shortcomings and would profit from several changes and additional experiments that support the claims.”

We would like to point out that our current study does not underscore a metabolic adaptation in sepsis but more an immune regulation and a specific blockade of the non-canonical inflammasome signaling machinery.

“Conceptually, the definition of sepsis is outdated. Sepsis is not SIRS, as in sepsis-2. Sepsis-3 defines sepsis as infection-associated organ dysfunction. This concept needs to be taken into account for the introduction and when describing the potential effects of NAD+ in sepsis. Also, LPS application cannot be considered a sepsis model, since it only recapitulates the consequence of TLR-4 activation. It is a model of endotoxemia. Also, the LPS data does not allow to draw conclusions about bacterial clearance (L135).”

Our study uses highly lethal doses of E. Coli or LPS. These doses have been shown to result in multiple organ failure (1, 2). For many decades until now an un-numerable number of studies have used LPS as a model of sepsis (3, 4, 5). We have used LPS animal model based on a study published in 2013 by Kayagaki et al. (1), where the authors reported a novel TLR4-independent mechanism but mediated via activate caspase-11. We used the same animal model to demonstrate the specific role of NAD+ in targeting this TLR4-independent mechanism but mediated via activate caspase-11 and underscore NAD+’s mode of protection.

Moreover, we have not only used LPS but bacterial infection as well using E. Coli. We have also previously published an additional research article demonstrating the protective effect against Listeria Monocytogenes (6). The only model we currently did not use in our current study, is a cecal ligation puncture (CLP) model which is also another common animal model for sepsis.

Our conclusions regarding bacterial clearance are based not only on LPS results but also based on the bacterial load measurement and survival (Figure 1B&C) following E. Coli administration in different tissues (kidney and liver) and not LPS.

“The authors state that protective effects by NAD were independent of the host pathogen load. This clearly indicates that NAD confers protection via enhancing a disease tolerance mechanism, potentially via reducing immunopathology. This aspect is not considered by the authors. The authors should incorporate the concept of disease tolerance in their work, cite the relevant literature on the topic and discuss it their findings in light of the published evidence for metabolic alteration sand adaptations in sepsis.”

We respectfully disagree with the reviewer’s comment and do not believe that NAD+ enhances disease tolerance. We have supporting data indicating that NAD+ mediates protection via a specific blockade of the non-canonical inflammasome pathway, which prevents an over-zealous immune response that results in organ damage and multiple organ failure (MOF). Moreover, we demonstrate that not only NAD+ mediates protection via a specific blockade of the non-canonical inflammasome pathway but prevents septic shock induced death by an additional immunosuppression mediated by the systemic production of IL-10.

Both Caspase-11 and IL-10 pathways are crucial in NAD+ mediated protection against lethal doses of E. Coli and LPS administration. Figure 5A indicates that caspase-11-/- mice treated with PBS have a modest survival rate (~40% survival) when compared to the group of mice treated with NAD+ (>80% survival). These data indicate that NAD+ promotes survival via a caspase-11independent mechanism. Similarly, wild type mice subjected to NAD+ administration exhibited >80% survival, while NAD+ administration to IL-10-/- mice resulted only in a 40% survival rate. Based on these findings, we believe that NAD+ mediated protection against septic shock via a blockade of caspase-11 blockade and by IL-10 cytokine production that dampened the overzealous immune response rather than a disease tolerance.

“For the in vitro data, the manuscript would benefit from additional experiments using in vitro infection models.”

In the current study we have used two in vivo models using LPS and E. Coli a gram-negative bacterium. We have also previously reported the protective role of NAD+ in the context of Listeria Monocytogenes (6) a gram-positive bacterium. In the current study, our aim was to demonstrate the inhibitory role of NAD+ on the non-canonical pathway specifically. We believe that additional in vitro experiments for this study are out of scope.

“In the merge manuscript, the authors provide two different versions of the figures. In one, bar plots are shown without individual data and in the other with scatter blots. All bar plots need to be provided as scatter plots showing individual values.”

As requested by reviewer #2 all bar plots are now provided as scatter plots showing individual values.

“The authors should show further serology data for kidney and liver failure etc. as well as further cytokine data such as IL-6 and TNF to better characterize their models.”

We did not perform further serology analysis, but we did measure IL-6 and TNFα in mice treated with NAD+ or PBS. Mice treated with NAD+ had a reduced systemic level of both cytokines IL-6 and TNFα. We have now added the figures (Figure 1F). In addition, we performed a long-term survival, and all mice treated with NAD+ recovered fully after 10 days and survived over a year after infection. In addition, the mice that survived following NAD+ treatment died of old age.

“Careful revision of the entire manuscript, the figure legends and figures is required. The figure legend should not repeat the methods and materials section. The nomenclature for mouse protein and genes needs to be thoroughly revised.”

A Careful revision of the entire manuscript has been performed.

“L350. The authors write that they dissect the capacity of NAD+ to dampen auto- and alloimmunity. In this work, no data that supports this statement is shown and experiments with autoantigens or alloantigens are not performed.”

We thank the reviewer for this comment. We have now re-phrased our last sentence in the discussion and included references for our previous work. We have now stated:” We have previously reported that NAD+ administration can block auto- (7) and allo-immunity (8) via IL10 cytokine production. Here, we unveiled the capacity of NAD+ to protect against sepsisinduced death via a specific blockade of the non-canonical inflammasome pathway and a robust immunosuppression mediated by IL-10 cytokine production.

L163 The authors describe pyroptosis but in the figure legend call it apoptosis. Specific markers for each cell death should be measured and determined which cell death mechanisms is involved.

We thank the reviewer for this comment. We have focuses on pyoptosis-mediated cell death and not apoptosis. We have now replaced the term “apoptosis” by “pyroptosis-mediated to cell death”.

“Animal data comes from an infection model and LPS application. The RNAseq data is obtained from cells primed with Pam3CSK4 and subsequently subjected to LPS. It is unclear how the cell culture model reflects the animal model. As such the link between IFN signaling and the bacterial infection/LPS model are not convincing and need to be further elaborated.”

Our findings, depicted in Figure 3, pertain exclusively to in vitro investigations rather than in vivo examinations. Our research has demonstrated the selective inhibition of the non-canonical inflammasome pathway by NAD+, with a primary focus on unraveling the specific signaling pathway influenced by NAD+. Our in vitro outcomes indicate that the introduction of recombinant IFN-β counteracted the inhibitory effect of NAD+ on the non-canonical pathway. However, it's important to note that we have not evaluated the IFN-β pathway within our E. Coli and LPS in vivo models. Our primary intention was to exclusively decipher the roles of IFN-β and NAD+ in the context of inhibiting the non-canonical inflammasome, without extending our investigation to the broader in vivo scenarios.

“Figure 5: It is unclear how many independent survival experiments were done, how many mice per group were used and whether the difference between groups was statistical significant. This information should be added.”

We have now included the number of experiments, p values and number of animals used in Figure 5.

“Further experiments with primary cells from Il10 k.o. and Caspase11 k.o. animals should be provided that support the findings in macrophages.”

We concur with the reviewer's suggestion regarding the need for further experiments involving primary cells from IL-10-/- and Caspase-11-/- mice. However, we are uncertain about the potential contribution of these experiments in generating novel or supplementary findings to the existing study.

Recommendations For The Authors:

Besides the comments made in the public section, there are further issues that need to be considered by the authors.

“It is unclear what signifies „impressive, L106" or „dramatic, L257"”

“impressive” meant that we were surprised by the results since to the best of our knowledge prior this study there exists no report/study claiming such survival (>80%) following such high dose of E. Coli. In this aspect protective effects of NAD+ are unique. “dramatic” We (8) and others (9, 10) have previously used this term to describe a robust increase of cytokine production.

“L116. The authors describe „symptoms". It should be clarified what symptoms they observed and the data should be shown. If only temperature is available, then this should be said. It would be interesting to see effects of NAD+ on the glucose levels of the animals during sepsis.”

We thank the reviewer’s comment. We have measured only temperature. We believe that glucose level is beyond the scope of this study.

“L29. Sepsis is not restricted to bacterial and viral pathogens. Also fungi and protozoa can cause sepsis.”

We have now included fungi and protozoa.

“Suppl.Fig.1. A scale should be added.”

Scale has been added

“L822. Lethal dose of LPS would mean that this was lethal for all mice. However, the data suggests that NAD+ treated animals would not have died. This should be clarified.”

Here we meant lethal dose in absence of NAD+ treatment. Our study focuses on the protective role of NAD+ in a lethal context (bacterial and LPS).

“L823/824. The part of the sentence: ... IHC was performed staining for H&E.. is incomplete.”

We thank the reviewer’s comment. We have re-phrased our sentence.

“L804. IL-10 is not a pathway. This should be revised.”

We have replaced “pathway” by” mechanism”.

“The graphical abstract should be the last figure summarizing all findings.”

Figure 4 isn't the final illustration, as it doesn't encompass an overarching graphical summary of our discoveries. Instead, it exclusively highlights the findings related to NAD+'s impact on noncanonical inflammasome inhibition. Notably, this figure omits NAD+-mediated IL-10 cytokine generation and its crucial role in mitigating septic shock.

“The authors report that they used a dosage of 54mg/kg LPS (l.502). This is a rather unusual concentration. How was this determined?”

This was initially based on the first study reporting the role of casapase-11 in septic shock induced death published in 2013 by Kayagaki et al. (1). Many other have used this dosage for septic shock induced death animal model (11, 12, 13).

References:

1. Kayagaki N, et al. Noncanonical inflammasome activation by intracellular LPS independ ent of TLR4. Science 341, 1246‐1249 (2013).

2. Qin, X., Jiang, X., Jiang, X. et al. Micheliolide inhibits LPS-induced inflammatory response and protects mice from LPS challenge. Sci Rep 6, 23240 (2016).

3. Li Z, Qu W, Zhang D, Sun Y, Shang D. The antimicrobial peptide chensinin-1b alleviates the inflammatory response by targeting the TLR4/NF-κB signaling pathway and inhibits Pseudomonas aeruginosa infection and LPS-mediated sepsis. Biomed Pharmacother. 2023 Aug 1; 165:115227.

4. Ramani V, Madhusoodhanan R, Kosanke S, Awasthi S. A TLR4-interacting SPA4 peptide inhibits LPS-induced lung inflammation. Innate Immun. 2013 Dec;19(6):596610.

5. Zhang Y, Lu Y, Ma L, Cao X, Xiao J, Chen J, Jiao S, Gao Y, Liu C, Duan Z, Li D, He Y, Wei B, Wang H. Activation of vascular endothelial growth factor receptor-3 in macrophages restrains TLR4-NF-κB signaling and protects against endotoxin shock. Immunity. 2014 Apr 17;40(4):501-14.

6. Rodriguez Cetina Biefer H, Heinbokel T, Uehara H, Camacho V, Minami K, Nian Y, Koduru S, El Fatimy R, Ghiran I, Trachtenberg AJ, de la Fuente MA, Azuma H, Akbari O, Tullius SG, Vasudevan A, Elkhal A. Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation. J Allergy Clin Immunol. 2018 Dec;142(6):18941908.e7.

7. Tullius SG, Biefer HR, Li S, Trachtenberg AJ, Edtinger K, Quante M, Krenzien F, Uehara H, Yang X, Kissick HT, Kuo WP, Ghiran I, de la Fuente MA, Arredouani MS, Camacho V, Tigges JC, Toxavidis V, El Fatimy R, Smith BD, Vasudevan A, ElKhal A. NAD+ protects against EAE by regulating CD4+ T-cell differentiation. Nat Commun. 2014 Oct 7;5:5101.

8. Elkhal A, et al. NAD(+) regulates Treg cell fate and promotes allograft survival via a systemic IL‐10 production that is CD4(+) CD25(+) Foxp3(+) T cells independent. Sci Rep 6, 22325 (2016).

9. Natalia Garcia-Becerra, Marco Ulises Aguila-Estrada, Luis Arturo Palafox-Mariscal, Georgina Hernandez-Flores, Adriana Aguilar-Lemarroy, Luis Felipe Jave-Suarez, FOXP3 Isoforms Expression in Cervical Cancer: Evidence about the Cancer-Related Properties of FOXP3Δ2Δ7 in Keratinocytes, Cancers, 15, 2, (347), (2023).

10. Estelle Bettelli, Maryam Dastrange, Mohamed Oukka. Foxp3 interacts with nuclear factor of activated T cells and NF-κB to repress cytokine gene expression and effector functions of T helper cells. Proceedings of the National Academy of Sciences. 2005.102; 14; 5138-5143.

11. Han Gyung Kim, Chaeyoung Lee, Ji Hye Yoon, Ji Hye Kim, Jae Youl Cho,BN82002 alleviated tissue damage of septic mice by reducing inflammatory response through inhibiting AKT2/NF-κB signaling pathway,Biomedicine & Pharmacotherapy,Volume 148,2022,112740.

12. Tao Q, Zhang Z-D, Qin Z, Liu X-W, Li S-H, Bai L-X, Ge W-B, Li J-Y and Yang Y-J (2022) Aspirin eugenol ester alleviates lipopolysaccharide-induced acute lung injury in rats while stabilizing serum metabolites levels. Front. Immunol. 13:939106.

13. Chen, N, Ou, Z, Zhang, W, Zhu, X, Li, P, Gong, J. Cathepsin B regulate non-canonical NLRP3 inflammasome pathway by modulating activation of caspase-11 in Kupffer cells. Cell Prolif. 2018; 51:e12487.

Segundo esses autores um sistema é definido por entrada, gerenciamento dos dados que entraram e saída (distribuição).

Hans Steinmüller: Comparaciones implícitas o por qué es inevitable estudiar a China en perspectiva comparada

site:: [[Facebook]] date:: 2023-04-18 author:: [[Hans Steinmüller]] url:: https://www.facebook.com/IDAES/videos/1399579184127619 accessed:: 2023-12-29

:o

falta cerrar la frase... se caracterizó en espacio y tiempo para identificar los momentos del año y las regiones del territorio donde los niveles exeden los recomendados (o algo así)

esto explicalo un poco mejor, es decir tienes que explicar que nos interesan los viajes que van de casa a trabajo o alreves porque nos permite suponer que esos viajes incluyen personas que pasan un tercio de su día en el área trabajo y 2/3 en el area casa y esto permite estimar las poblaciones dinámicas. Para los otros tipos de movilidad no se puede hacer esta supocisión.. en el caso de los otros-trbajo, da ejmeplos de dejar a los niños en la escuela, o hacer la compra, para que se entienda de que se está hablando.

DOI: 10.26508/lsa.202302168

Resource: (R and D Systems Cat# AF1002, RRID:AB_2077789)

Curator: @scibot

SciCrunch record: RRID:AB_2077789

What is this?

Explain what O, O, P switches are?

"Итак, как взаимное сопоставление противоположностей придает красоту речи, так из сопоставления противоположностей, из своего рода красноречия не слов, а вещей, образуется красота мира. Это весьма ясно выражено и в книге Екклесиаста, когда говорится о том, что как злому противопоставляется благое и смерти – жизнь, так и грешник – добродетельному: одному всегда противопоставляется другое"

Se necesita que las conclusiones hablen de modo más explícito con los objetivos. Al menos debería haber una por cada objetivo, hablando de manera explícita de cómo esta tesis permitió alcanzar o no dicho objetivo.

Esto sería posible una vez el análisis de la calidad de los microdatos extraídos esté realizado en Pharo.

Sobran asteriscos.

create flashcard for this term - set 1 - words built from word parts. (this term is not in edition 1) Xerostomia (zēr-ŏ-STŌ-mē-ă) - condition of dry mouth.

Also add xer/o to the word root list - xer/o - dry, dryness

add xer/o to the word root list - xer/o - dry, dryness

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This important study extends insights on NAFLD and NASH regarding the role of plasma lactate levels using mice haplo-insufficient for the gene encoding lactate transporter MCT-1. While the evidence is largely convincing and the work significantly advances our understanding of the roles of distinct hepatic cell types in steatosis, a number of issues require attention and would best be solved by further experimentation.

RESPONSE: We agree with this assessment by eLife, and appreciate the reviewers’ view that the study is important and extends insights into liver disease.

Public Reviews:

Reviewer #1 (Public Review):

The authors put forth the hypothesis that hepatocyte and/or non-parenchymal liver MCT1 may be responsible for physiologic effects (lower body weight gain and less hepatic steatosis) in MCT1 global heterozygote mice. They generate multiple tools to test this hypothesis, which they combine with mouse diets that induce fatty liver, steatohepatitis and fibrosis. Novel findings include that deletion of hepatocyte MCT1 does not change liver lipid content, but increases liver fibrosis. Deletion of hepatic stellate cell (HSC) MCT1 does not substantially affect any liver parameter, but concomitant HSC MCT1 deletion does reverse fibrosis seen with hepatocyte MCT1 knockout or knockdown. In both models, plasma lactate levels do not change, suggesting that liver MCT1 does not substantially affect systemic lactate. In general, the data match the conclusions of the manuscript, and the studies are well-conducted and well-described. Further work would be necessary to dissect mechanism of fibrosis with hepatocyte MCT1, and whether this is due to changes in local lactate (as speculated by the authors) or another MCT1 substrate. This would be important to understand this novel potential cross-talk between hepatocytes and HSCs.

A parallel and perhaps more important advance is the generation of new methodology to target HSC in mice, using modified siRNA and by transduction of AAV9-Lrat-Cre. Both methods would reduce the need to cross floxed mice with the Lrat-Cre allele, saving time and resources. These tools were validated to an extent by the authors, but not sufficiently to ensure that there is no cross-reactivity with other liver cell types. For example, AAV9-LratCre-transduced MCT1 floxed mice show compelling HSC but not hepatocyte Mct1 knockdown, but other liver cell types should be assessed to ensure specificity. This is particularly important as overall liver Mct1 decreased by ~30% in AAV9-Lrat-Cre-transduced mice, which may exceed HSC content of these mice, especially when considering a 60-70% knockdown efficiency. This same issue also affects Chol-MCT1-siRNA, which the authors demonstrate to affect hepatocytes and HSC, but likely affects other cell types not tested. As this is a new and potentially valuable tool, it would be important to assess Mct1 expression across more non-parenchymal cells (i.e. endothelial, cholangiocytes, immune cells) to determine penetration and efficacy.

RESPONSE: We appreciate the reviewer’s view that the new methods we describe represent an important advance. To ensure the specificity of our novel AAV-Lrat-Cre construct, it would be fair to test its distribution among all possible hepatic cell types, including endothelial cells, cholangiocytes, and other immune cells, as suggested. Our efforts in this study were primarily focused on the major cell types thought to contribute to NASH, namely hepatocytes, Kupffer cells, and in particular hepatic stellate cells. The reasons for this focus were:

1) Our primary goal was to investigate the role of MCT1 in hepatic fibrogenesis. According to Manderacke et al. (2013, Nature Comm), hepatic stellate cells account for the dominant proportion (82-96%) of myofibroblast progenitors, which produce collagen fibers. While there may be interesting roles of MCT1 in those other cell types, to elucidate MCT1's role in fibrogenesis, focusing on the dominant fibrogenic cell type, hepatic stellate cells, was the most appropriate approach for this goal.

2) Considering the proportion of each hepatic cell type in the liver, hepatocytes constitute the majority (60-70%), followed by endothelial cells (15%), immune cells (10%), and stellate cells (5%), among others.

3) The AAV-Cre system is highly specific to its promoter, in this case, Lrat, which has been well established in multiple previous studies to exhibit high specificity for hepatic stellate cells in the liver. We will certainly conduct more comprehensive biodistribution studies in the future, as we believe that our AAV-Lrat-Cre system could be a valuable tool in this field.

