Soluble P‐tau217 reflects amyloid and tau pathology and mediates the association of amyloid with tau

Abstract Alzheimer’s disease is characterized by β‐amyloid plaques and tau tangles. Plasma levels of phospho‐tau217 (P‐tau217) accurately differentiate Alzheimer’s disease dementia from other dementias, but it is unclear to what degree this reflects β‐amyloid plaque accumulation, tau tangle accumulation, or both. In a cohort with post‐mortem neuropathological data (N = 88), both plaque and tangle density contributed independently to higher P‐tau217, but P‐tau217 was not elevated in patients with non‐Alzheimer’s disease tauopathies (N = 9). Several findings were replicated in a cohort with PET imaging (“BioFINDER‐2”, N = 426), where β‐amyloid and tau PET were independently associated with P‐tau217. P‐tau217 concentrations correlated with β‐amyloid PET (but not tau PET) in early disease stages and with both β‐amyloid and (more strongly) tau PET in late disease stages. Finally, P‐tau217 mediated the association between β‐amyloid and tau in both cohorts, especially for tau outside of the medial temporal lobe. These findings support the hypothesis that plasma P‐tau217 concentration is increased by both β‐amyloid plaques and tau tangles and is congruent with the hypothesis that P‐tau is involved in β‐amyloid‐dependent formation of neocortical tau tangles.

Thank you again for submitting your work to EMBO Molecular Medicine. One of the referees who initially accepted to review the manuscript finally dropped out. We have now heard back from the other two referees who evaluated your manuscript. As you will see from the reports below, the referees acknowledge the potential interest of the study. However, they also raise a series of concerns about your work, which should be convincingly addressed in a major revision of the present manuscript.
The referees' recommendations are rather clear and there is no need to reiterate their comments. Importantly, alternative explanations need to be considered (as suggested by Referee #1) and overstatements should be avoided (commented by Referee #3). Referee #3 mentioned that additional data on pure tauopathy would strengthen the study, which we would encourage you to address.
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Sincerely, Jingyi
Jingyi Hou Editor EMBO Molecular Medicine *** Instructions to submit your revised manuscript *** ** PLEASE NOTE ** As part of the EMBO Publications transparent editorial process initiative (see our Editorial at https://www.embopress.org/doi/pdf/10.1002/emmm.201000094), EMBO Molecular Medicine will publish online a Review Process File to accompany accepted manuscripts.
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10) A Conflict of Interest statement should be provided in the main text 11) Please note that we now mandate that all corresponding authors list an ORCID digital identifier. This takes <90 seconds to complete. We encourage all authors to supply an ORCID identifier, which will be linked to their name for unambiguous name identification.
Currently, our records indicate that the ORCID for your account is 0000-0002-8885-7724.
Please click the link below to modify this ORCID: Link Not Available 12) The system will prompt you to fill in your funding and payment information. This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance. This quote takes into account any reduction or fee waivers that you may be eligible for. Authors do not need to pay any fees before their manuscript is accepted and transferred to our publisher.  The dataset presented is exceptional, in particular the combination of neuropathological data with in vivo biomarker analysis in the same subjects. The addition of the in vivo PET data further increases the translational value of the study. Third, everything is nicely put together in a coherent model of AD pathogenesis.