Reviewer #2 (Public Review):

In this study, the authors seek to answer two main questions: 1) Whether interfering with lactate availability in hepatocytes through depletion of hepatocyte specific MCT-1 depletion would reduce steatosis, and 2) Whether MCT-1 in stellate cells promote fibrogenesis. While the first question is based on the observation that haploinsufficiency of MCT-1 makes mice resistant to steatosis, the rationale behind how MCT-1 could impact fibrogenesis in stellate cells is not clear. A more detailed discussion regarding how lactate availability would regulate two different processes in two different cell types would be helpful. The authors employ several mouse models and in vitro systems to show that MCT1 inhibition in hepatic stellate cells reduces the expression of COL-1. The significance of the findings is moderately impacted due to the following considerations:

RESPONSE: We have included additional in vitro data in order to provide a more comprehensive discussion of MCT1's potential role in regulating collagen production. Please refer to the new Figure 8, Supplementary Figure 6, and the results section (Potential Mechanism). Also note that our original hypothesis was that depleting MCT1 specifically in hepatocytes would protect mice with MCT1 haploinsufficiency from liver lactate overload and NAFLD. Furthermore, we postulated that this protection might prevent NASH progression since lipotoxicity-driven hepatocyte damage is a central factor in NASH pathogenesis. However, our findings did not support this hypothesis. We found only one brief article (2015, Z Gastroenterol et al., "Functional effects of monocarboxylate transporter 1 expression in activated hepatic stellate cells") that discussed the potential role of MCT1 depletion in hepatic stellate cells in regulating collagen production or fibrosis, as mentioned in their abstract. Unfortunately, the DOI for this article is not functional, and the data cannot be located. Moreover, when we attempted to replicate their results, we were unable to do so, leading us to report our own findings in the current paper.

a. Fibrosis in human NAFLD is a significant problem as a predictor of liver related mortality and is associated with type 1 and type 3 collagen. However, the reduction in COL1 in stellate cells did not amount to a reduction in liver fibrosis even in cell specific KO (in Fig 7E, there is no indication of whether Sirius red staining was different between HSC KO and control mice- the authors mention a downward trend in the text). The authors postulate that type 1 COL may not be the more predominant form of fibrosis in the model. This does not seem likely, since the same ob/ob mouse model was used to determine that fibrosis was enhanced with hepatocyte specific MCT-1 KO and decreased with Chol MCT-1KO. Measurements of different types of collagens in their model and the effect of MCT-1 on different types could be more informative. In particular, although collagens are the structural building blocks for hepatic fibrosis, fibrosis can also be controlled by matrix remodeling factors such as Timp1, Serpine 1, PAI-1 and Lox.

RESPONSE: We monitored the expression levels of matrix remodeling factors, such as Timp1 (Figure 5C, 5F). There was no change in expression upon Chol-MCT1-siRNA treatment, while a significant increase was observed upon GN-MCT1-siRNA treatment. This trend was similar to collagen expression in both cases. Regarding the different types of collagen, instead of measuring each individual type of collagen, we conducted Sirius red and trichrome staining, which enabled us to detect multiple types of collagen simultaneously (Figure 5G, Figure 7D).

b. The authors use multiple animal models including cell specific KO to conclude that stellate cell MCT-1 inhibition decreases COL-1. However, the mechanisms behind this reduced expression of COL-1 are not discussed or explored, making it descriptive.

RESPONSE: We agree that the mechanisms involved are not fully defined but have added new data (Figure 8, Supplement Figure 6) and text to discuss possibilities.

c. Different types of diets are used in this study which could impact lactate availability. Choline deficiency diets are reported to cause weight loss, and importantly have none of the metabolic features of human NASH. Therefore, their utility is doubtful, especially for this study which proposes to investigate if metabolic dysregulation and substrate availability could be a tool for therapy.

RESPONSE: Unfortunately, none of the rodent models used to study NASH completely replicate the condition in human patients, each having its own set of advantages and drawbacks. In line with the concern raised by reviewer #2, there has been a shift away from the use of severely detrimental methionine and choline-deficient diets in contemporary NASH research. Instead, diets that combine methionine and other amino acids with cholinedeficient diets, in conjunction with high-fat diets, have become more popular. The diet we employed in our study consists of high-fat diet combined with choline-deficient diets. We believe that our findings, which are consistent and established across two distinct NASH pathogenesis models and genetic backgrounds, lend additional robustness to our results.

d. Hepatocyte specific MCT-1 KO mice seem to have increased COL-1 production, despite no noticeable difference in hepatocyte steatosis. The reasons for this are not discussed. Fibrosis in NASH is thought to be from stellate cell activation secondary to signals from hepatocellular damage. There is no evidence that there was a difference in either of these parameters in the mouse models used.

RESPONSE: While lipotoxicity-driven liver damage remains a central aspect of NASH pathogenesis, the traditional two-hit theory has become less tractable, giving way to the multi-hit theory in the NASH field. The current debate revolves around whether steatosis is a decisive factor and requirement for NASH fibrogenesis. Our previous publication (Yenilmez et al., 2022, Mol Ther) demonstrated that nearly complete resolution of steatosis did not prevent other NASH features like inflammation and fibrosis, indicating the existence of multiple factors beyond steatosis in NASH pathogenesis. We believe that steatosis and fibrosis influence each other but can also develop independently.

e. The authors report that serum lactate levels did not rise after MCT-1 silencing, but the reasons behind this are unclear. There is insufficient data about lactate production and utilization in this model, which would be useful to interpret data regarding steatosis and fibrosis development. For example, does the MCT-1 KO prevent hepatocyte and stellate cell net import or export of lactate? What is the downstream metabolic consequence in terms of pyruvate, acetylCoA and the NAD/NADH levels. Does the KO have downstream effects on mitochondrial TCA cycling?

RESPONSE: Due to both biological and technical challenges (which are described in the new draft), conducting a comprehensive metabolomics study comparing hepatocyte MCT1 KO to hepatic stellate cell MCT1 KO was not feasible. It is important to note that MCT1 can also transport other substrates that are often overlooked, including pyruvate, short-chain fatty acids, and ketone bodies. Also, in addition to MCT1, there are at least two other functional isoforms of MCT: MCT2 and MCT4. Regrettably, due to these biological and technical complications, conducting a comprehensive metabolomic analysis is extremely complicated and difficult to interpret. Nevertheless, some insights are gained from a study involving MCT1 chaperone protein Basigin/CD147 knockout (KO) mice in a high-fat diet- induced hepatic steatosis model. Basigin acts as an auxiliary protein for MCT1, and its absence leads to improper localization and stabilization of MCT1, effectively simulating a state of MCT1 deficiency. In this context, hepatic lactate levels were reduced by half, and other metabolites such as pyruvate, citrate, α-ketoglutarate, fumarate, and malate were significantly decreased. While we must exercise caution when extrapolating these findings to our MCT1 study, they suggest that multiple metabolites, particularly pyruvate, may play a crucial role in the context of MCT1 deficiency.

f. MCT-1 protein expression is measured only in the in vitro assay. Similar quantitation through western blot is not shown in the animal models.

RESPONSE: We monitored MCT1 protein expression with either Western blot (Fig 2D, 2E (in vitro)) or immune-histology (Fig 4B, 4C (in vivo, ob/ob + GAN diet NASH model), Sup Fig 5F, 5G (in vivo, MCT1 f/f + CDHFD model)).

Reviewer #3 (Public Review):

A major finding of this work is that loss of monocarboxylate transporter 1 (MCT1), specifically in stellate cells, can decrease fibrosis in the liver. However, the underlying mechanism whereby MCT1 influences stellate cells is not addressed. It is unclear if upstream/downstream metabolic flux within different cell types leads to fibrotic outcomes. Ultimately, the paper opens more questions than it answers: why does decreasing MCT1 expression in hepatocytes exacerbate disease, while silencing MCT1 in fibroblasts seems to alleviate collagen deposition? Mechanistic studies in isolated hepatocytes and stellate cells could enhance the work further to show the disparate pathways that mediate these opposing effects. The work highlights the complexity of cellular behavior and metabolism within a disease environment but does little to mechanistically explain it.

RESPONSE: Described above to Reviewer #2

The observations presented are compelling and rigorous, but their impact is limited by the nearly complete lack of mechanistic insight presented in the manuscript. As also mentioned elsewhere, it is important to know whether lactate import or export (or the transport of another molecule-like ketone bodies, for example) is the decisive role of MCT1 for this phenotype. Beyond that, it would be interesting, albeit more difficult, to determine how that metabolic change leads to these fibrotic effects.

RESPONSE: Described above to Reviewer #2

Kuppfer cells are initially analyzed and targeted. These cells may play a major role in fibrotic response. It will be interesting to determine the effects of lactate metabolism in other cells within the microenvironment, like Kuppfer cells, to gain a complete understanding of how metabolism is altered during fibrotic change.

RESPONSE: To address the potential involvement of inflammatory cells, we added new data to the manuscript (Supplement Figure 4). Given the distinct hepatic cellular distribution of Chol-MCT1-siRNA and GN-MCT1-siRNA, the opposite fibrogenic phenotype observed may be attributed to MCT1’s role in non-hepatocyte cell types such as the inflammatory Kupffer cells and the fibrogenic hepatic stellate cells. To determine which hepatic cell type drives the opposite fibrotic phenotypes, we first hypothesized that GN-MCT1-siRNA activates M2 pro-fibrogenic macrophages more than Chol-MCT1-siRNA does. The representative M1/ M2 macrophage polarization gene markers were monitored in Kupffer cells. However, GN-MCT1-siRNA treatment caused comparable M1/M2 macrophage activation levels to Chol-MCT1-siRNA treatment (Supplement Figure 4A, 4B). These data suggest that the opposite fibrotic phenotypes caused by the different siRNA constructs are not due to M1/M2 macrophage polarization.

The timing of MCT1 depletion raises concern, as this is a largely prophylactic experiment, and it remains unclear if altering MCT1 would aid in the regression of established fibrosis. Given the proposal for translation to clinical practice, this will be an important question to answer.

RESPONSE: Agree these are important experiments for future evaluation.

Reviewer #1 (Recommendations For The Authors):

As above, in general, the conclusions match the data presented. The one exception is the authors discussion point that these data show the importance of lactate flux in fibrosis. As MCT1 has other substrates, it does not seem this is definitively due to lactate flux. It would be helpful to have additional experiments to clarify mechanism by which loss of hepatocyte MCT1 leads to increased fibrosis, while loss of HSC MCT1 reverses this finding. This may aid in concluding that altered fibrosis is in fact due to lactate flux in these cell types.

RESPONSE: Described above to Reviewer #2

In addition, it is unclear why the authors switched NASH models for the two tools generated (GAN diet for siRNA, CDHFD for AAV). Similarly, methodology to assess fibrosis switched between these two experiments - i.e. Sirius Red staining for siRNA-treated GAN diet-fed mice vs. Trichrome staining for AAV-transduced CDHFD-fed mice. These changes make it difficult to perform cross-comparisons of the data, to explain (for example), why GN-siRNA to Mct1 reduced body weight but AAV8-TBG-Cre did not. Similarly, GN-siRNA increased liver Col1a1 protein but AAV8-TBG-Cre did not. These differences could be explained by model system, or tool efficacy/off-target effects.

RESPONSE: We agree that different model systems can explain difference in results, but there is also an advantage of using different models and various methodologies as preclinical tests of consistency of data on NASH under different conditions. There are no perfect mouse models for human NASH.

• Phenotyping is also incomplete for the latter experiment, in particular amount of liver lipid content –

RESPONSE: We estimated lipid content by H&E (Fig 6E, F). In some experiments, we focused mostly on COL1 protein expression, as this rather than mRNA is the functional aspect of fibrosis.

Reviewer #2 (Recommendations For The Authors):

This study could benefit from standardization of the types of diet used across all animal models and a more comprehensive focus on the metabolic/substrate availability and utilization aspects of NAFLD and NASH affected in the mouse models with MCT-1 dependent lactate transport deficiency. Since hepatic fibrogenesis in NASH is impacted by signals following hepatocyte damage, the extent of cell death in these models could also be better characterized.

RESPONSE: Our ALT data provides indirect insight into hepatocyte damage. Our histology images did not reveal significant changes in cell morphology or integrity and there were no notable changes in caspase protein levels.

Other comments:

In Fig 4G, there is an increase in the number of lipid droplets with Chol- MCT-1 siRNA compared to GN-MCT1-sirRNA, suggesting that the stellate cell component might be responsible for this finding. The possible reasons for this are not discussed.

RESPONSE: The effects in Fig 4G were exceedingly small and there is no difference in total TG in these experiments, so it is hard to interpret these data and provide logical explanations.

In Fig 5A. A western Blot for aSMA and COL 1 is shown but the sample labeling is unclear i.e, do the lanes belong to different mice of the same condition? HFD mice vs Ctr mice?

RESPONSE: Both groups of ob/ob mice were fed a GAN diet. The graph in Fig 5 is a direct comparison between NTC-siRNA and MCT1-siRNA. To enhance clarity, this is indicated in the figure legends, and the data in Fig 5 is a continuation of the data presented in Fig 4

In Fig 5E, COL1 densitometry data should also be provided for non-silenced mice on HFD and Chow diet for appropriate comparison

RES\PONSE: Both groups of ob/ob mice were fed a GAN diet. The graph in Fig 5 represents a comparison between NTC-siRNA and MCT1-siRNA. It's important to note that, typically, ob/ob mice fed either a chow diet or a high-fat diet do not exhibit fibrogenic phenotypes within this time frame (3 weeks of dietary intervention).

There are many mis-statements throughout the text.Page 6 - "MCT1 silencing significantly inhibited Tgf1β-stimulated ACTA2 mRNA expression as well as collagen 1 protein production" but it is not stated that CO1A1 mRNA is unchanged in Fig 1C.

RESPONSE: We observed no change in CO1A1 mRNA levels (Fig 1C), so we focused on collagen 1 protein production (Fig 1B) on page 6. Given the consistent trend observed in Chol-MCT1-siRNA (Fig 5C), we proposed the possibility of MCT1's influence on collagen translation or protein turnover on page 11.

Page 7- ".......our Chol-MCT1-siRNA does not require transfection reagents as it is fully chemically modified". What does fully chemically modified mean and why does this mean in terms of transfection efficiency.

RESPONSE: One of the primary challenges in utilizing RNAi as a therapeutic approach has been the effective in vivo delivery strategy, particularly concerning stability and longevity against systemic nucleases. Recent developments in siRNA duplex chemical modification strategies, such as 2-Fluoro and 2-O-Methyl ribose substitutions, as well as phosphorothioate backbone replacements, have addressed these challenges (Please see Figure 3. In our current study, we employed 'chemically fully modified' siRNA, featuring several key modifications: (1) every single ribose is chemically modified to 2-F or 2-OMeribose, (2) phosphorothioate backbone replacement, (3) 5'-end of the antisense strand modification to (E)-Vinyl-phosphonate, and (4) 3'-end of the sense strand linkers such as Cholesterol or Tri-N-Acetyl-galactosamine. These chemical enhancements significantly improve transfection efficiency, longevity, and selectivity, setting it apart from traditional siRNA lacking such chemical modifications. A prior study from the Khvorova lab has demonstrated substantial efficiency differences between partially and fully modified siRNA in vivo.

Page 7- the results present for Fig 2 ignores Fig, 2C, if this is important it needs to be described if not, please delete.

RESPONSE: The dose-response potency results, crucial for identifying the most potent Chol-MCT1-siRNA compound, are depicted in Figure 2C. The wording "(Figure 2C)" has been inserted in the sentence as follows. “The silencing effect on Mct1 mRNA was monitored after 72 hours (Figure 2B). Several compounds elicited a silencing effect greater than 80% compared to the NTC-siRNA. The two most potent Chol-MCT1-siRNA, Chol- MCT1-2060 (IC50: 59.6nM, KD%: 87.2), and Chol-MCT1-3160 (IC50: 32.4nM, KD%: 87.7) (Figure 2C) were evaluated for their inhibitory effect on MCT1 protein levels (Figure 2D, 2E). Based on its IC50 value and silencing potency, Chol-MCT1-3160 construct was chosen for further studies in vivo (Table 2).”

Supplement Fig 1A-F should be analyzed by multiple comparisons not by paired t-tests.

RESPONSE: We performed t-tests for every comparison between two groups. However, for Sup Fig 1A-F, which involved a comparison among three different groups, we applied oneway ANOVA.

The x-axis in supplement Fig 2A and B are not labeled, and I assume are in weeks. The Fig 2B x-axis numbers also mis-labeled and should also be 0-3 and not 10-13.

RESPONSE: The x-axis is now appropriately labeled.

Page 10 - the description of supplement Fig 4A is not accurate. Srebf1 mRNA is unchanged by the GN-MCT1-siRNA treatment and Mlxipl mRNA is unchanged by Chol-MCT1-siRNA treatment. Is this total Mlxipl mRNA or can you distinguish between the alpha and beta variants.

RESPONSE: We adhered to NCBI nomenclature, where 'SREBP1' and 'ChREBP' represent proteins, not mRNA. The Mlxipl mRNA we tested pertains to total Mlxipl mRNA. Original draft shown below.

“To investigate the underlying mechanism by which lipid droplet morphological dynamics change, we monitored the effect of hepatic MCT1 depletion on DNL-related gene expression. Both GN-MCT1-siRNA and Chol-MCT1-siRNA strongly decreased the mRNA and protein levels related to representative DNL genes (Supplement Figure 4A-4D). Intriguingly, both modes of hepatic MCT1 depletion also inhibited expression of the upstream regulatory transcription factors SREBP1 and ChREBP.”

There are no molecular weight markers in supplement Fig 4C and D. Is the Srebp1c blot for the nuclear or precursor form?

RESPONSE: The Srebp1c blot presented represents the precursor form. I have edited the figure legend accordingly. It's worth noting that the cleaved form of Srebp1c either exhibited significantly lower expression compared to its precursor form or displayed comparable expression between the control group and the MCT1 depletion group.

Changes in mRNA and protein do not always reflect changes in activity (allosteric regulation). If you want to draw any conclusions about de novo lipogenesis you need to directly measure fatty acid synthesis rates from a carbohydrate precursor.

RESPONSE: We completely agree. Therefore, in the current study, we emphasized two key points: (1) hepatic MCT1 depletion affects the expression levels of representative DNL genes, and (2) however, this regulation was insufficient to resolve the steatosis phenotypes in our NASH model. We have added the text “while recognizing that the decreased expression of DNL genes does not necessarily indicate inhibited fatty acid synthesis rate” on page 15.

Reviewer #3 (Recommendations For The Authors):

Figure 1 - Are there changes to fibroblast phenotype with TGF-beta stimulation and are these changes reversed with MCT1 siRNA-mediated silencing, or is this purely an expression phenomenon?

RESPONSE: This study was designed to assess the preventative effect of MCT1 silencing on Tgf1β-induced fibrosis, rather than a reversal study. As detailed in the methods section, LX2 cells were initially cultured in DMEM/high glucose media with 2% FBS. The following day, we transfected the cells with either NTC-siRNA or MCT1-siRNA (IDT, cat 308915476) using Lipofectamine RNAi Max (ThermoFisher, cat 13778075) for 6 hours in serum-reduced Opti-MEM media (ThermoFisher, cat 31985062). Subsequently, the cells were maintained in serum-starved media, with or without 10ng/ml of recombinant human Tgf1β (R&D Systems, cat 240-B/CF), for 48 hours before harvesting.

Is lactate import/export itself responsible for this phenotype? It is presumed that MCT1 depletion alters import/export of lactate and subsequently modulates this phenotype, but this is never shown experimentally. Does lactate accumulate in these cells or in the medium in culture? The foundation of the paper rests on this hypothesis, so we believe that this is critical to establish. This is particularly relevant as MCT1 has been proposed to function primarily as a lactate importer, so the availability of medium lactate could be easily modulated to determine whether that mimics MCT1 loss.

RESPONSE: To address the underlying mechanism of MCT1/Lactate in stellate cells, we added a new figure to the manuscript (Figure 8). We had previously conducted an experiment to determine whether MCT1 depletion in LX2 cells in vitro influences extracellular lactate concentrations in DMEM/high glucose (25mM glucose) media supplemented with 1mM sodium pyruvate but without sodium lactate. Interestingly, we found no significant difference in extracellular glucose and lactate concentrations, which remained at 25mM and 5mM, respectively. These concentrations were comparable between groups, regardless of MCT1 loss. Additionally, we investigated the effects of MCT1 silencing in the presence of potent fibrogenic inducer TGF-β1. Intriguingly, MCT1 depletion effectively prevented TGF-β1-induced collagen production, irrespective of lactate (+/- pyruvate) supply in the media. LX2 cells with MCT1 depletion exhibited reduced collagen 1 production when lactate was solely generated by endogenous glycolysis (Figure 8F) and when exogenous lactate was supplied (Figure 8G).

Figure 2 - It is compelling that the Chol-MCT1-siRNA compounds are effective at targeting MCT1. However, is it clear how specific the siRNA target is? Are other MCT genes affected as well (if the siRNAs target areas of homology, for example)? Given that this siRNA strategy is used going forward and proposed as a therapeutic, it would be important to discuss and perhaps characterize off-target effects. A simple BLAST search for homology for the chosen siRNAs could help answer this question.

RESPONSE:

1) We designed the siRNA to specifically avoid any potential off-target effects on MCT1's 14 isoforms, and this approach aligns with the results obtained from the NCBI-BLAST analysis.

2) While there are 14 isoforms of MCTs, only the first four are functional. To assess the off-target effect of Chol-MCT1-siRNA on MCT2 and MCT4 (MCT3 was excluded due to its limited expression in retinal pigment epithelium), we conducted in vivo experiments in ob/ob mice, which demonstrated a highly selective MCT1 silencing effect. We have also included MCT1, MCT2, and MCT4 rt-qPCR data in the manuscript (Supplement Figure 2A, 2B).

3) We plan to further optimize and validate the human MCT1-targeting siRNA sequence for use in humanized mouse studies. It's important to note that the MCT1-siRNA used in this study was designed for mice.

Supplemental Figure 1 - brain would be one other highly metabolic tissue wherein it would be important to show lack of activity/accumulation.

RESPONSE: Undoubtedly, the brain is one of the most metabolically active tissues, playing a pivotal role in regulating signaling pathways and metabolism in other tissues. However, it poses a significant challenge in terms of targeting due to the presence of the blood-brain barrier (BBB). Overcoming BBB penetration remains one of the foremost challenges in the field of therapeutic siRNA delivery. For many therapeutic oligonucleotides, including Cholesterol-conjugated siRNAs, systemic administration alone is normally insufficient to achieve BBB penetration. Direct local injection or transient disruption of the BBB is normally required.

Figure 4 - The image shown for chol-MCT1-siRNA seems to show variation in lipid droplet size. Is this just this single image? The authors quantify smaller lipid droplets in this group, so the image may not be representative as there are many large droplets. Ultimately, additional mechanisms as to how alterations in lactate metabolism could mediate this phenotype are missing. This hypothesis also rests upon the assumption that MCT1 is modulating lactate, which is not shown experimentally, as discussed above.

RESPONSE: We changed the representative images (Fig 4B). We agree this aspect of the study is not resolved, and we have related text in the manuscript on this point: “neither GNMCT1-siRNA nor Chol-MCT1-siRNA decreased total hepatic TG levels (Figure 4H), although quantitative analysis of H&E images showed a small decrease in mean lipid droplet size and increased number of lipid droplets upon MCT1 silencing (Figure 4F, 4G). These data suggest the possibility that hepatic MCT1 depletion either 1) inhibits formation or fusion of lipid droplets, or 2) enhances lipolysis to diminish lipid droplet size.”

Figure 5 provides evidence that Chol-MCT1-siRNA expression decreases fibrosis but this is attributed to the effects on stellate cells. While GN-MCT1-siRNA and subsequent MCT1 silencing in hepatocytes has an opposite effect. The cell population that is not discussed, however, is the Kupffer cell. Could MCT1 silencing in this cell population be mediating part of the phenotype observed? How does MCT1 silencing affect Kupffer cell phenotype and activity?

This extends into Figure 6 where Kupffer cells are not given consideration in targeted experiments.

RESPONSE: Described above to Reviewer #3

Figure 6 and 7 use a different model to show that stellate cell depletion of MCT1, specifically, decreases collagen 1 protein levels in NASH, which reinforces the authors claims. Given the cell specificity of this experiment, it is more compelling data. It would be nice to show that Kupffer cell depletion of MCT1 does not have any affect (or perhaps show that it does.

RESPONSE: We agree, but Kupffer selective depletion is not possible to do with this siRNA technology. Please see the response above as our most recent attempt to address this question.

Figure 7 shows that even with decreased collagen deposition, there is no effect on liver stiffness or chronic liver injury as measure by ALT. This may suggest that the decreased level of fibrosis is either not significant to overall clinical outcome or that there are other fibroinflammatory mechanisms compensating for lack of COL1 deposition. Is there increased reticulin fibrosis when MCT1 is knocked down? This could be assessed with IHC or monitoring type 3 collogen (COL3A1).

RESPONSE: Reticulin fibrosis results from the excessive deposition of reticular fibers, primarily composed of type 3 collagen. However, based on our observation of trichrome staining in whole liver histology data (Fig 7D-E), which exhibited nearly identical trends to collagen type 1 expression (Fig 7A-C), it seems unlikely that type 3 collagen compensated for the decrease in type 1 collagen protein expression upon hepatic stellate cell MCT1 KO. We plan to perform detailed analysis of a more comprehensive list of ECM proteins including type 3 collagen in our humanized mouse model with engrafted human liver cells in future experiments.

Additional considerations:

It may be useful to know if inhibition of fibrosis affects survival/progression in these NASH models over a longer timeframe, although this may understandably be beyond the scope of the current work. The timing of MCT1 depletion is prophylactic and given the proposal to translate this research, it would be important to determine whether MCT1 inhibition reversed fibrosis, and if so, by what metabolic mechanism?

RESPONSE: We have observed that extending the duration of the NASH model increases the likelihood of hepatocarcinoma development. Exploring the aim to include survival and disease progression as well as reversal of fibrosis would be important in future experiments.

Summary of new Figures and Figures modified:

• Fig 1B: added "and" (significance) between the first and the third group, and the second and the last group.

• Fig 4B: replaced images with more representative ones as the mean lipid size was questioned by the reviewer.

• Fig 7D: made the images bigger (original images cropped and enlarged → 5X)

• Fig 8: newly created to explain the underlying pathway of lactate, and MCT1 regulating collagen production. Please find the results sections.

• Sup fig 2A, B: newly added to show our compounds’ selective silencing effect. - Sup Fig 2C-D: Added missing x-axis (moved from previous Figure 2A, 2B) - Sup Fig 2E-F: moved from sup Fig 3 not to have too many sup figures.

• Sup Fig 3C-D: showed both precursor and cleaved form of SREBP1 bands as requested (moved from previous sup Figure 4)

• Sup Fig 4: newly created, as questioned many times for the effect on Kupffer cells or other inflammatory cells.

• Sup Fig 6: newly created to explain the potential underlying mechanism of MCT1 depletion on collagen production.

• Sup Fig 7: moved from previous sup Fig 6.

• Sup Fig 8: moved from previous sup Fig 7.

That is a not lowest there is for a single YES/NO answer.

Fast path of ConsensusOnDemand requires O(n), it's only used when many peers approve a tx as not conflicting with others. And this is often the case for cryptocurrency, where conflict occurs only when txes on the same account are issued in parallel, which is rare.

Hashgraph's YES/NO on famous witnesses, which totally orders many txes, is O(n * log n).

Message complexity of fast path of ConsensusOnDemand is O(n).

Hashgraphs YES/NO about fame is O(n * log n)

https://blumm.blog/2022/07/31/el-metodo-zettelkasten-o-como-combatir-nuestra-mediocridad-cuando-leemos/

Bernardo Munuera Montero' review of Ahren's Smart Notes

Baraka published a book of poems called "S O S: Poems 1961-2013" where he has more "Heathen" poems, with titles such as "Heathens in Evolution," "Heathen Bliss," "Heathen Technology & Media," and "Heathens Think Fascism is Civilization." In these poems, Baraka uses the term "heathen" similarly to how he does here, with it criticizing whites.

As i had troubles understating this description here's the explanation of a user on reddit that surely knows more about African culture: A Q (sometimes spelled Que) is a member of the Omega Psi Phi fraternity. Omega Psi Phi was the first black fraternity to be formed at a Historically Black College or University (HBCU) and is a member of the "Divine 9", a group of historically black fraternities and sororities. Their colors are purple and gold, hence the purple windbreakers. https://www.reddit.com/r/grammar/comments/j8gyvw/on_a_book_of_tanehisi_coates/

Não faz muito sentido em relação à Reiko pq ela se sacrifica pelo filho no fim, esse pode ser um bom contra argumento

basicamente, se vc tá se perguntando isso, é pq n tá alienado o suficiente pelos prazeres da vida. O bonito nem disfarça

p

[os ateus]

O autor parte de um princípio diferente, mas é um questionamento interessante. Como seria a consciência dos parasitas? Teoricamente, as consciências da IAs seguiriam o propósito de seus criadores, mas n temos como aplicar isso aos parasitas. A consciência da Reiko é bem similar à humana. Poderia ser por conviver com humanos ou por habitar o corpod e uma humana?

How might this equate to the time at which Rome extended its citizen franchise to larger swaths of people and the attendant results which came about? particularly the shift towards an empire versus a republic?

These seem to have been happening in the case of America with Donald Trump attempting to become a modern day Julius Caesar. To whom is Trump indebted?

Эту хуйня просто в качестве примера

Author Response

The following is the authors’ response to the original reviews.

eLife Assessment

This valuable manuscript investigates the roles of DKK3 in AD synapse integrity. Although previous work has identified the involvement of Wnt and DKK1 in synaptic physiology, this study provides compelling evidence that suppression of DKK3 rescues the changes in excitatory synapse numbers, as well as memory deficits in an established AD model mice. The authors provide both gain and loss of function data that support the main conclusion and advance our understanding of the mechanisms by which Wnt pathway mediates early synaptic dysfunction in AD models.

Public Reviews:

Reviewer #1 (Public Review):

In this study, Nuria Martin-Flores, Marina Podpolny and colleagues investigate the role of Dickkopf-3 (DKK3), a Wnt antagonist in synaptic dysfunction in Alzheimer's disease. Loss of synapses is a feature of Alzheimer's and other forms of dementia such as frontotemporal dementia and linked amyotrophic lateral sclerosis (FTD). The authors utilise a broad range of experimental approaches. They show that DKK3 levels are increased in Alzheimer's disease and that this occurs early in disease. This is an important finding since early disease changes are believed to be the most important. They also show increases in DKK3 in transgenic mouse models of Alzheimer's disease and that DKK3 knockdown restores synapse number and memory in one such model. Finally, they link these DKK3 increases to loss of excitatory synapses via the blockade of the Wnt pathway and subsequent activation of GSK3B; GSK3B is strongly linked to both Alzheimer's disease and FTD. The quality of the data is good and the conclusions well supported by these data. There are no major weaknesses. The findings support studies that target the Wnt pathway as a potential therapeutic for Alzheimer's disease.

Reviewer #2 (Public Review):

This manuscript by Martin-Flores et al., has examined the role of DKK3 in Alzheimer's disease, focusing on the regulation of synaptic numbers. By using human AD brain databases and tissue samples, the authors showed that DKK3 protein and mRNA levels are increased in the brains of AD patients. DKK3 is expressed in the excitatory neurons in WT mouse brains and accumulates at atrophic neurites around amyloid plaques in AD mouse brains. Interestingly, secretion of DKK3 appears to be regulated by NMDAR antagonist as well as chemical LTD. Through gain and loss of function studies, the authors showed that DKK3 regulates the number of excitatory as well as inhibitory synapses with distinct downstream pathways. Finally, the authors investigated the contribution of DKK3 to synaptic changes in AD and found that DKK3 loss of function rescues both the excitatory and inhibitory synaptic defects, resulting in the improvement of memory function in J20 mice.

Overall, the data is clearly presented and deals with novel roles of DKK3 in controlling excitatory and inhibitory synapses. The finding that shRNA expression of DKK3 in AD model mice rescues synaptic phenotypes and memory impairment is potentially interesting and may provide a new strategy for AD treatment.

We would like to thank the Editors and the Reviewers for their very insightful suggestions. We are delighted to receive very positive reviews of our manuscript. In response to the comments made by the reviewers, we have carried out an extensive revision of our manuscript. In the revised manuscript, we have addressed all the comments made by the reviewers.

Recommendations for the authors:

Reviewer #1:

My only comment regards the role of GSK3B activation in synaptic dysfunction and its targets. GSK3B is a Tau kinase but is also involved in IP3 receptor delivery of Ca2+ to mitochondria. This delivery is major regulator of mitochondrial ATP production and synaptic function is heavily dependent on ATP. Both Alzheimer's disease and FTD insults have been linked to GSK3B activation -see for e.g. Szabo EMBO R 2023, Gomez-Suaga Aging Cell 2022. It might be valuable to readers for the authors to speculate briefly on potential GSK3B synaptic targets in the Discussion.

We appreciate the reviewer for this suggestion. In the Discussion, we now included how GSK3β may contribute to synaptic dysfunction and loss in the context of increased DKK3 levels and in Alzheimer’s disease.

Reviewer #2:

1. In Fig 1B, the authors showed that soluble DKK3 levels were increased in Braak 1-3 patients, while no changes were observed in Braak 4-5. If the secretion of DKK3 is dependent on NMDAR activity, does this data imply that Braak 4-5 patients have reduced NMDAR activity in general, resulting in the reduced DKK3 release even with the increased mRNA levels? It would be interesting to test this hypothesis in a mouse AD model.

In Figure 1B, we analyzed the levels of soluble and insoluble DKK3 in the hippocampus of AD patients at different disease stages based on their Braak stages. As the reviewer indicated, soluble levels of DKK3 were increased in patients with Braak I-III but not at later stages. Importantly, DKK3 levels were also elevated in Braak IV-VI patients, but only in the insoluble fraction (Figure 1C), suggesting that DKK3 could accumulate within Aβ aggregates. Based on these findings, we cannot conclude that DKK3 release is reduced at later stages of the disease in patients.

To explore the underlying mechanisms regulating DKK3 levels, we used cultured hippocampal neurons and AD mouse brain slices. In mouse models, we have demonstrated that extracellular DKK3 levels (secreted DKK3 fraction) depends on NMDAR activation early in the disease progression (Figure 2E, F). Moreover, we also provide new data showing that antagonizing NMDAR partially blocks the increase of DKK3 extracellular levels induced by oligomeric Aβ (see response to question 4 of this reviewer and Figure S2G, H). It is well established that oligomeric Aβ promotes hyperexcitability through, in part, the aberrant activation of NMDAR (Li S et al., 2011, PMID: 21543591; Mucke L and Selkoe DJ et al., 2012, PMID: 22762015). In line with this, NMDAR blockers prevent Aβ-induced synapse loss and improve cognition in AD models (Hu NW et al., 2009, PMID: 19918059; Ye C et al., 2004, PMID: 15288443). In addition, an NMDAR antagonist is currently approved as a drug treatment for AD patients (Cumming J 2021, PMID: 33441154). Together, our findings in dissociated neurons, AD mouse brain and human samples indicate that soluble Aβ oligomers promote the release of DKK3 through NMDAR activation and suggest that this mechanism might also be occurring in the brain of AD patients.

1. Recent work (Yuan et al., 2022, Nature) has shown that dystrophic neurites/axonal spheroids found around Aβ deposits are filled with neuronal endolysosomes. Are DKK3 in ThioS positive amyloid plaques located in endolysosomes of these axonal spheroids? If so, does this data mean that DKK3 in Fig 2B-D represents the entrapped DKK3 protein population that fails to be secreted from dystrophic neurites?

The reviewer points an interesting question. Our results show that secretion of DKK3 is increased in two AD models before substantial plaque load. Later in the disease, DKK3 accumulates in dystrophic neurites (visualized as axonal spheroids) surrounding amyloid plaques. To address if DKK3 protein is located in vesicles of the endolysosomal pathway within axonal spheroids, we performed co-localization analyses of DKK3 and the endolysosomal marker LAMP1. We found that DKK3 colocalized with LAMP1 (Figure 2D) indicating the presence of DKK3 in axonal spheroids. These results indeed suggest that DKK3 is present in abnormally enlarged vesicles in dystrophic neurites around Aβ plaques. This could affect the axonal transport of DKK3. Given that proteins present in dystrophic neurites have been correlated with defects in bidirectional transport in the axon (Stokin GB et al., 2005, PMID: 15731448; Sadleir KR et al., 2016, PMID: 26993139), both DKK3 turnover and secretion could be affected.

1. Why does only LTD induce DKK3 release? Why not general activation of neuronal activity? It would be important to test the relationship between DKK3 secretion and neuronal activity with optogenetics and chemogenetics.

We tested whether neuronal activity triggered increased extracellular DKK3 levels by subjecting neurons to chemical long-term potentiation (cLTP) or long-term depression (cLTD). However, only cLTD increased extracellular DKK3, which we then confirmed in brain slices (Figure S3). This finding is not unexpected as it is well described that different patterns of activity can lead to different molecular outcomes. For example, high-frequency stimulation (HFS; an activity pattern that resembles LTP) and low-frequency stimulation (LFS; a different activity pattern resembling LTD) leads to opposing effects on surface levels of the Wnt receptor Frizzled-5 (Fz5) (Sahores M et al., 2010, PMID: 20530549). Furthermore, cLTP increases Fz5 s-acylation, an important post-translational modification that regulates the surface levels of Fz5, whereas cLTD decreases it (Teo S et al., 2023, PMID: 37557176). Another example is the BDNF receptor TrkB. Surface TrkB is increased by tetanic stimulation, which also induces LTP as HFS or cLTP, but not by LFS (Du J et al., 2000, PMID: 10995446). Our findings suggest that DKK3 might contribute to synaptic changes underlying cLTD. Future experiments using chemogenetics or optogenetics might elucidate the role of DKK3 in activity-induced synaptic changes.

1. Are Abeta oligomer treatment-dependent increases in DKK3 protein levels in the cellular lysate and the extracellular fraction also suppressed by APV?

Our results in AD mice indicate that increased DKK3 release is dependent on NMDAR activation. To investigate if amyloid-β oligomers (Aβo) increase DKK3 levels in the cell lysate and extracellular fractions through NMDAR, we blocked these receptors in hippocampal neurons using AP-V (Figure S2G, H). In these experiments, we use a lower concentration of Aβo (200nM of Aβ1-42) to avoid any potential cytotoxic effect. In line with our previous results using a higher concentration of Aβo, we observed that Aβo markedly increased DKK3 levels both in the cell lysate and in the extracellular fraction compared to the reverse Aβ42-1 control peptide. Kruskal-Wallis with Dunn’s test showed a trend to a reduced levels of DKK3 in the extracellular fraction when we compared neurons treated with Aβo and APV with those neurons treated with Aβ and vehicle (p = 0.0726). However, this reduced levels of DKK3 in the extracellular fraction reached statistical significance using a t-test (p = 0.0384). No differences were observed between the reverse control peptide and Aβo and APV conditions. These results suggest that blockade of the NMDAR partially occludes the ability of Aβo to increase DKK3 levels in the extracellular fraction.

1. Why does DKK3 shRNA only downregulate inhibitory synapses but not excitatory synapses in the WT brain slice? Does this mean that in the WT brain, other DKK proteins (without changes in their expression as shown in Fig S6) are sufficiently expressed and compensate for the roles of DKK3 in excitatory synapse integrity?

The reviewer points out an interesting result. In J20 mice, DKK3 knockdown affects both excitatory and inhibitory synapse density (Figure 6B, C). In Figure 3B, D, we show that in vivo downregulation of DKK3 leads to an increased number of inhibitory synapses without affecting excitatory ones in the brain of WT animals. These results indicate that in a healthy brain (WT), DKK3 is required for the maintenance of inhibitory synapses but not for excitatory synapses under our experimental conditions. Furthermore, DKK3 partially shares the mechanism of action with DKK1 as both DKK proteins promote excitatory synapse loss through the Wnt/GSK3β pathway (Figure 4A-C) (Marzo A et al., 2016, PMID: 27593374). Therefore, it is possible that endogenous DKK1 levels in the hippocampus could compensate for the reduced expression of DKK3 resulting in the lack of changes in excitatory synapse number when DKK3 is knockdown in WT animals.

1. Manipulating DKK3 in WT brains only affects Gephyrin but not VGAT, but in J20, both Gephyrin and VGAT seem to be affected by DKK3 shRNA (Fig 6). The authors need to provide the pre vs post synapse number in Fig 6 and discuss the potential differences.

We have now included the quantification of excitatory and inhibitory pre- and postsynaptic puncta for 4-months old (Figure S6B, C) and 9-months old (Figure S6D, E) WT and J20 mice. At 4-months old, the density of Homer1 puncta for excitatory synapses and both vGAT and Gephyrin for inhibitory synapses was increased and decreased respectively by knocking down DKK3 in the J20 mice. At 9-months, strong trends were observed in all the synaptic markers when downregulating DKK3, but significance was only reached for Homer1 puncta.

1. Where are the Wnt receptors expressed? Are they exclusively expressed in neurons? Can the authors exclude the potential involvement of glial cells in this process?

In neurons, Wnt receptors can be expressed in the synaptic terminals. For example, Wnt receptor Frizzled-5 is located at the presynaptic terminal and the dendritic shaft but not at spines (Sahores M et al., 2010, PMID: 20530549; McLeod F et al., 2018, PMID: 29694885), whereas Frizzled-7 is located at the dendritic shaft and spines (McLeod F et al., 2018, PMID: 29694885). In addition, the Wnt co-receptor LRP6 is present at both pre- and postsynaptic sites in excitatory synapses (Jones ME et al., 2023, PMID: 36638182). Kremen1, another receptor for Dkk proteins, is also highly expressed in the brain and our unpublished superresolution results show that this receptor is present in both pre- and postsynaptic sites of 53% of excitatory and 30% of inhibitory synapses. However, these receptors are not exclusively expressed in neurons and many of them are also highly expressed in astrocytes (Zhang Y et al., 2016, PMID: 25186741). Based on the literature and our findings, we cannot rule out the possibility that DKK3 may signal to other cell types such as astrocytes, which could also contribute to changes in synapse density. However, recombinant DKK3 induces structural and functional changes in excitatory and inhibitory synapses within 3-4h (Figure 3), suggesting that DKK3 acts on neurons leading to synaptic changes.

1. Does the shRNA treatment of DKK3 affect the size and number of amyloid plaques in the AD mice?

We thank the reviewer for raising this very important question. We have now evaluated the impact of DKK3 knockdown in Aβ pathology in the J20 mice. We did not observe differences in the Aβ coverage nor the averaged number and size of Aβ plaques when DKK3 was silenced in the CA3 (Figure S6F). Therefore, the changes we observe in excitatory and inhibitory synapse density around plaques after knocking down DKK3 are unlikely to be due to changes in Aβ plaques.

Recomiendo que se explique si sí o no single source == edición ramificada.

Para mí la edición ramificada es una clase de single source donde los materiales de partida (la fuente) dan pie a gráficas de árbol, mientras que en el single source convencional lo que se busca son gráficas de estrella. En este sentido, el single source sería una visión más simplificada, idealista y repetitiva (en una estrella, el html se reproduce dos veces para una versión en línea y un epub, mientras que en la edición ramificada un mismo html puede reutilizarse). La edición ramificada sería una visión más compleja y concreta de los desafíos editoriales del día a día, en el que se intenta tener el árbol más simple posible según las necesidades y las capacidades con las que se cuentan.

Hay que explicar mejor esto, porque la herencia de características es también importante para la edición ramificada. Por ejemplo, la herencia en un tamaño y familia de fuente por defecto para el documento, que se modifica si alguna etiqueta sobreescribe esta herencia, como en la etiqueta para fragmentos de código o verbatim.

No creo que sea necesario. Un editor WYSIWYG puede funcionar como mera interfaz gráfica donde el usuario edita de manera visual un texto que queda correctamente estructurado. Por ejemplo: LyX o ProseMirror.

En lo que sí estoy de acuerdo es que casi todos los WYSIWYG provocan dificultades porque permiten adición de atributos que afectan al diseño sin afectar la estructura (como cambio de fuentes o de colores).

Cambiaría por «escribir, editar, imprimir y distribuir» o «escribir y publicar» porque escribir es también producir.

Alberto, te recomiendo que reescribas el texto con estas indicaciones en cuenta:

• Corta oraciones en oraciones más pequeñas, principalmente las que abarcan un párrafo entero.
• No uses «y/o», en español la conjunción es inclusiva.

Si haces eso, en una oportunidad que tenga puedo ser corrector de estilo xd

of brede zijspier is een spier aan de voorzijde van het dijbeen

Na frase “Art Brut toma as correntes naïf como ponto de partida e os seus artistas são completamente autodidactas”, o significado de naïf é “que se caracteriza por refletir a realidade com deliberada ingenuidade, aparentemente infantil, e com poesia e simplicidade” 1. Essa palavra vem do francês naïf, que significa “ingênuo ou inocente” . O termo naïf é usado para designar um tipo de arte popular e espontânea, que valoriza a representação de temas cotidianos e manifestações culturais do povo 2. A arte naïf costuma ser mais associada à pintura e foi instituída no século XIX, com o pintor francês Henri Rousseau (1844-1910), considerado o precursor do estilo 2. A arte naïf é permeada por imagens do cotidiano, retratados de modo a lembrar desenhos infantis, dada a espontaneidade e pureza 2. Os artistas naïfs geralmente são autodidatas, ou seja, que não possuem conhecimento formal e técnico de arte, mas que exibem produções em que outros princípios são considerados, como a autenticidade 2.

Comentario 21/12/2023 público

A gestão da informação, de acordo com o estudioso Davenport (2002) consiste em algumas etapas: 4 ao todo, a 1 - Identificar como os gerentes e funcionários percebem os ambientes informacionais; 2 - Aquisião contínua das informações, obtenção, armazenamento e formatação das informações; 3 - Uso da informação: aplicação da informação atráves de uma ação símbolica; 4 - Distribuição da informação: Envolve o uso da informação entre os colaboradores da instituição, com o que precisam ou não.

Exemplos.

Conceito importante. Dados são a matéria, já a informação é o resultado desses dados organizamos a fim de transmitir uma ideia.

A informação é um conjunto de dados organizamos a fim de transmistir um conhecimento, o autor MCGEE também informa que a informação deve ter um limite, já os dados não.

Um dado não conduz a nenhum entendimento, é uma sequência de quantificados ou quantificáveis, representação dos fatos, conceitos ou instruções que se adaptem a relação, comunicação e processamento.

This is a ton of complexity per message.

Tuy nhiên, vì điểm tiêm nằm ở thượng nguồn của van nạp, màng nhiên liệu lỏng có thể hình thành trên thành cửa nạp và van nạp trong PFI động cơ.

Período em que a arte novamente volta-se ao sagrado.

Na grécia antiga a arte tinha mais liberdade, já que ideiais de valorização do homem eram pregados.

Não sabia dessa. As pinturas egipcias seguiam regras bem estabelicidas, exemplo: a lei da frontalidade.

A arte egipcia também era carregada de simbologia, religiosidade e significados, assim como na idade média.

A arte também se molda de acordo com o período e de acordo com as ferramentas da epóca..

Importante ressaltar que a arte aqui, para os indígenas, é algo partilhado, coletivo.

Read this to understand that "feminine" is not a biological idea for Young

DOI: 10.1681/ASN.2019040414

Resource: BDSC_32929

Curator: @Naa003

SciCrunch record: RRID:BDSC_32929

What is this?

DOI: 10.1158/0008-5472.CAN-23-1065

Resource: (BD Biosciences Cat# 566281, RRID:AB_2739655)

Curator: @scibot

SciCrunch record: RRID:AB_2739655

What is this?

• 65000 / min dažnio = max Ra period;
• 65000 / maz daznio = min Ra period

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1 Evidence, reproducibility and clarity: Bompierre et al have presented a set of interesting findings that demonstrate the interconnected roles of multiple PDEs in striatal cholinergic interneurons. They show that the regulation of neurons by PDEs differs between ChiNs and MSNs. Disentangling the complex and interconnected biology of PDE subtypes and calcium signalling is notoriously difficult and I commend the authors for not shying away from it. The data are interesting and compelling however I have a number of readily addressable concerns regarding their statistical analysis.

We are very grateful to the reviewer for the careful reading of our manuscript, positive comments and useful suggestions to enhance its readability. We also thank the reviewer for highlighting the difficulties resulting from the scarcity of these neurons. As described in the manuscript, during the course of several other projects, we noticed this sparse neuronal population that clearly departed from the vast majority of striatal neurons, the medium-sized spiny neurons. This particular neuronal population was identified as ChINs only later with immunohistochemistry. We were quite surprised that our visual identification was confirmed by immunohistochemistry in all cases, as described, now with more details, in the manuscript. We then performed a number of new experiments focused on ChINs while we could also re-analyze older experiments performed for other projects and in which ChINs were visually identified - all our experiments are terminated with the acquisition of a Z stack which allows a precise observation of each neuron in the imaging field. This explains some changes in the drugs presented in this manuscript.

Major concerns: • Statistics, general comments: o When performing multiple comparisons (as in figures 2-6) the authors should be using a one-way ANOVA or Friedman's test (for non-parametric).

Since data normality of a few samples was rejected by the Shapiro-Wilk test, and in line with our previous publication, we used non-parametric statistics for all of this study. The Friedman’s test is now applied to all situations comparing more than two conditions.

o When comparing an effect size between experimental conditions e.g. cAMP following NMDA with or without Lu AF64193, the conditions need to be compared directly, not just inferred from being 0.05. [ see Makin and Orban de Xivry 2019 PMID: 31596231 and Nieuwnhuis et al 2011 PMID: 31596231 for more details].

We agree with this comment and the recommended tests have been performed.

o Please include P values and degrees of freedom for each analysis, rather than just * indicating PP values are now indicated in the text - although a smaller P value does not imply that the difference is “more” significant. Degrees of freedom do not apply with Friedman and Wilcoxon rank tests.

• Introduction: o Page 3 para, describing the various PDEs reported to operate in ChiNs vs MSNs, is very convoluted and hard to follow. I recommend replacing this paragraph with a table: column 1: PDE subtype, 2: neuron type, 3: effect reported 4: ref.

We agree that this paragraph was difficult to understand. We tried to build a table as suggested, but such table could not convey all the aspects that we think should be made clear (mRNA or protein data, for example). Instead, we propose a revised paragraph that we hope will be easier to read.

• Methods: o Please include explanation for estimated cAMP concentration (right axis on figs 2c, 3b,5c-f

This was described in “Methods / Estimates of biosensor activation level”. We also added a similar estimate for cGMP concentration based on our previous work with the cGMP biosensor cyGNAL in Figures 4 and 6.

o Please specify slice thickness and age of mice

Slice thickness was missing and now added in methods (300 µm). The age was indicated in the first paragraph of Methods: “7 to 12 days”.

• Figure 1: o describe if 13 uM fsk is supposed to be a maximal concentration, what is the justification for this concentration. Is there a previously published dose-response curve?

A biochemical study of adenylyl cyclase intracellular domains (VC1 and IIC2 heteromer) reports a Kd for forskolin of 0.1 µM {Dessauer et al., 1997, J Biol Chem, 272, 22272-7}, well below the dose we used. To our knowledge, there is no published dose-response analysis of forskolin effect in ChINs and we did not perform this measurement. We routinely use 13 µM for practical reasons, our stock being 25 mM diluted 2,000-fold. ChINs display a much lower cAMP response than MSNs at this dose: we think that this interesting observation deserves to be reported (but we would like to point out that this is only a marginal aspect of our study). We totally agree that this point deserves more explanation and a paragraph has been added in the discussion: “The low responsiveness of ChINs to cAMP-activating signals such as forskolin is striking but the underlying cellular mechanisms remain to be determined. All adenylyl cyclases except AC9 are activated by forskolin {Defer et al., 2000, Am J Physiol Renal Physiol, 279, F400-16}. AC1, AC2 and AC5 are widely expressed in the brain, including the striatum {Matsuoka et al., 1997, J Neurochem, 68, 498-506}. Cluster analysis of mRNA transcript suggest that AC1 and AC2 predominate in ChINs whereas AC5 is mainly expressed in MSNs {Saunders et al., 2018, Cell, 174, 1015-1030.e16}. This indicates that the reduced responsiveness to forskolin in ChINs compared to MSNs does not result from ChINs lacking adenylyl cyclases sensitive to forskolin.”.

• Figure 2c: o Clarify which replicates are shown on the graph (I assume it's the n=11 i.e. number of slices)

Yes, the plot shows the 11 replicates of the same protocol, with each set of 3 points of a same color linked with a line showing the measurements for one experiment. This is now stated more clearly in the figure legend.

o You say "PDE3 thus contributes importantly to the regulation of cAMP level, and when this phosphodiesterase is inactivated pharmacologically, cAMP level becomes controlled by PDE4." But in fig 2c panel 2 inhibition of PDE4 with Picla increases cAMP without PDE3 being already inhibited. This doesn't agree with your statement, which implies that PDE4 only controls cAMP once PDE3 has been inhibited.

This is unfortunate since we did not want to suggest that PDE4 has no effect unless PDE3 is inhibited. Indeed, this experiment together with the next actually demonstrate that PDE3 and 4 are simultaneously engaged in cAMP regulation. This ambiguity was also noted by reviewer 3. We thus rephrased the link between between these two paragraphs.

o Data described but not shown: "In the absence of forskolin, application of PDE3 inhibitor (cilostamide, 1 µM, N=3; n=3; A=3) or PDE4 inhibitor (rolipram, 1 µM, N=2; n=2; A=2) or their combination (N=6; n=8; A=3) induced no significant ratio change in ChINs (as well as in MSNs)".

We removed the mention to the experiments with cilostamide and rolipram alone since N was less than 5. The effect of rolipram and cilostamide together are displayed in Figure 2, with proper statistics indicated in the text.

Also please explain why you have changed PDE inhibitor?

This project developed over several years and the phosphodiesterase inhibitors used in the team changed depending on their availability. In addition, some data such as the lack of effects on baseline cAMP level were extracted from experiments performed for other purposes, hence some differences in the inhibitors we used. Please note that given the scarcity of this neuronal population, many more trials and mice would be required to reproduce these experiments with a single inhibitor. We further consider that our approach contributes to the desired reduction in the use of animals in research (the 3-R).

• Figure 4: o Please clarify in text that data in 4b is from ChiNs and not pooled with data from MSNs? i.e. is the summary data from MSNs not shown?

Figure 4B and C only show average calcium responses in ChINs. This is now indicated in the results section “Figure 4B shows the calcium response to NMDA in ChINs with the average trace (left), and baseline and peak amplitude (right) for individual ChINs.” The calcium response to NMDA uncaging in MSNs has already been published (Betolngar 2019) and is not shown in this study.

o Response to DHPG is not formally compared between MSNs and ChiNs

In MSNs, the calcium response to DHPG showed variability, with a lack of response in most experiments but a clear calcium signal in a few MSNs. The cause of this variability was not the focus of this study but, nevertheless, we wanted to mention this qualitative observation to stimulate future studies on this subject. However, if a quantitative measurement of this variability is required, the description of the DHPG effect on MSNs will be removed.

• Figure 5: o Explain reasoning for switching PDE4 inhibitor (roflumilast).

At the time these experiments were performed, we were using roflumilast to inhibit PDE4.

Explain change in fsk concentration (0.5 uM in figure 5 vs 13 uM in earlier figs)

We wanted to study PDE1 in isolation, i.e. in conditions in which both PDE3 and PDE4 were inhibited. Simultaneous inhibition of PDE3 and 4 leads to biosensor saturation (Figure 2), a situation in which a PDE1-mediated decrease in cAMP level might be difficult to resolve. The forskolin concentration was therefore reduced to decrease adenylyl cyclase activation. This is now explained in the manuscript: ”In order to stimulate a moderate cAMP production and thus maintain the visibility of PDE1 action, a lower concentration of forskolin (0.5 µM) was employed in these experiments.“

o Text states: "These effects were blocked with Lu AF64196 (1-10 µM), a potent and selective PDE1 inhibitor" but this was not formally tested and in the figure a response is still visible (smaller than before Lu, but not blocked)

Indeed, a small change in the average ratio trace is still visible, so we changed “blocked” by “largely reduced”. The effect of Lu AF64196 in the NMDA condition was tested as follows: the change in ratio level induced by NMDA uncaging was calculated in control and Lu AF64196 conditions. This ratio change was compared between control and Lu condition with a Wilcoxon rank test. The same test was applied to compare control and DHPG condition. This is now indicated in the manuscript.

o Please explain the reason for the different time courses in figure 5ab vs 5c-f)

Figure 5A,B are illustrative experiments. Figure 5C-F are average traces from several experiments. This is indicated in the figure legend.

o Data not shown: "Of note, the addition of the PDE1 inhibitor did not increase the steady-state cAMP level, demonstrating that PDE1 did not exhibit a tonic activity before its activation by the calcium signal"

In our experimental conditions, the cAMP level is too high to faithfully report an increase in cAMP upon Lu AF64196 application, so we removed this sentence. However, the cGMP level in the presence of DEANO is farther from saturation, allowing to perform this control: the application of Lu AF64196 produced no significant increase in cGMP level, indicating that PDE1 is not active in our experimental conditions. This has been added in the results.

• Figure 6: o Comment on why using quisqualate (mixed AMPA and mGLuR) rather than DHPG as used previously?

These early experiments were performed with these drugs until we made sure that group 1 mGlu was responsible for this effect and the more specific agonist DHPG was used. The drug combination, however, is specific of group I mGlu activation.

o Again, effect reported as being blocked, but not formally tested and a response is still evident on the graph. If the authors believe that response is an artefact from the stim then please show data with NMDAR antagonists.

We agree that a small change in the average ratio trace is still visible, so we changed “blocked” by “largely reduced”. The effect of Lu AF64196 was tested as described above for cAMP, which is now indicated in the manuscript. We agree that NMDA as well as mGlu stimulation, by increasing calcium, can affect cyclic nucleotides by other mechanisms than PDE1 activation. This was already reported in our previous work, in particular in the hippocampus and cortex (Betolngar 2019). We are not sure that NMDAR antagonists would clarify the situation since NMDA receptor blockade would probably suppress the cAMP change that is still visible in the presence of the PDE1 inhibitor. Nonetheless our manuscript reports experimental conditions in which the vast majority of the effect that we focus on is blocked by the PDE1 selective inhibitor.

• Discussion: o Add references in para 2 describing the PDEs expressed by ChiNs

Done

o Figure 7: cartoon indicates that calcium will exclusively activate PDE1A, is this for simplicity or is their evidence to support this?

Single-cell RT-PCR data {Saunders et al., 2018, #42891} indicate that ChINs express PDE1A but not PDE1B. This is now indicated in the introduction. The cartoon has been changed accordingly.

o Please comment on the specificity of the pharmacological tools used throughout the study.

Phosphodiesterases bear a cleft-shaped catalytic site that is particularly amenable to chemical inhibition, and phosphodiesterase thus constitute a therapeutic target of great interest: a large number of highly specific phosphodiesterase inhibitors have been developed and tested by pharmaceutical companies. It nonetheless remains that our demonstration relies on the specificity of the phosphodiesterase inhibitors. We added a sentence in the discussion “We used highly specific phosphodiesterase inhibitors to acutely test the functional contribution of these phosphodiesterases.” to acknowledge this.

o In the context of regulation of striatal cholinergic and dopaminergic signalling by NO (via cGMP), I believe the authors should cite Hartung et al PMID: 21508928

This very valuable citation has been added in the discussion.

Minor comments: • Page 3 paragraph 1 and 3 the authors have used ellipsis instead of finishing their sentences.

Done.

• In figure 1a please indicate you are recording in dorsomedial region of the striatum (you state in methods this is your recording location, but it would be helpful to show it here)

We thought of improving the cartoon in Figure 1 by drawing a rectangle over the dorso-medial striatum on the image of the brain slice, but that would suggest that the slices had been cut at this stage of the preparation, which was not the case. Adding another image of a brain slice would clutter the figure. We leave it to the Editor to decide how this should be handled.

• The authors use a lot of different drugs, I think a table listing the drugs, concentrations and their targets would help limit confusion.

This will certainly make it easier for the reader. This table has been added in Methods / Chemicals and drugs.

• I found the graphs with each replicate in a different colour quite difficult to read. My personal preference would be for graphs to show each replicate as a transparent line/small symbol, with the mean and SEM shown larger and in bold.

We tried to enhance the visibility as suggested. SEM should not be shown since our statistics are non-parametric.

Significance: General strengths: The question of how PDEs interact to regulate striatal output is extremely interesting and notoriously difficult to tackle. The authors have relied upon pharmacological manipulation of PDEs. A pharmacological approach has both strengths (intact system with little compensation occurring between PDEs, which would occur with a genetic strategy) and weaknesses (relying on each of the drugs to act selectively and specifically). By investigating multiple PDEs in the same system in two neuron types, I believe the authors are illustrating interesting findings. For these findings to be more concrete I believe they need a more robust statistical approach, but the experiments and questions are valid. PDEs are of interest to most neuroscientists and understanding cholinergic function is of interest to anyone studying the striatum of basal ganglia more broadly. My expertise is investigating the interactions between striatal cholinergic and dopaminergic signalling.

Reviewer #2 Evidence, reproducibility and clarity : The work characterizes in depth the dynamics of the cyclic nucleotide signaling in cholinergic interneurons (ChINs) in the striatum and the interconnection with calcium signaling. The study is ambitious and risky since it targets a minority of neurons representing only 1% of the total population of striatal neurons. For that they used genetically encoded biosensors, at a very low infection rate, and highly specific phosphodiesterase inhibitors. With these tools they defined PDE1, PDE3 and PDE4 as the key regulators of cAMP levels in ChINs and the interplay with incoming signals raising cGMP and free-calcium levels after nitric oxide or glutamate activation of NMDA or mGlu1/5 receptors, respectively. The conclusions of the study are solid and well supported by the experimental results.

We would like to thank the reviewer for his/her positive comments about our work.

The team has the necessary technical and conceptual background in the field. This is very important to trust the criteria they used to identify ChINs, a fundamental hallmark in this study.

Again, we are very grateful to the reviewer for this very positive comment.

Still, confirmation by immunohistochemical labeling with ChAT antibodies sounds important and perhaps it should had been performed in more experiments.

We agree that our qualitative immunostaining validation of ChAT expression was too terse. We re-analyzed our archived data to provide a more precise account of our observations. We first identified ChINs from their morphology in the biosensor image stack, then checked whether these neurons were positive for ChAT. This is now explained in detail in “Identification of Cholinergic Interneurons in a brain slice”: “11 brain slices were fixed after the biosensor experiment and later processed for ChAT immunoreactivity. In these slices, 15 neurons were visually identified as ChINs during the biosensor recording session. All of these neurons showed a positive ChAT labelling.”

Significance: The results represent an important conceptual advance in the field. To understand better the signaling that regulates firing of cholinergic neurons in the striatum might be relevant to explain pathological responses and they could be useful to define better strategies for the treatment of Parkinson's patients, for instance. In this regard, this study fills an existing gap since this elusive neuronal population was not functionally characterized before. The basic aspects of the study could be of interest to a broad audience.

Reviewer #3 Evidence, reproducibility and clarity: Summary: The author's present very elegant findings regarding how NO regulates cAMP in striatal cholinergic interneurons (ChINs). The major strength of the manuscript lies in the approach, which enables single-cell imaging of neuronal signaling in acute brain slices. A clever combination of pharmacological tools were then utilized to dissect PDE contribution toward cAMP alterations in ChINs. While the manuscript is high-quality, there are a few controls and points of discussion that need to be considered.

We would like to thank the reviewer for his/her careful reading of our manuscript and very positive comments about our study.

Major: There are numerous references to results seemingly missing from the figures. - Figure 2 TP-10 - Figure 2 PF-05 traces - Figure 2 data in absence of FSK (rolipram, cilo) - Figure 3 ODQ

We thought that these experiments showing a lack of effect were of little interest to the reader and therefore omitted raw traces and statistics from the manuscript. However, we fully agree to display more of our data, as long as it does not clutter the main points of our manuscript. We now illustrate the lack of effect of PDE2A inhibition with a typical experiment in Figure 2C. The ratio level is now shown in Figure 2D for roli-cilo and TP-10, with matching statistics in the text. Figure 3 now shows the lack of effect of ODQ.

Have the author's considered an alternative perspective that slow cAMP detection in ChIN, relative to MSN, could be due to the size of neuron? The significantly greater volume of ChIN soma could conceivably require more cAMP to reach the detection threshold of the biosensor. Therefore, how do the author's reconcile such technical caveats?

This is a very interesting hypothesis that is supported by many theoretical and experimental data: it takes longer for membrane adenylyl cyclases to fill up a void volume in which both phosphodiesterases and the biosensor reside. We could rule-out the buffering effect of the biosensor by the experiment described in Figure 1B, but a lower surface to volume ratio such as that observed for ChINs vs MSNs could indeed explain a biologically slower onset in cAMP level. However, a lower surface to volume ratio should not affect the steady-state level that will be eventually reached upon continuous forskolin application: it takes longer to fill up the volume but, if waiting long enough, the final level will be only determined by the equilibrium between cyclase and phosphodiesterase. Forskolin applications of more than 10 min (Figure 2) led to steady-state levels that were far below biosensor saturation, while it did reach saturation in MSNs. Therefore, while we certainly acknowledge the importance of the peculiar neuronal morphology of ChINs, there must be additional specific differences between ChINs and MSNs. In any case, we agree that this important point was missing in our manuscript and we added a paragraph in the discussion to discuss differences in adenylyl cyclases and cell geometry.

In the traces from Figure 2, it is unclear why PDE3 and PDE4 have differential contributions toward cAMP elevation depending on the order of inhibition.

Thank you for pointing out our poor wording, which has also been noted by the first reviewer. Indeed, the data in Figure 2 shows that PDE3 and PDE4 are simultaneously engaged in cAMP regulation. This part has been rewritten.

Moreover, we cannot conclude that PDE3 and PDE4 the major PDEs in ChIN from such experiment based on the result that IBMX did not further raise cAMP. Likely, the biosensor has reached the detection ceiling. This should be discussed as a possibility.

It is an interesting possibility that cAMP levels higher than biosensor saturation level ([cAMP] above 100 µM) could be modulated after PDE3 and PDE4 inhibition, in concentration ranges that go beyond biosensor saturation level. However, our experiments clearly demonstrate that the combined action of PDE3 and PDE4 constitutes the first line of cAMP control since the concomitant inhibition of PDE3 and PDE4 raises [cAMP] beyond physiological relevance. When both PDE3 and PDE4 were inhibited, cAMP indeed reached the level of biosensor saturation which led us to state “The ratio was not further raised by the non-specific phosphodiesterase inhibitor IBMX (200 µM), indicating the saturation level of the biosensor by cAMP (Rmax)”, a conclusion that remains valid.

The author's intepretation ignores the influence of calcium on the activity of various types of ACs, which seems to be a critical feature given the experimental design that first broadly stimulates ACs with forskolin.

We agree with the Reviewer that calcium will certainly activate AC1 (possibly present in ChINs) and inhibit AC5 (expressed in MSNs and possibly also in ChINs). Our experimental design relies on the highly specific PDE1 inhibitor to isolate the selective contribution of PDE1, but minor changes in cAMP and cGMP remained even after PDE1 inhibition, as also pointed out by the first Reviewer. We found experimental conditions in which the contribution of PDE1 could be largely visible, which was the point of this study. However, we certainly agree with the Reviewer that a calcium signal will affect cyclic nucleotide levels through a number of other mechanisms. Therefore, we added the sentence “It should also be noted that, in the presence of the PDE1 inhibitor Lu AF64196, some changes in cAMP level still remained, which can result either from incomplete PDE1A inhibition and/or from NMDA effects on other targets, such as calcium-modulated adenylyl cyclases.”

A missing control in Figure 5 is the effect of Lu AF64196 by itself on cAMP (and in the presence of FSK pre stimulation).

We agree that this is an important control. However, this could not be tested on this protocol with cAMP since the ratio was too close to Rmax, and an increase in cAMP level following PDE1 inhibition would be undetectable. However, with cGMP imaging, the ratio reached with DEANO was farther from saturation such that an increase in cGMP resulting from the inhibition of PDE1 should be detectable. However, Lu AF64196 showed no significant effect. This measurement was added in the manuscript: “As a control, we verified that PDE1inhibition had no effect on the steady-state cGMP level elicited by 10 µM DEANO (Figure 7G: in DEANO: 0.78 of Rmax; in DEANO and Lu AF64196: 0.81; N=5, n=6, a=5; Wilcoxon P=0.094).”

The mechanism would be signicantly bolstered by measuring cAMP from PDE4 inhibition following forskolin and DEANO (Figure 3).

It is true that, in the presence of forskolin and DEANO, the only PDE that remains is PDE4: its inhibition should increase cAMP to the maximal level. Unfortunately, this experimental scheme was not tested. However, Figure 3 is focusing on the regulation of PDE3 by cGMP and at this stage of the reasoning, it might be confusing to get back to the question of which PDE remains after PDE3 has been blocked. In this manuscript, we want to highlight that PDE3 is blocked via the NO-cGMP signaling pathway, and we think that the data in Figure 3 demonstrates this point clearly.

Minor: The introduction should discuss the critical role of ChIN in striatum rather than simply stating "critical role in striatal functions"

A paragraph has been added in the Introduction to highlight the importance of ChINs in striatal function.

PDE should be included as abbreviation in introduction after first mention of phosphodiesterases.

We agree that this was inducing confusion. We keep PDE1-11 as it is the official names of proteins, but we replaced all occurrences of “PDE” by “phosphodiesterases” when alluding to the general concept of this class of enzymes.

Light sources (e.g. laser) for excitation during imaging are missing from the methods.

This was indicated in Methods / Biosensor imaging: “LED light sources (420 nm with a 436 nm excitation filter and 360 nm) were purchased from Mightex (Toronto, Canada).”

The study certainly provides implication for diseases associated with striatal dysfunction such as Parkinson's disease. However, it may be important to note that experiments were performed on slice preparations from very young animals, which could have inherent differences in functionality relative to an aged or diseased context.

We agree that our preparation of brain slices from mice pups is not representative of what could be found in adults, and even less in pathological conditions. Nonetheless, we believe that we identified a crosstalk mechanism that has never been reported in neurons, and that is not taken into account in theories of striatal functions. We hope that this novel understanding will lead to the development of experiments on adult mice that might confirm the functional importance of this effect, in particular pharmacological studies in adult animals with PDE3 inhibitors.

Significance: General assessment: The study utilizes pharmacological tools to selectively target enzymes and receptors in the cAMP cascade to mechanistically dissect how cAMP is handled in ChINs. The major strength is ability to perform such experiments in acute brain slices, i.e. a "native" neuronal context. An exciting aspect is stimulation of NMDAR by agonist-uncaging, thereby revealing an endogenous signaling route that modulates cAMP. A major physiological limitation is reliance on fsk to induce AC activity, however the approach is suitable to obtain mechanistic information. Moreover, conducting experiments in a Parkinsonian disease model would provide tremendous value, although such pursuits are beyond the scope of the work here. Advance: The study builds off robust studies previously published by the author's. The work is also similar to AC-cAMP investigations on acute brain slices performed by the Sabatini (24765076, 29154125) and Martemyanov (29298426, 31644915) labs, which unfortunately were not discussed/cited here. This is perhaps a missed opportunity to highlight the significance of the author's study. For instance, the Sabatini lab investigated calcium influence on cAMP but in the hippocampus. The Martemyanov lab investigated striatal cAMP, but not in ChINs or through calcium or cGMP mechanisms. Therefore there is a gap toward understanding striatal ChINs, which is clearly demonstrated in the author's work here.

These very important references have been left out of our manuscript intentionally since not pertaining directly to the topic of the manuscript. We would like to point out that we also omitted citations to our own work which had also described dopamine, adenosine and acetylcholine responses measured with various biosensors in striatal neurons (Castro 2013 23551948; Yapo 2017 28782235; Nair 2019 24560149). Indeed, this research field is flourishing and we hope that people interested in the general question of cAMP signal integration in neurons will easily find many such relevant publications.

Audience: I find this basic research to have a relatively broad appeal. For example, my lab is working on behavioral aspects of motor dysfunction. Therefore, the pharmacological insight here is very intriguingly. The mechanistic nature of the work may also appeal to those working on the signaling aspect of such diseases.

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Author Response

Reviewer #1 (Public Review):

Sun et al. investigated the circuit mechanism of a novel type of synaptic plasticity in the projection from the visual cortex to the auditory cortex (VC-AC), which is thought to play an important role in visuo-auditory associative learning. The key question behind this paper is what is the role of CCK positive projection from the entorhinal cortex in the plasticity of VC-AC projections? They discover that the strength of VC-AC projections does not change when pairing the stimulation of this pathway with the acoustic stimulation of the auditory cortex (AC) unless CCK is applied to the AC or CCK positive projection from the entorhinal cortex to auditory cortex (EC-AC) is optogenetically stimulated. In contrast, optogenetically stimulating VC-AC projections, which express a lower level of CCK than the EC-AC projection, do not induce such synaptic plasticity. Interestingly, the data also indicates that even if the EC-AC pathway is stimulated 500ms ahead of the pairing of stimulating VC-AC pathway and the AC, the VC-AC synaptic strength can still be potentiated, consistent with the long-lasting nature of CCK as a neuropeptide. By performing a fear conditioning assay, the authors demonstrate that the CCK signaling is indeed required for the association of visual and auditory cues.

The proposed mechanism is interesting because it not only helps explain the heterosynaptic plasticity of the visual-auditory projection but also will provide insight into how the entorhinal cortex as an association area contributes to the association of visual and auditory cues. Nevertheless, this study suffers from the lack of a few key experiments, which prevents drawing a conclusion on the contribution of CCK release from the EC-AC projection to the plasticity of the VC→AC projection.

We are grateful for the constructive comments provided by the reviewers and appreciate the significant effort they have dedicated to reviewing our manuscript. To enhance our study and strengthen our conclusions, we have made the following revisions in response to their feedback.

1) One main conclusion from figures 1-3 is that CCK released from the EC-AC projection is required for the plasticity of VC-AC projection in addition to pairing VALS with noise/electrical stimulation. But the data in those figures cannot exclude alternative explanations that CCK alone or the pairing CCK with either VALS or noise are sufficient to make the VC-AC synaptic connection more potent. It concerns the mechanism underlying the effect of CCK: CCK may function simply as a neuromodulator to regulate the excitatory synaptic transmission, but not to promote long term synaptic plasticity.

Thanks for the valuable comment and pointing out the weakness. In response to the comment, we have conducted additional control experiments to reinforce our conclusions. These include: For Figure 1G, we introduced three control groups: CCK alone (Figure1-figure supplement 1F-G), CCK + presynaptic activation of VC-to-AC inputs (Figure 1-figure supplement 1H-I), and CCK + postsynaptic firing induced by noise (Figure 1-figure supplement 1J-K). Our findings from these control experiments indicate that in all three scenarios, there was no potentiation of the VC-to-AC inputs. Further details can be found in Figure 1-figure supplement 1F-K.

For Figure 2E, we introduced three control groups: HFS laser EC-to-AC alone (Figure 2-figure supplement 1H-I), HFS laser EC-to-AC + presynaptic activation of VC-to-AC inputs (Figure 2-figure supplement 1L-M), and HFS laser + postsynaptic firing induced by noise (Figure 2-figure supplement 1P-Q). And we found that in all three scenarios, the VC-to-AC inputs were not significantly potentiated. Please see details in Figure 2-figure supplement 1.

Given that our in vivo results already demonstrated that neither HFS laser EC-to-AC alone, nor its combination with presynaptic or postsynaptic activation, potentiated the VC-to-AC inputs, we did not replicate these control groups in our ex vivo setup. These additional experiments enhance the robustness of our findings and address the initial concerns raised.

2) Similar issue exists in Fig. 2H and 3J. Without proper controls, it is impossible to tell whether all three conditions (HFLSEA, VALA, noise/electrical stimulation) are necessary for potentiated AC responses to acoustic/electrical stimulation.

Same as above, we have conducted additional control experiments to reinforce our conclusions. These include:

For Figure 2H, we also tested the noise response in the above three control groups: HFS laser EC to AC alone (Figure 2-figure supplement 1J-K), HFS laser EC-to-AC + presynaptic activation of VC-to-AC inputs (Figure 2-figure supplement 1N-O), and HFS laser + postsynaptic firing induced by noise (Figure 2-figure supplement 1R-S). And we found that fEPSPs evoked by noise stimuli were significantly potentiated after HFS laser EC-to-AC + Post (Figure 2-figure supplement 1R-S). However, there was no potentiation observed following HFS laser EC-to-AC alone (Figure 2-figure supplement 1J-K) and HFS laser EC-to-AC + Pre (Figure 2-figure supplement 1N-O).

These results suggest that both HFS laser targeting the EC-to-AC projection and noise-induced AC firing are required to potentiate the AC's response to acoustic stimuli. In contrast, activation of the VC-to-AC projection is not necessary. This finding aligns with our previous research (Li et al., 2014).

Given the similarity in experimental design, we opted not to replicate these specific control groups in our ex vivo setup.

These additional control experiments have been crucial in reinforcing the conclusions of our study.

3) Fig. 2E and 3G show that the stimulation of CCK-positive EC-AC projection is required for the plasticity of VC-AC projection. Considering most EC-AC projection neurons co-release glutamate and CCK, however, we cannot tell if CCK or glutamate or both matter to this type of plasticity. Even though the long delay in Fig 5B is consistent with the neuropeptide nature of CCK, direct experimental evidence is needed, since it is where the novelty of the paper is.

Thank you for your constructive feedback. In response to the suggestions, for Figure 2E, we have incorporated two additional experiments: one with a CCKB receptor (CCKBR) antagonist and another with ACSF infused into the AC prior to HFS laser EC-to-AC + Pre/Post Pairing (Figures 2N-P). Our findings demonstrate that the CCKBR antagonist effectively inhibited the potentiation of the VC-to-AC inputs following the HFS laser EC-to-AC + Pre/Post Pairing. Conversely, ACSF did not exhibit this inhibitory effect. For further information, please refer to Figures 2N-P. Given the similarity in experimental design, we opted not to replicate these groups in our ex vivo setup.

4) In Fig. 6, the authors examined the necessity of CCK for the generation of the visuo-auditory association. The experimental approach of injection CCK receptor blocker or CCK-4 is not specific to the EC-AC pathway. There is neither a link between VC-AC plasticity nor this behavioral result. Thus, the explanatory power of this experiment is limited in the context set up by the first 5 figures.

Thank you for highlighting this area for improvement. To enhance the explanatory power of our behavioral experiments, we conducted the following additional studies:

1) Assessing the Necessity of CCK+ EC-to-AC Projection in Establishing Visuo-Auditory Association:

We bilaterally injected AAV9-syn-DIO-hM4Di-eYFP or AAV9-syn-DIO-eYFP into the EC and implanted cannulae in the AC of Cck Ires-Cre mice. During the encoding phase, we inactivated the CCK+ EC-to-AC pathway via CNO infusion into the AC. Our results show that this inactivation prevents the behavioral establishment of an association between the visual stimulus (VS) and auditory stimulus (AS), without affecting the fear conditioning memory to the AS (Figure 6B, beige).

2) Determining the Role of VC-to-AC Projection in Establishing Visuo-Auditory Association: We bilaterally injected AAV9-syn-hM4Di-eYFP or AAV9-syn-eYFP into the visual cortex (VC) and also implanted cannulae in the AC of Cck Ires-Cre mice. Inactivating the VC-to-AC pathway during the encoding phase with CNO infusion in the AC, we observed that this inactivation hinders the establishment of a behavioral association between VS and AS, but does not interfere with the fear conditioning memory to the AS (Figure 6B, red).

3) Investigating the Importance of CCK+ EC-to-AC Projection in Recalling Recent Visuo-Auditory Association:

Again, AAV9-syn-DIO-hM4Di-eYFP or AAV9-syn-DIO-eYFP was injected bilaterally into the EC, and cannulae were implanted in the AC of Cck Ires-Cre mice. By inactivating the CCK+ EC-AC pathway during the retrieval phase with CNO infusion into the AC, we found that such inactivation disrupted the recall of the recent association between VS and AS behaviorally, yet did not affect the fear conditioning memory to the AS (Figure 6D, beige).

4) Assessing the Necessity of VC-to-AC Projection in Recalling Recent Association Memory: For this experiment, AAV9-syn-hM4Di-eYFP or AAV9-syn-DIO-eYFP was injected bilaterally into the VC, and cannulae were placed in the AC of Cck Ires-Cre mice. Inactivating the VC-AC pathway during the retrieval phase with CNO infusion in the AC led to the discovery that this inactivation disrupted the behavioral recall of the recent association between VS and AS but did not disrupt the fear conditioning memory to the AS (Figure 6D, red).

These additional experiments significantly contribute to our understanding of the roles played by the CCK+ EC-AC and VC-AC projections in both the establishment and recall of visuo-auditory associative memories.

5) In page 16, line 322-326, the authors concluded that to induce the plasticity of VC→AC projection, Delay 1 should be longer than 10 ms and Delay 2 should be longer than 0 ms. This conclusion was not fully supported by the data from Figure 5B-D, because there is no data point between -65 ms and 10 ms for Delay 1 (for example 0 ms), and no negative values for Delay 2.

We rewrote this paragraph and hope it is more accurate now.

“Taken together, our study indicates that significant potentiation of the VC-to-AC inputs can be observed (Figure 5D, black cube) across five pairing trials with a 10-second inter-trial interval, under certain tested conditions: (i) the frequency of repetitive laser stimulation of the CCK+ entorhinal cortex (EC) to AC projection was maintained at 10 Hz or higher (as we did not test frequencies between 1 to 10 Hz), (ii) Delay 1 was set within the tested range of 10 to 535 ms (noting the absence of data between -65 to 10 ms), and (iii) Delay 2 was within the range of 0 to 200 ms (acknowledging that negative values for Delay 2 were not explored).”

Reviewer #2 (Public Review):

The manuscript by Sun et al., investigates the synaptic plasticity underlying visuo-auditory association. Through a series of in vivo and ex vivo electrophysiology recordings, the authors show that high-frequency stimulation (HFLS) of the cholecystokinin (CCK) positive neurons in the entorhino-auditory projection paired with an auditory stimulus can evoke long-term potentiation (LTP) of the visuo-auditory projection. However, LTP of the visuo-auditory projection could not be elicited by HFLS of the visuo-auditory projection itself or by an unpaired stimulus. They further demonstrate that auditory stimulus pairing with CCK is required to elicit LTP of the visuo-auditory projection as well as visuo-auditory association in a fear conditioning behavioral experiment. As they found elevated expression of CCK in entorhinal neurons which project to the auditory cortex, they conclude that HFLS of the entorhino-auditory projection causes CCK release.

Strengths:

The authors use an elegant approach with Chrimson and Chronos to stimulate different auditory inputs in the same mouse in vivo and also in slice and demonstrate that potentiation of the visuo-auditory projection is dependent on HFLS of the entorhino-auditory projection paired with auditory stimulus. Furthermore, they test several parameters in a systematic fashion, generating a comprehensive analysis of the plasticity changes that regulate visuo-auditory association.

Weaknesses:

In their previous publications (Chen et al., 2019; Li et al., 2014; Zhang et al., 2020), it has been established that HFLS of the entorhino-auditory projection and CKK release are important for visuo-auditory association via electrophysiology and behavioral experiments. The Chrimson and Chronos approach was applied by Zhang et al., 2020, where they already found that the visuo-auditory projection was potentiated through HFLS of entorhino-neocortical fibers. This manuscript extends those findings by testing different parameters of pairing, which may not represent a major conceptual advance. Unlike the electrophysiological recordings, drug infusion is used in behavioral manipulations to show that HFLS of the entorhino-auditory projection is important for visuo-auditory association. While the use of drugs to inhibit CKK receptors is important, it does not directly demonstrate that CCK release from the entorhino-auditory is necessary.

We deeply appreciate the reviewer's constructive and insightful feedback. Building on our previous work (Zhang et al., 2020), which highlighted the potentiation of the VC-to-AC projection through high-frequency laser stimulation (HFS laser) of entorhino-neocortical fibers, our current study probes further into the intricacies of this process. We have thoroughly explored the specific conditions necessary for the potentiation of the VC-to-AC projection, assessing a wide range of parameters.

A significant advancement in our current research is the elucidation of why HFS of the VC-to-AC pathway alone fails to induce potentiation, whereas HFS of the EC-to-AC pathway, coupled with Pre/Post Pairing, is effective. This critical distinction is linked to the heightened expression of CCK in EC neurons projecting to the AC, in contrast to those from the VC. In this revised version of our study, we have also demonstrated that HFS laser stimulation of the EC-to-AC CCK+ projection induces the release of endogenous CCK in the AC using a combination of a CCK sensor and fiber photometry.

Behaviorally, our revised research emphasizes the vital role of the CCK+ EC-AC projection in both establishing and retrieving visuo-auditory memories, thereby highlighting its fundamental importance in memory processing. Moreover, our study confirms that the CCK+ EC-AC projection is not only crucial for memory formation and retrieval but also indicates that the VC-to-AC projection is the anatomical basis for establishing visuo-auditory associations and serves as the principal storage site for visuo-auditory associative memory. These findings represent significant strides in our understanding of synaptic plasticity and memory mechanisms.

For the behavioral part, to build the link that HFS laser of the EC-to-AC CCK+ projection is important for visuo-auditory association in the behavioral context, we conducted the following additional behavioral studies (for details please see the response to comment 4 of reviewer 1):

1) Assessing the Necessity of CCK+ EC-to-AC Projection in Establishing Visuo-Auditory Associative memories, by inactivating the pathway with inhibitory DREADD during the encoding phase.

2) Investigating the Importance of CCK+ EC-to-AC Projection in Recalling Visuo-Auditory Association, by inactivating the pathway with inhibitory DREADD during the retrieving phase.

1. Use multiprocessing when you need to do many heavy calculations and you can split them up.
2. Use asyncio or threading when you're performing I/O operations -- communicating with external resources or reading/writing from/to files.
3. Multiprocessing and asyncio can be used together, but a good rule of thumb is to fork a process before you thread/use asyncio instead of the other way around -- threads are relatively cheap compared to processes.

"Non-blocking" means a program will allow other threads to continue running while it's waiting. This is opposed to "blocking" code, which stops execution of your program completely. Normal, synchronous I/O operations suffer from this limitation.

• for: adjacency - Thomas Homer-Dixon - Ernest Becker, terror management theory, immortality project

INCIDENTE DE DESLOCAMENTO DE COMPETÊNCIA Nº 1 - PA

CONSTITUCIONAL. PENAL E PROCESSUAL PENAL. HOMICÍDIO DOLOSO QUALIFICADO. (VÍTIMA IRMÃ DOROTHY STANG). CRIME PRATICADO COM GRAVE VIOLAÇÃO AOS DIREITOS HUMANOS. INCIDENTE DE DESLOCAMENTO DE COMPETÊNCIA – IDC. INÉPCIA DA PEÇA INAUGURAL. NORMA CONSTITUCIONAL DE EFICÁCIA CONTIDA. PRELIMINARES REJEITADAS. VIOLAÇÃO AO PRINCÍPIO DO JUIZ NATURAL E À AUTONOMIA DA UNIDADE DA FEDERAÇÃO. APLICAÇÃO DO PRINCÍPIO DA PROPORCIONALIDADE. RISCO DE DESCUMPRIMENTO DE TRATADO INTERNACIONAL FIRMADO PELO BRASIL SOBRE A MATÉRIA NÃO CONFIGURADO NA HIPÓTESE. INDEFERIMENTO DO PEDIDO. 1. Todo homicídio doloso, independentemente da condição pessoal da vítima e/ou da repercussão do fato no cenário nacional ou internacional, representa grave violação ao maior e mais importante de todos os direitos do ser humano, que é o direito à vida, previsto no art. 4º, nº 1, da Convenção Americana sobre Direitos Humanos, da qual o Brasil é signatário por força do Decreto nº 678, de 6/11/1992, razão por que não há falar em inépcia da peça inaugural. 2. Dada a amplitude e a magnitude da expressão “direitos humanos”, é verossímil que o constituinte derivado tenha optado por não definir o rol dos crimes que passariam para a competência da Justiça Federal, sob pena de restringir os casos de incidência do dispositivo (CF, art. 109, § 5º), afastando-o de sua finalidade precípua, que é assegurar o cumprimento de obrigações decorrentes de tratados internacionais firmados pelo Brasil sobre a matéria, examinando-se cada situação de fato, suas circunstâncias e peculiaridades detidamente, motivo pelo qual não há falar em norma de eficácia limitada. Ademais, não é próprio de texto constitucional tais definições. 3. Aparente incompatibilidade do IDC, criado pela Emenda Constitucional nº 45/2004, com qualquer outro princípio constitucional ou com a sistemática processual em vigor deve ser resolvida aplicando-se os princípios da proporcionalidade e da razoabilidade. 4. Na espécie, as autoridades estaduais encontram-se empenhadas na apuração dos fatos que resultaram na morte da missionária norte-americana Dorothy Stang, com o objetivo de punir os responsáveis, refletindo a intenção de o Estado do Pará dar resposta eficiente à violação do maior e mais importante dos direitos humanos, o que afasta a necessidade de deslocamento da competência originária para a Justiça Federal, de forma subsidiária, sob pena, inclusive, de dificultar o andamento do processo criminal e atrasar o seu desfecho, utilizando-se o instrumento criado pela aludida norma em desfavor de seu fim, que é combater a impunidade dos crimes praticados com grave violação de direitos humanos. 5. O deslocamento de competência – em que a existência de crime praticado com grave violação aos direitos humanos é pressuposto de admissibilidade do pedido – deve atender ao princípio da proporcionalidade (adequação, necessidade e proporcionalidade em sentido estrito), compreendido na demonstração concreta de risco de descumprimento de obrigações decorrentes de tratados internacionais firmados pelo Brasil, resultante da inércia, negligência, falta de vontade política ou de condições reais do Estado-membro, por suas instituições, em proceder à devida persecução penal. No caso, não há a cumulatividade de tais requisitos, a justificar que se acolha o incidente. 6. Pedido indeferido, sem prejuízo do disposto no art. 1º, inc. III, da Lei nº 10.446, de 8/5/2002.

• Informativo nº 790
• 10 de outubro de 2023.
• TERCEIRA SEÇÃO
• Processo: IDC 22-RO, Rel. Ministro Messod Azulay Neto, Terceira Seção, por unanimidade, julgado em 23/8/2023, DJe 25/8/2023.

Ramo do Direito DIREITO CONSTITUCIONAL, DIREITO PROCESSUAL PENAL

Paz, Justiça e Instituições EficazesTema <br /> Incidente de deslocamento de competência (IDC). Deferimento parcial. Art. 109, §5°, da CF/1988. Medida constitucional excepcional. Requisitos cumulativos. Presença. Conflito agrário em Rondônia. Grave violação a direitos humanos. Ineficácia das instâncias locais e risco de responsabilização internacional.

DESTAQUE - A Terceira Seção deferiu, parcialmente, o incidente de deslocamento de competência para que a investigação, o processamento e o julgamento dos mandantes, intermediários e executores dos assassinatos de vítimas, em sua maioria, lideranças de movimentos em prol dos trabalhadores rurais, e responsáveis por denúncias de grilagem de terras e de extração ilegal de madeira, ocorridos em contexto de conflito agrário instalado no Estado de Rondônia, sejam deslocados para o âmbito da Justiça Federal daquele Estado.

INFORMAÇÕES DO INTEIRO TEOR - O art. 109, § 5º, da Constituição Federal, estabelece que, nas "hipóteses de grave violação de direitos humanos, o Procurador-Geral da República, com a finalidade de assegurar o cumprimento de obrigações decorrentes de tratados internacionais de direitos humanos dos quais o Brasil seja parte, poderá suscitar, perante o Superior Tribunal de Justiça, em qualquer fase do inquérito ou processo, incidente de deslocamento de competência para a Justiça Federal".

• Conforme se extrai do IDC n. 1, os requisitos do incidente de deslocamento de competência são: a) grave violação de direitos humanos; b) necessidade de assegurar o cumprimento, pelo Brasil, de obrigações decorrentes de tratados internacionais; c) incapacidade - oriunda de inércia, omissão, ineficácia, negligência, falta de vontade política, de condições pessoais e/ou materiais, etc. - de o Estado Membro, por suas instituições e autoridades, levar a cabo, em toda a sua extensão, a persecução penal (IDC n. 1/PA, Terceira Seção do STJ).

Colhe-se da doutrina relativa ao tema, bem como da análise dos casos de deslocamento de competência decididos neste Superior Tribunal de Justiça, que os requisitos são cumulativos, não bastando a constatação de ineficiência dos mecanismos existentes para apuração e punição por parte dos órgãos persecutórios estaduais. É imprescindível que se demonstrem a gravidade das violações aos direitos humanos, a incapacidade de o Estado-Membro atuar, bem como, o risco de responsabilização do país perante órgãos internacionais. - Tudo isso emoldurado pela proporcionalidade, sob pena de se banalizar a medida constitucional e de se incorrer em risco de violar o princípio do juiz e do promotor natural, criando-se verdadeiros tribunais de exceção. Além de ferir o art. 34 da Constituição Federal, por se proceder à intervenção da União nos Estados Membros fora das situações previstas no mencionado dispositivo constitucional.

• Estão preenchidos todos os requisitos de ordem constitucional, legal e aqueles irradiados da jurisprudência deste STJ, que autorizam o deslocamento de competência da esfera estadual para a federal (relativamente a seis inquéritos não solucionados), eis que evidenciada a grave violação de direitos humanos, a possibilidade de responsabilização do Brasil em razão de descumprimento a obrigações contraídas em tratados internacionais e a incapacidade de órgão locais darem respostas efetivas às demandas.

Note que aos municípios não foi dada a competência para legislar concorrente.

O município somente poderá, nos termos do art. 30, editar lei municipal que vise suplementar as leis federal e estadual. Ou seja, exerce a competência legislativa suplementar complementar, mas jamais a suplementar supletiva.

Very sad to know that struggle and the process Native Americans had to g o through in order to keep sacred items and important valuable such as the remains of their ancestors.

Una vez que seleccionas una categoría, no hay más indicaciones o acciones a seguir. ¿Nos ayudan a revisar si hizo falta alguna pieza del prototipo? Pareciera que hace falta algún botón que diga "solicitar" o "continuar"...

En página de "Mis solicitudes", cuando das click a "solicitud general" actualmente se despliega dos veces la frase "solicitud general", un "número" y atrás se alcanza a ver "SEAT IBIZA". Nos ayudan confirmando o corriendo ese elemento colapsable?

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This paper reports the fundamental discovery of adrenergic modulation of spontaneous firing through the inhibition of the Na+ leak channel NALCN in cartwheel cells in the dorsal cochlear nucleus. This study provides unequivocal evidence that the activation of alpha-2 adrenergic or GABA-B receptors inhibit NALCN currents to reduce neuronal excitability. The evidence supporting the conclusions is compelling, the electrophysiological data is high quality and the experimental design is rigorous.

Public Reviews:

Reviewer #1 (Public Review):

This study uses electrophysiological techniques in vitro to address the role of the Na+ leak channel NALCN in various physiological functions in cartwheel interneurons of the dorsal cochlear nucleus. Comparing wild type and glycinergic neuron-specific knockout mice for NALCN, the authors show that these channels 1) are required for spontaneous firing, 2) are modulated by noradrenaline (NA, via alpha2 receptors) and GABA (through GABAB receptors), 3) how the modulation by NA enhances IPSCs in these neurons.

This work builds on previous results from the Trussell's lab in terms of the physiology of cartwheel cells, and from other labs in terms of the role of NALCN channels, that have been characterized in more and more brain areas somewhat recently; for this reason, this study could be of interest for researchers that work in other preparations as well. The general conclusions are strongly supported by results that are clearly and elegantly presented.

I have a few comments that, in my opinion, might help clarify some aspects of the manuscript.

1. It is mentioned throughout the manuscript, including the abstract, that the results suggest a closed apposition of NALCN channels and alpha2 and GABAB receptors. From what I understand, this conclusion comes from the fact that GABAB receptors activate GIRK channels through a membrane-delimited mechanism. Is it possible that these receptors converge on other effectors, for example adenylate cyclase (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374141/).

We have now tested the role of adenylyl cyclase modulation in the control of NALCN, by saturating the cells with a cAMP analogue 8-Br-cAMP and found no effect on the NA response. These data are included in the paper. While further experiments are necessary, these results argue in favor of a direct gating by G-proteins.

1. In Figure 2G, the neurons from NALCN KO mice appear to reach a significantly higher frequency than those from WT (figure 2E, 110 vs. 70 spikes/s). Was this higher frequency a feature of all experiments? The results mention a rundown of peak firing rate due to whole-cell dialysis, but, from what I understand, the control conditions should be similar for all experiments.

The peak firing rates in control solutions for WT and KO CWC are not statistically different.

1. Also in Figure 2, the firing patterns for neurons from WT and NALCN KO mice appear to be quite different, with spikes appearing to be generated during the hyperpolarization of the bursts in the second half of the current step for WT neurons but always during the depolarization in KO neurons. Was this always the case? If so, could NALCN channels be involved in this type of firing? Along these lines, it would be interesting to show an example of a firing pattern of neurons from WT mice in the presence of NA, which inhibits NALCN channels.

The specific pattern of spikes in CWC is quite variable from trial-to-trial or cell-to-cell, as it is dependent on multiple CaV and calcium dependent K channels subtypes, and is not dependent on the genotypes used here. The primary effects observed in the KO are in background firing and sensitivity to NA, both reflected alterations in rheobase. The firing pattern example requested was shown in the raster plot of fig 2B2.

1. It might be interesting to discuss how the hyperpolarization induced by the activation of GIRK channels and inhibition of NALCN channels could have different consequences due to their opposite effect on the input resistance.

We considered this as a point of discussion, but decided that making sense of it would depend on assumptions about the location of the channels (dendritic vs somatic, distance to AIS) that we do not have data for. For example, a dendritic increase in resistance through NALCN block, leading to a hyperpolarization of the soma, might have actions similar to a somatic hyperpolarizing conductance increase by GIRK, as far as the voltage at the AIS is concerned.

Reviewer #2 (Public Review):

This is a very interesting paper with several important findings related to the working mechanism of the cartwheel cells (CWC) in the dorsal cochlear nucleus (DCN). These cells generate spontaneous firing that is inhibited by the activation of α2-adrenergic receptors, which also enhances the synaptic strength in the cells, but the mechanisms underlying the spontaneous firing and the dual regulation by α2-adrenergic receptor activation have remained elusive. By recording these cells with the NALCN sodium-leak channel conditionally knocked, the authors discovered that both the spontaneous firing and the regulation by noradrenaline (NA) require NALCN. Mechanistically, the authors found that activation of the adrenergic receptor or GABAB receptor inhibits NALCN. Interestingly, these receptor activations also suppress the low [Ca2+] "activation" of NALCN currents, suggesting crosstalk between the pathways. The finding of such dominant contribution of the NALCN conductance to the regulation of firing by NA is somewhat surprising considering that NA is known to regulate K+ conductances in many other neurons.

The studies reveal the molecular mechanisms underlying well known regulations of the neuronal processes in the auditory pathway. The results will be important to the understanding of auditory information processing in particular, and, more generally, to the understanding of the regulation of inhibitory neurons and ion channels. The results are convincing and are clearly presented.

Reviewer #3 (Public Review):

The study by Ngodup and colleagues describes the contribution of sodium leak NALCN conductance on the effects of noradrenaline on cartwheel interneurons of the DCN. The manuscript is very well-written and the experiments are well-controlled. The scope of the study is of high biological relevance and recapitulates a primary finding of the Khaliq lab (Philippart et al., eLife, 2018) in ventral midbrain dopamine neurons, that Gi/o-coupled receptors inhibit NALCN current to reduce neuronal excitability. Together these studies provide unequivocable evidence for NALCN as a downstream target of these receptors. There are no major concerns. I have only minor suggestions:

Minor

1. As introduced in the introduction, NALCN is inhibited by extracellular calcium which has led to some discourse of the relevance of NALCN when recorded in 0.1 mM calcium. A strength of this study is the effect of NA on NALCN is recorded in physiological levels of calcium (1.2 mM). I suggest including the concentration of extracellular calcium in the aCSF in the Results section instead of relying on the reader to look to the Methods.

Done.

1. It would be interesting to include the basal membrane properties of the KO compared to wildtype, including membrane resistance and resting membrane potential. From the example recording in Figure 2, one might think that the KOs have lower membrane resistance, so it is interesting that the 2 mV hyperpolarization produced similar effects on rheobase. In addition, from the example in Figure 2G, it appears that NA has an effect on firing frequency with large current injection in the KO. Is this true in grouped data and if so, is there any speculation into how this occurs?

We have included in the text a comparison of the input resistance in WT and KO. These were not different. This should not be too surprising given the wide range of values between animals, and the necessity to compare populations. Measurements of resting potential are complicated by the fact that CWC are normally spontaneously active. As was discussed in the text, peak firing frequency declined with time during recording in both control and KO, necessitating normalization as shown in Fig 2E-H.

1. Please expand on the rationale for why GABAB and alpha2 must be physically close to NALCN. To my knowledge, the mechanism by which these receptors inhibit NALCN is not known. Must it be membrane-delimited?

Given the known membrane delimited modulation of GIRK by GABAB, and that alpha2 and GABAB receptors appear to share the same population of NALCN channels, and that alpha2 receptors do not appear to target GIRK channels, we felt the simplest explanation would be coupling through G-proteins, with spatial segregation of different receptor/channel pools providing the means for separating GIRK and NALCN effects. Given that the alpha2 receptor is a Gi/o GPCR, we have now included in the revision new experiments using 8-Br-cAMP, as discussed above. These showed no effect on the NA response, consistent with a direct effect membrane delimited of G-proteins. We acknowledge however that further experiments are warranted.

Reviewer #1 (Recommendations For The Authors):

1. I suggest labeling the voltage traces in Figure 2 with WT and KO for easier comprehension; in addition, I suggest adding the average data to the plots in Figure 2, as in Figure 2-supplementary Figure 1 panel F.

We have added the figure labels as requested. We chose not to add the average data as we noticed that averaging the full FI plots led to a smearing of the curves and a distortion in the apparent rheobase. Thus, we instead measured the rheobase for individual cells and report their average.

1. For readers that are not familiar with the field, more details should be given about the electrical stimulation to evoke IPSCs in cartwheel cells, and what they represent.

Done.

1. The methods should mention if and how the concentrations of divalents were adjusted in the experiments with 0.1 extracellular Ca2+

Done.

Reviewer #2 (Recommendations For The Authors):

I only have several minor comments.

1. The total lack of spontaneous firing in CWCs in the NALCN KO (Fig. 1) is interesting and provides an opportunity to probe the in vivo function of such spontaneous firing. Besides being a little smaller, do the mutant mice have any sign of abnormality in sound signal processing?

Figure 1 – Figure supplement 1 showed that there are no effects on auditory brainstem responses in the KO.

1. Figs. 3&4 (and several other figures with voltage-clamp recordings), a line indicating zero current level would be useful.

Done

1. page 7, "Outward current generated by suppression of NALCN": it might be better to state as "Outward response generated by suppression of NALCN", as the authors correctly pointed out that the NA-induced apparently outward current response is largely a result of an inhibition of NALCN-mediated inward Na+ current. One way to clarify this might be to record at the Nernst potential of K+ to isolate the contribution of Na+ currents (unclear if K+- or Cs+-based pipette was used in the experiment in Fig 3).

Text has been modified.

1. Figs. 5,6&7: do the dashed lines indicate initial current level or zero current level?

Initial current. See legends.

1. The labeling of some of the bar graphs can be made more clear. For example, in Fig. 2K, the right two columns should be labeled as WT as well. Fig. 3C & Fig. 4C, the left two columns should be labeled as WT and the right two as KO.

Added labels to Fig 2 as requested.

1. Figs. 5-7: The suppression of low extracellular [Ca2+]-induced NALCN-dependent current by NA and baclofen is very interesting. As the tonic inhibition of NALCN by extracellular Ca2+ is likely through a Ca2+-sensing GPCR (CaSR) and G-proteins (lowering [Ca2+] releases the inhibition and generates inward current) (Lu et al. 2010), the action of NA and baclofen may all converge onto the same G-protein dependent pathway of the Ca2+-sensing receptor. I'd include this in the discussion to provide a potential mechanistic explanation of the interesting observation.

This is indeed an interesting idea. We prefer not to discuss here, as 1) the source of Ca2+ sensitivity of the channel seems to be controversial (Chua et al 2020), and 2) the effect of Ca2+ reduction is enormously slower than the effect of the modulators (Fig 5-7), implying distinct mechanisms.

Reviewer #3 (Recommendations For The Authors):

Typos/general comments

1. Figure 2 would be easier to comprehend with WT and KO labels as in the other figures. Done

2. Page 11, size of the IPSCs in NA is missing the minus sign.

Corrected.

1. Is the y-axis correct on Figure 8B? This looks like it is doubling the size of the IPSC.

Thank you for catching this mistake. The formula used to calculate % change was in error. We have corrected all the data analysis in the figure, which fortunately did not change the conclusion. Regarding the axis, note that the measurement was % change, not ratio of drug vs control.

Reviewer #3 (Public Review):

The study by Ngodup and colleagues describes the contribution of sodium leak NALCN conductance on the effects of noradrenaline on cartwheel interneurons of the DCN. The manuscript is very well-written and the experiments are well-controlled. The scope of the study is of high biological relevance and recapitulates a primary finding of the Khaliq lab (Philippart et al., eLife, 2018) in ventral midbrain dopamine neurons, that Gi/o-coupled receptors inhibit NALCN current to reduce neuronal excitability. Together these studies provide unequivocable evidence for NALCN as a downstream target of these receptors.

In re-review of this study, the authors have addressed the concerns sufficiently. This is a very nice study.

Author Response

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

In this very strong and interesting paper the authors present a convincing series of experiments that reveal molecular mechanism of neuronal cell type diversification in the nervous system of Drosophila. The authors show that a homeodomain transcription factor, Bsh, fulfills several critical functions - repressing an alternative fate and inducing downstream homeodomain transcription factors with whom Bsh may collaborate to induce L4 and L5 fates (the author's accompanying paper reveals how Bsh can induce two distinct fates). The authors make elegant use of powerful genetic tools and an arsenal of satisfying cell identity markers.

Thanks!

I believe that this is an important study because it provides some fundamental insights into the conservation of neuronal diversification programs. It is very satisfying to see that similar organizational principles apply in different organisms to generate cell type diversity. The authors should also be commended for contextualizing their work very well, giving a broad, scholarly background to the problem of neuronal cell type diversification.

Thanks!

My one suggestion for the authors is to perhaps address in the Discussion (or experimentally address if they wish) how they reconcile that Bsh is on the one hand: (a) continuously expressed in L4/L4, (b) binding directly to a cohort of terminal effectors that are also continuously expressed but then, on the other hand, is not required for their maintaining L4 fate? A few questions: Is Bsh only NOT required for maintaining Ap expression or is it also NOT required for maintaining other terminal markers of L4? The former could be easily explained - Bsh simply kicks of Ap, Ap then autoregulates, but Bsh and Ap then continuously activate terminal effector genes. The second scenario would require a little more complex mechanism: Bsh binding of targets (with Notch) may open chromatin, but then once that's done, Bsh is no longer needed and Ap alone can continue to express genes. I feel that the authors should be at least discussing this. The postmitotic Bsh removal experiment in which they only checked Ap and depression of other markers is a little unsatisfying without further discussion (or experiments, such as testing terminal L4 markers). I hasten to add that this comment does not take away from my overall appreciation for the depth and quality of the data and the importance of their conclusions.

Great suggestions, we will discuss these two hypotheses as requested.

Bsh initiates Ap expression in L4 neurons which then maintain Ap expression independently of Bsh expression, likely through Ap autoregulation. During the synaptogenesis window, Ap expression becomes independent from Bsh expression, but Bsh and Ap are both still required to activate the synapse recognition molecule DIP-beta. Additionally, Bsh also shows putative binding to other L4 identity genes, e.g., those required for neurotransmitter choice, and electrophysiological properties, suggesting Bsh may initiate L4 identity genes as a suite of genes. The mechanism of maintaining identity features (e.g., morphology, synaptic connectivity, and functional properties) in the adult remains poorly understood. It is a great question whether primary HDTF Bsh maintains the expression of L4 identity genes in the adult. To test this, in our next project, we will specifically knock out Bsh in L4 neurons of the adult fly and examine the effect on L4 morphology, connectivity, and function properties.

Reviewer #2 (Public Review):

Summary:

In this paper, the authors explore the role of the Homeodomain Transcription Factor Bsh in the specification of Lamina neuronal types in the optic lobe of Drosophila. Using the framework of terminal selector genes and compelling data, they investigate whether the same factor that establishes early cell identity is responsible for the acquisition of terminal features of the neuron (i.e., cell connectivity and synaptogenesis).

Thanks for the positive words!

The authors convincingly describe the sequential expression and activity of Bsh, termed here as 'primary HDTF', and of Ap in L4 or Pdm3 in L5 as 'secondary HDTFs' during the specification of these two neurons. The study demonstrates the requirement of Bsh to activate either Ap and Pdm3, and therefore to generate the L4 and L5 fates. Moreover, the authors show that in the absence of Bsh, L4 and L5 fates are transformed into a L1 or L3-like fates.

Thanks!

Finally, the authors used DamID and Bsh:DamID to profile the open chromatin signature and the Bsh binding sites in L4 neurons at the synaptogenesis stage. This allows the identification of putative Bsh target genes in L4, many of which were also found to be upregulated in L4 in a previous single-cell transcriptomic analysis. Among these genes, the paper focuses on Dip-β, a known regulator of L4 connectivity. They demonstrate that both Bsh and Ap are required for Dip-β, forming a feed-forward loop. Indeed, the loss of Bsh causes abnormal L4 synaptogenesis and therefore defects in several visual behaviors. The authors also propose the intriguing hypothesis that the expression of Bsh expanded the diversity of Lamina neurons from a 3 cell-type state to the current 5 cell-type state in the optic lobe.

Thanks for the excellent summary of our findings!

Strengths:

Overall, this work presents a beautiful practical example of the framework of terminal selectors: Bsh acts hierarchically with Ap or Pdm3 to establish the L4 or L5 cell fates and, at least in L4, participates in the expression of terminal features of the neuron (i.e., synaptogenesis through Dip-β regulation).

Thanks!

The hierarchical interactions among Bsh and the activation of Ap and Pdm3 expression in L4 and L5, respectively, are well established experimentally. Using different genetic drivers, the authors show a window of competence during L4 neuron specification during which Bsh activates Ap expression. Later, as the neuron matures, Ap becomes independent of Bsh. This allows the authors to propose a coherent and well-supported model in which Bsh acts as a 'primary' selector that activates the expression of L4specific (Ap) and L5-specific (Pdm3) 'secondary' selector genes, that together establish neuronal fate.

Thanks again!

Importantly, the authors describe a striking cell fate change when Bsh is knocked down from L4/L5 progenitor cells. In such cases, L1 and L3 neurons are generated at the expense of L4 and L5. The paper demonstrates that Bsh in L4/L5 represses Zfh1, which in turn acts as the primary selector for L1/L3 fates. These results point to a model where the acquisition of Bsh during evolution might have provided the grounds for the generation of new cell types, L4 and L5, expanding lamina neuronal diversity for a more refined visual behaviors in flies. This is an intriguing and novel hypothesis that should be tested from an evo-devo standpoint, for instance by identifying a species when L4 and L5 do not exist and/or Bsh is not expressed in L neurons.

Thanks for the appreciation of our findings!

To gain insight into how Bsh regulates neuronal fate and terminal features, the authors have profiled the open chromatin landscape and Bsh binding sites in L4 neurons at mid-pupation using the DamID technique. The paper describes a number of genes that have Bsh binding peaks in their regulatory regions and that are differentially expressed in L4 neurons, based on available scRNAseq data. Although the manuscript does not explore this candidate list in depth, many of these genes belong to classes that might explain terminal features of L4 neurons, such as neurotransmitter identity, neuropeptides or cytoskeletal regulators. Interestingly, one of these upregulated genes with a Bsh peak is Dip-β, an immunoglobulin superfamily protein that has been described by previous work from the author's lab to be relevant to establish L4 proper connectivity. This work proves that Bsh and Ap work in a feed-forward loop to regulate Dip-β expression, and therefore to establish normal L4 synapses. Furthermore, Bsh loss of function in L4 causes impairs visual behaviors.<br /> Thanks for the excellent summary of our findings.

Weaknesses:

● The last paragraph of the introduction is written using rhetorical questions and does not read well. I suggest rewriting it in a more conventional direct style to improve readability.

We agree and have updated the text as suggested.

● A significant concern is the way in which information is conveyed in the Figures. Throughout the paper, understanding of the experimental results is hindered by the lack of information in the Figure headers. Specifically, the genetic driver used for each panel should be adequately noted, together with the age of the brain and the experimental condition. For example, R27G05-Gal4 drives early expression in LPCs and L4/L5, while the 31C06-AD, 34G07-DBD Split-Gal4 combination drives expression in older L4 neurons, and the use of one or the other to drive Bsh-KD has dramatic differences in Ap expression. The indication of the driver used in each panel will facilitate the reader's grasp of the experimental results.

We agree and have updated the figure annotation.

● Bsh role in L4/L5 cell fate: o It is not clear whether Tll+/Bsh+ LPCs are the precursors of L4/L5. Morphologically, these cells sit very close to L5, but are much more distant from L4.

Our current data show L4 and L5 neurons are generated by different LPCs. However, currently, we don’t have tools to demonstrate which subset of LPCs generate which lamina neuron type. We are currently working on a follow-up manuscript on LPC heterogeneity, but those experiments have just barely been started.

● Somatic CRISPR knockout of Bsh seems to have a weaker phenotype than the knockdown using RNAi. However, in several experiments down the line, the authors use CRISPR-KO rather than RNAi to knock down Bsh activity: it should be explained why the authors made this decision. Alternatively, a null mutant could be used to consolidate the loss of function phenotype, although this is not strictly necessary given that the RNAi is highly efficient and almost completely abolishes Bsh protein.

The reason we chose CRISPR-KO (L4-specific Gal4, uas-Cas9, and uas-Bsh-sgRNAs) is that it effectively removed Bsh expression from the majority of L4 neurons. However, it failed to knock down Bsh in L4 neurons using L4-split Gal4 and Bsh-RNAi because L4-split Gal4 expression depends on Bsh. We have updated this explanation in the text.

● Line 102: Rephrase "R27G05-Gal4 is expressed in all LPCs and turned off in lamina neurons" to "is turned off as lamina neurons mature", as it is kept on for a significant amount of time after the neurons have already been specified.

Thanks; we have made that change.

● Line 121: "(a) that all known lamina neuron markers become independent of Bsh regulation in neurons" is not an accurate statement, as the markers tested were not shown to be dependent on Bsh in the first place.

Good point. We have rephrased it as “that all known lamina neuron markers are independent of Bsh regulation in neurons”.

● Lines 129-134: Make explicit that the LPC-Gal4 was used in this experiment. This is especially important here, as these results are opposite to the Bsh Loss of Function in L4 neurons described in the previous section. This will help clarify the window of competence in which Bsh establishes L4/L5 neuronal identities through ap/pdm3 expression.

Thanks! We have updated Gal4 information in the text for every manipulation.

● DamID and Bsh binding profile:

● Figure 5 - figure supplement 1C-E: The genotype of the Control in (C) has to be described within the panel. As it is, it can be confused with a wild type brain, when it is in fact a Bsh-KO mutant.

Great point! Thank you for catching this and we have updated it.

● It Is not clear how L4-specific Differentially Expressed Genes were found. Are these genes DEG between Lamina neurons types, or are they upregulated genes with respect to all neuronal clusters? If the latter is the case, it could explain the discrepancy between scRNAseq DEGs and Bsh peaks in L4 neurons.

We did not use “L4-specific Differentially Expressed Genes”. Instead, we used all genes that are significantly transcribed in L4 neurons (line 209-213).

● Dip-β regulation:

● Line 234: It is not clear why CRISPR KO is used in this case, when Bsh-RNAi presents a stronger phenotype.

As we explained above, the reason we chose CRISPR-KO (L4-specific Gal4, uas-Cas9, and uas-BshsgRNAs) is that it effectively removed Bsh expression from the majority of L4 neurons. However, it failed to knock down Bsh in L4 neurons using L4-split Gal4 and Bsh-RNAi because L4-split Gal4 expression depends on Bsh. We have updated this explanation in the text.

● Figure 6N-R shows results using LPC-Gal4. It is not clear why this driver was used, as it makes a less accurate comparison with the other panels in the figure, which use L4-Split-Gal4. This discrepancy should be acknowledged and explained, or the experiment repeated with L4-Split-Gal4>Ap-RNAi.

I think you mean 6J-M shows results using LPC-Gal4. We first tried L4-Split-Gal4>Ap-RNAi but it failed to knock down Ap because L4-Split-Gal4 expression depends on Ap. We have added this to the text.

● Line 271: It is also possible that L4 activity is dispensable for motion detection and only L5 is required.

Thanks! Work from Tuthill et al, 2013 showed that L5 is not required for any motion detection. We have included this citation in the text.

● Discussion: It is necessary to de-emphasize the relevance of HDTFs, or at least acknowledge that other, non-homeodomain TFs, can act as selector genes to determine neuronal identity. By restricting the discussion to HDTFs, it is not mentioned that other classes of TFs could follow the same PrimarySecondary selector activation logic.

That is a great point, thank you! We have included this in the discussion.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, the authors explore the role of the Homeodomain Transcription Factor Bsh in the specification of Lamina neuronal types in the optic lobe of Drosophila. Using the framework of terminal selector genes and compelling data, they investigate whether the same factor that establishes early cell identity is responsible for the acquisition of terminal features of the neuron (i.e., cell connectivity and synaptogenesis).

The authors convincingly describe the sequential expression and activity of Bsh, termed here as 'primary HDTF', and of Ap in L4 or Pdm3 in L5 as 'secondary HDTFs' during the specification of these two neurons. The study demonstrates the requirement of Bsh to activate either Ap and Pdm3, and therefore to generate the L4 and L5 fates. Moreover, the authors show that in the absence of Bsh, L4 and L5 fates are transformed into a L1 or L3-like fates.

Finally, the authors used DamID and Bsh:DamID to profile the open chromatin signature and the Bsh binding sites in L4 neurons at the synaptogenesis stage. This allows the identification of putative Bsh target genes in L4, many of which were also found to be upregulated in L4 in a previous single-cell transcriptomic analysis. Among these genes, the paper focuses on Dip-β, a known regulator of L4 connectivity. They demonstrate that both Bsh and Ap are required for Dip-β, forming a feed-forward loop. Indeed, the loss of Bsh causes abnormal L4 synaptogenesis and therefore defects in several visual behaviors.

The authors also propose the intriguing hypothesis that the expression of Bsh expanded the diversity of Lamina neurons from a 3 cell-type state to the current 5 cell-type state in the optic lobe.

Strengths:<br /> Overall, this work presents a beautiful practical example of the framework of terminal selectors: Bsh acts hierarchically with Ap or Pdm3 to establish the L4 or L5 cell fates and, at least in L4, participates in the expression of terminal features of the neuron (i.e., synaptogenesis through Dip-β regulation).

The hierarchical interactions among Bsh and the activation of Ap and Pdm3 expression in L4 and L5, respectively, are well established experimentally. Using different genetic drivers, the authors show a window of competence during L4 neuron specification during which Bsh activates Ap expression. Later, as the neuron matures, Ap becomes independent of Bsh. This allows the authors to propose a coherent and well-supported model in which Bsh acts as a 'primary' selector that activates the expression of L4-specific (Ap) and L5-specific (Pdm3) 'secondary' selector genes, that together establish neuronal fate.

Importantly, the authors describe a striking cell fate change when Bsh is knocked down from L4/L5 progenitor cells. In such case, L1 and L3 neurons are generated at the expense of L4 and L5. The paper demonstrates that Bsh in L4/L5 represses Zfh1, which in turn acts as the primary selector for L1/L3 fates. These results point to a model where the acquisition of Bsh during evolution might have provided the grounds for the generation of new cell types, L4 and L5, expanding lamina neuronal diversity for a more refined visual behaviors in flies. This is an intriguing and novel hypothesis that should be tested from an evo-devo standpoint, for instance by identifying a species when L4 and L5 do not exist and/or Bsh is not expressed in L neurons.

To gain insight into how Bsh regulates neuronal fate and terminal features, the authors have profiled the open chromatin landscape and Bsh binding sites in L4 neurons at mid-pupation using the DamID technique. The paper describes a number of genes that have Bsh binding peaks in their regulatory regions and that are differentially expressed in L4 neurons, based on available scRNAseq data. Although the manuscript does not explore this candidate list in depth, many of these genes belong to classes that might explain terminal features of L4 neurons, such as neurotransmitter identity, neuropeptides or cytoskeletal regulators. Interestingly, one of these upregulated genes with a Bsh peak is Dip-β, an immunoglobulin superfamily protein that has been described by previous work from the author's lab to be relevant to establish L4 proper connectivity. This work proves that Bsh and Ap work in a feed-forward loop to regulate Dip-β expression, and therefore to establish normal L4 synapses. Furthermore, Bsh loss of function in L4 causes impairs visual behaviors.

Weaknesses:<br /> ● The last paragraph of the introduction is written using rhetorical questions and does not read well. I suggest rewriting it in a more conventional direct style to improve readability.

● A significant concern is the way in which information is conveyed in the Figures. Throughout the paper, understanding of the experimental results is hindered by the lack of information in the Figure headers. Specifically, the genetic driver used for each panel should be adequately noted, together with the age of the brain and the experimental condition. For example, R27G05-Gal4 drives early expression in LPCs and L4/L5, while the 31C06-AD, 34G07-DBD Split-Gal4 combination drives expression in older L4 neurons, and the use of one or the other to drive Bsh-KD has dramatic differences in Ap expression. The indication of the driver used in each panel will facilitate the reader's grasp of the experimental results.

● Bsh role in L4/L5 cell fate:<br /> o It is not clear whether Tll+/Bsh+ LPCs are the precursors of L4/L5. Morphologically, these cells sit very close to L5, but are much more distant from L4.<br /> o Somatic CRISPR knockout of Bsh seems to have a weaker phenotype than the knockdown using RNAi. However, in several experiments down the line, the authors use CRISPR-KO rather than RNAi to knock down Bsh activity: it should be explained why the authors made this decision. Alternatively, a null mutant could be used to consolidate the loss of function phenotype, although this is not strictly necessary given that the RNAi is highly efficient and almost completely abolishes Bsh protein.<br /> o Line 102: Rephrase "R27G05-Gal4 is expressed in all LPCs and turned off in lamina neurons" to "is turned off as lamina neurons mature", as it is kept on for a significant amount of time after the neurons have already been specified.<br /> o Line 121: "(a) that all known lamina neuron markers become independent of Bsh regulation in neurons" is not an accurate statement, as the markers tested were not shown to be dependent on Bsh in the first place.<br /> o Lines 129-134: Make explicit that the LPC-Gal4 was used in this experiment. This is especially important here, as these results are opposite to the Bsh Loss of Function in L4 neurons described in the previous section. This will help clarify the window of competence in which Bsh establishes L4/L5 neuronal identities through ap/pdm3 expression.

● DamID and Bsh binding profile:<br /> ○ Figure 5 - figure supplement 1C-E: The genotype of the Control in (C) has to be described within the panel. As it is, it can be confused with a wild type brain, when it is in fact a Bsh-KO mutant.<br /> ○ It Is not clear how L4-specific Differentially Expressed Genes were found. Are these genes DEG between Lamina neurons types, or are they upregulated genes with respect to all neuronal clusters? If the latter is the case, it could explain the discrepancy between scRNAseq DEGs and Bsh peaks in L4 neurons.

● Dip-β regulation:<br /> ○ Line 234: It is not clear why CRISPR KO is used in this case, when Bsh-RNAi presents a stronger phenotype.<br /> ○ Figure 6N-R shows results using LPC-Gal4. It is not clear why this driver was used, as it makes a less accurate comparison with the other panels in the figure, which use L4-Split-Gal4. This discrepancy should be acknowledged and explained, or the experiment repeated with L4-Split-Gal4>Ap-RNAi.<br /> ○ Line 271: It is also possible that L4 activity is dispensable for motion detection and only L5 is required.

● Discussion: It is necessary to de-emphasize the relevance of HDTFs, or at least acknowledge that other, non-homeodomain TFs, can act as selector genes to determine neuronal identity. By restricting the discussion to HDTFs, it is not mentioned that other classes of TFs could follow the same Primary-Secondary selector activation logic.

Reviewer #1 (Public Review):

Summary:

This study aims to provide imaging methods for users of the field of human layer-fMRI. This is an emerging field with 240 papers published so far. Different than implied in the manuscript, 3T is well represented among those papers. E.g. see the papers below that are not cited in the manuscript. Thus, the claim on the impact of developing 3T methodology for wider dissemination is not justified. Specifically, because some of the previous papers perform whole brain layer-fMRI (also at 3T) in more efficient, and more established procedures.

The authors implemented a sequence with lots of nice features. Including their own SMS EPI, diffusion bipolar pulses, eye-saturation bands, and they built their own reconstruction around it. This is not trivial. Only a few labs around the world have this level of engineering expertise. I applaud this technical achievement. However, I doubt that any of this is the right tool for layer-fMRI, nor does it represent an advancement for the field. In the thermal noise dominated regime of sub-millimeter fMRI (especially at 3T), it is established to use 3D readouts over 2D (SMS) readouts. While it is not trivial to implement SMS, the vendor implementations (as well as the CMRR and MGH implementations) are most widely applied across the majority of current fMRI studies already. The author's work on this does not serve any previous shortcomings in the field.

The mechanism to use bi-polar gradients to increase the localization specificity is doubtful to me. In my understanding, killing the intra-vascular BOLD should make it less specific. Also, the empirical data do not suggest a higher localization specificity to me.

Embedding this work in the literature of previous methods is incomplete. Recent trends of vessel signal manipulation with ABC or VAPER are not mentioned. Comparisons with VASO are outdated and incorrect.

The reproducibility of the methods and the result is doubtful (see below).

I don't think that this manuscript is in the top 50% of the 240 layer-fmri papers out there.

3T layer-fMRI papers that are not cited:<br /> Taso, M., Munsch, F., Zhao, L., Alsop, D.C., 2021. Regional and depth-dependence of cortical blood-flow assessed with high-resolution Arterial Spin Labeling (ASL). Journal of Cerebral Blood Flow and Metabolism. https://doi.org/10.1177/0271678X20982382

Wu, P.Y., Chu, Y.H., Lin, J.F.L., Kuo, W.J., Lin, F.H., 2018. Feature-dependent intrinsic functional connectivity across cortical depths in the human auditory cortex. Scientific Reports 8, 1-14. https://doi.org/10.1038/s41598-018-31292-x

Lifshits, S., Tomer, O., Shamir, I., Barazany, D., Tsarfaty, G., Rosset, S., Assaf, Y., 2018. Resolution considerations in imaging of the cortical layers. NeuroImage 164, 112-120. https://doi.org/10.1016/j.neuroimage.2017.02.086

Puckett, A.M., Aquino, K.M., Robinson, P.A., Breakspear, M., Schira, M.M., 2016. The spatiotemporal hemodynamic response function for depth-dependent functional imaging of human cortex. NeuroImage 139, 240-248. https://doi.org/10.1016/j.neuroimage.2016.06.019

Olman, C.A., Inati, S., Heeger, D.J., 2007. The effect of large veins on spatial localization with GE BOLD at 3 T: Displacement, not blurring. NeuroImage 34, 1126-1135. https://doi.org/10.1016/j.neuroimage.2006.08.045

Ress, D., Glover, G.H., Liu, J., Wandell, B., 2007. Laminar profiles of functional activity in the human brain. NeuroImage 34, 74-84. https://doi.org/10.1016/j.neuroimage.2006.08.020

Huber, L., Kronbichler, L., Stirnberg, R., Ehses, P., Stocker, T., Fernández-Cabello, S., Poser, B.A., Kronbichler, M., 2023. Evaluating the capabilities and challenges of layer-fMRI VASO at 3T. Aperture Neuro 3. https://doi.org/10.52294/001c.85117

Scheeringa, R., Bonnefond, M., van Mourik, T., Jensen, O., Norris, D.G., Koopmans, P.J., 2022. Relating neural oscillations to laminar fMRI connectivity in visual cortex. Cerebral Cortex. https://doi.org/10.1093/cercor/bhac154

Strengths:

See above. The authors developed their own SMS sequence with many features. This is important to the field. And does not leave sequence development work to view isolated monopoly labs. This work democratises SMS.<br /> The questions addressed here are of high relevance to the field: getting tools with good sensitivity, user-friendly applicability, and locally specific brain activity mapping is an important topic in the field of layer-fMRI.

Weaknesses:

1. I feel the authors need to justify why flow-crushing helps localization specificity. There is an entire family of recent papers that aim to achieve higher localization specificity by doing the exact opposite. Namely, MT or ABC fRMRI aims to increase the localization specificity by highlighting the intravascular BOLD by means of suppressing non-flowing tissue. To name a few:

Priovoulos, N., de Oliveira, I.A.F., Poser, B.A., Norris, D.G., van der Zwaag, W., 2023. Combining arterial blood contrast with BOLD increases fMRI intracortical contrast. Human Brain Mapping hbm.26227. https://doi.org/10.1002/hbm.26227.

Pfaffenrot, V., Koopmans, P.J., 2022. Magnetization Transfer weighted laminar fMRI with multi-echo FLASH. NeuroImage 119725. https://doi.org/10.1016/j.neuroimage.2022.119725

Schulz, J., Fazal, Z., Metere, R., Marques, J.P., Norris, D.G., 2020. Arterial blood contrast ( ABC ) enabled by magnetization transfer ( MT ): a novel MRI technique for enhancing the measurement of brain activation changes. bioRxiv. https://doi.org/10.1101/2020.05.20.106666

Based on this literature, it seems that the proposed method will make the vein problem worse, not better. The authors could make it clearer how they reason that making GE-BOLD signals more extra-vascular weighted should help to reduce large vein effects.

The empirical evidence for the claim that flow crushing helps with the localization specificity should be made clearer. The response magnitude with and without flow crushing looks pretty much identical to me (see Fig, 6d).<br /> It's unclear to me what to look for in Fig. 5. I cannot discern any layer patterns in these maps. It's too noisy. The two maps of TE=43ms look like identical copies from each other. Maybe an editorial error?

The authors discuss bipolar crushing with respect to SE-BOLD where it has been previously applied. For SE-BOLD at UHF, a substantial portion of the vein signal comes from the intravascular compartment. So I agree that for SE-BOLD, it makes sense to crush the intravascular signal. For GE-BOLD however, this reasoning does not hold. For GE-BOLD (even at 3T), most of the vein signal comes from extravascular dephasing around large unspecific veins, and the bipolar crushing is not expected to help with this.

2. The bipolar crushing is limited to one single direction of flow. This introduces a lot of artificial variance across the cortical folding pattern. This is not mentioned in the manuscript. There is an entire family of papers that perform layer-fmri with black-blood imaging that solves this with a 3D contrast preparation (VAPER) that is applied across a longer time period, thus killing the blood signal while it flows across all directions of the vascular tree. Here, the signal cruising is happening with a 2D readout as a "snap-shot" crushing. This does not allow the blood to flow in multiple directions.<br /> VAPER also accounts for BOLD contaminations of larger draining veins by means of a tag-control sampling. The proposed approach here does not account for this contamination.

Chai, Y., Li, L., Huber, L., Poser, B.A., Bandettini, P.A., 2020. Integrated VASO and perfusion contrast: A new tool for laminar functional MRI. NeuroImage 207, 116358. https://doi.org/10.1016/j.neuroimage.2019.116358

Chai, Y., Liu, T.T., Marrett, S., Li, L., Khojandi, A., Handwerker, D.A., Alink, A., Muckli, L., Bandettini, P.A., 2021. Topographical and laminar distribution of audiovisual processing within human planum temporale. Progress in Neurobiology 102121. https://doi.org/10.1016/j.pneurobio.2021.102121

If I would recommend anyone to perform layer-fMRI with blood crushing, it seems that VAPER is the superior approach. The authors could make it clearer why users might want to use the unidirectional crushing instead.

3. The comparison with VASO is misleading.<br /> The authors claim that previous VASO approaches were limited by TRs of 8.2s. The authors might be advised to check the latest literature of the last years.<br /> Koiso et al. performed whole brain layer-fMRI VASO at 0.8mm at 3.9 seconds (with reliable activation), 2.7 seconds (with unconvincing activation pattern, though), and 2.3 (without activation).<br /> Also, whole brain layer-fMRI BOLD at 0.5mm and 0.7mm has been previously performed by the Juelich group at TRs of 3.5s (their TR definition is 'fishy' though).

Koiso, K., Müller, A.K., Akamatsu, K., Dresbach, S., Gulban, O.F., Goebel, R., Miyawaki, Y., Poser, B.A., Huber, L., 2023. Acquisition and processing methods of whole-brain layer-fMRI VASO and BOLD: The Kenshu dataset. Aperture Neuro 34. https://doi.org/10.1101/2022.08.19.504502

Yun, S.D., Pais‐Roldán, P., Palomero‐Gallagher, N., Shah, N.J., 2022. Mapping of whole‐cerebrum resting‐state networks using ultra‐high resolution acquisition protocols. Human Brain Mapping. https://doi.org/10.1002/hbm.25855

Pais-Roldan, P., Yun, S.D., Palomero-Gallagher, N., Shah, N.J., 2023. Cortical depth-dependent human fMRI of resting-state networks using EPIK. Front. Neurosci. 17, 1151544. https://doi.org/10.3389/fnins.2023.1151544

The authors are correct that VASO is not advised as a turn-key method for lower brain areas, incl. Hippocampus and subcortex. However, the authors use this word of caution that is intended for inexperienced "users" as a statement that this cannot be performed. This statement is taken out of context. This statement is not from the academic literature. It's advice for the 40+ user base that wants to perform layer-fMRI as a plug-and-play routine tool in neuroscience usage. In fact, sub-millimeter VASO is routinely being performed by MRI-physicists across all brain areas (including deep brain structures, hippocampus etc). E.g. see Koiso et al. and an overview lecture from a layer-fMRI workshop that I had recently attended: https://youtu.be/kzh-nWXd54s?si=hoIJjLLIxFUJ4g20&t=2401

Thus, the authors could embed this phrasing into the context of their own method that they are proposing in the manuscript. E.g. the authors could state whether they think that their sequence has the potential to be disseminated across sites, considering that it requires slow offline reconstruction in Matlab?<br /> Do the authors think that the results shown in Fig. 6c are suggesting turn-key acquisition of a routine mapping tool? In my humble opinion, it looks like random noise, with most of the activation outside the ROI (in white matter).

4. The repeatability of the results is questionable.<br /> The authors perform experiments about the robustness of the method (line 620). The corresponding results are not suggesting any robustness to me. In fact, the layer profiles in Fig. 4c vs. Fig 4d are completely opposite. The location of peaks turns into locations of dips and vice versa.<br /> The methods are not described in enough detail to reproduce these results.<br /> The authors mention that their image reconstruction is done "using in-house MATLAB code" (line 634). They do not post a link to github, nor do they say if they share this code.

It is not trivial to get good phase data for fMRI. The authors do not mention how they perform the respective coil-combination.<br /> No data are shared for reproduction of the analysis.

5. The application of NODRIC is not validated.<br /> Previous applications of NORDIC at 3T layer-fMRI have resulted in mixed success. When not adjusted for the right SNR regime it can result in artifactual reductions of beta scores, depending on the SNR across layers. The authors could validate their application of NORDIC and confirm that the average layer-profiles are unaffected by the application of NORDIC. Also, the NORDIC version should be explicitly mentioned in the manuscript.

Akbari, A., Gati, J.S., Zeman, P., Liem, B., Menon, R.S., 2023. Layer Dependence of Monocular and Binocular Responses in Human Ocular Dominance Columns at 7T using VASO and BOLD (preprint). Neuroscience. https://doi.org/10.1101/2023.04.06.535924

Knudsen, L., Guo, F., Huang, J., Blicher, J.U., Lund, T.E., Zhou, Y., Zhang, P., Yang, Y., 2023. The laminar pattern of proprioceptive activation in human primary motor cortex. bioRxiv. https://doi.org/10.1101/2023.10.29.564658

DOI: 10.7554/eLife.87098

Resource: (Proteintech Cat# 18843-1-AP, RRID:AB_10603469)

Curator: @scibot

SciCrunch record: RRID:AB_10603469

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B L A C K SWAN

T h e I m p a c t o f t h e H I G H L Y I M P R O B A B L E Nassim Nicholas Taleb

from - https://hyp.is/Vb9oCJqaEe6nE6eI09UnJQ/archive.org/details/10.1.1.695.4305

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