Referee #1 (Remarks for Author):
Mattsson-Carlgren et al evaluated how 217phosphotau in plasma relates to neuropathological measures of tangles, plaques and their interaction and also to PET measures of these variables. They demonstrate that 217phosphotau depends on the amount of plaques, tangles and the interaction between the two. Medial temporal tangle density does not correlate with 217phosphotau levels while neocortical tangle density does. The results are interpreted within a model that is shown in Fig 7. This is an exceptionally rich dataset with appropriate sample sizes. The report combines a set of postmortem cases of whom blood biomarker analysis pre-mortem is available with the well-known BioFinder cohort. The combination of neuropathology and in vivo blood biomarker analysis within the same subjects is particularly valuable. The data are impressive, in particular the neuropath+blood biomarker and the verification in vivo. My only concern relates to the interpretation in terms of the model outlined in figure 7. Other, more trivial explanations must be entertained. Major comments: 1. Figure 3: The data are analysed with a linear regression but the distribution of the datapoints in panel A & B does not seem to be linear. The same is true for figure 1B for the pooled data. The authors should report whether the assumptions for linear regression are met. 2. The authors interpret their 217phosphotau data within a mainstream model that neocortical amyloidosis is a trigger for inducing the spread of medial temporal tangles to the neocortex. Here is an alternative explanation: Obviously the total amount of aggregated tau is much lower when tangles are limited to the medial temporal cortex than when tangles are also spreading into neocortex. Hence, if 217phosphotau reflects aggregated tau, it is more likely that a relationship will be found with neocortical than with medial temporal tau. Under this hypothesis the difference in the relationship of phosphotau to medial temporal versus neocortical tau is due to 1. a quantitative difference in total brain tangle load 2. the fact that neocortical spread of tau is rarely seen in amyloid negative individuals. 3. Likewise, on p 6 the interpretation of the observation that "Higher tau PET was associated with higher plasma P-tau217 in those with high (but not in those with low) Aβ PET", must take into account that those with a high Abeta PET have a much wider range in tau PET levels. When the range of the predictor variable is wide, the likelihood to find a significant association if there is one is much higher than when the range of the predictor variable is narrow (as in the amyloid negative cases). The authors' interpretation that 'plasma P-tau217 is independently related to both Aβ and tau build-up' is interesting but an alternative, more trivial explanation should also be entertained.
Very minor comments: 1. Figure  This is a well done study using state of the art biomarkers in humans it is sufficiently powered.
Referee #3 (Remarks for Author): I think the data speaks for itself that pTau217 in blood is strongly associated with amyloid and tau pathology. This extends previous studies of this and other pTau epitopes.
However, association does not prove causality and the manuscript at times goes a litel to far or is not precise enough with inference. One example is the statement: "We also found that plasma P-tau217 levels mediated the effect of Aβ load on tau load." This and a number of other similar interpretative statements should be modified to reflect that it is an association and as a they did in the abstract say they potentially support a hypothesis.
A missing link here is data on a pure tauopathy (PSP, FTDP-17)it would strengthen the manuscript to include data on this group.
Overall I think a more conservative discussion is warranted but in genral the study will be impactful on the field

Referee #1 (Comments on Novelty/Model System for Author):
The dataset presented is exceptional, in particular the combination of neuropathological data with in vivo biomarker analysis in the same subjects. The addition of the in vivo PET data further increases the translational value of the study. Third, everything is nicely put together in a coherent model of AD pathogenesis.
We are thankful to the reviewer for these positive comments.

Referee #1 (Remarks for Author):
Mattsson-Carlgren et al evaluated how 217phosphotau in plasma relates to neuropathological measures of tangles, plaques and their interaction and also to PET measures of these variables. They demonstrate that 217phosphotau depends on the amount of plaques, tangles and the interaction between the two. Medial temporal tangle density does not correlate with 217phosphotau levels while neocortical tangle density does. The results are interpreted within a model that is shown in Fig 7. This is an exceptionally rich dataset with appropriate sample sizes. The report combines a set of postmortem cases of whom blood biomarker analysis pre-mortem is available with the well-known BioFinder cohort. The combination of neuropathology and in vivo blood biomarker analysis within the same subjects is particularly valuable. The data are impressive, in particular the neuropath+blood biomarker and the verification in vivo. My only concern relates to the interpretation in terms of the model outlined in figure 7. Other, more trivial explanations must be entertained.
We agree, and we have added this text to the legend of figure 7: "We also acknowledge that other alternative explanations may be considered for our findings. One possibility is that lack of sensitivity of our methods to accurately quantify aggregated tau makes us unable to detect very early associations between aggregated tau and plasma P-tau217." We have also added other considerations throughout the text, as explained below.
Major comments: 1. Figure 3: The data are analysed with a linear regression but the distribution of the datapoints in panel A & B does not seem to be linear. The same is true for figure 1B for the pooled data. The authors should report whether the assumptions for linear regression are met.
We have inspected standard plots for diagnostic of the regression models. The linearity assumption was checked by inspection plots of model residuals versus fitted values. These did not indicate linearity problems. We include the plots below for the reviewer. We have also added text in the statistics section about inspection of diagnostic plots (page 15, line 11). Figure 1B: 1st Mar 2021 1st Authors' Response to Reviewers Figure 3A:

Residuals vs fitter values for model shown in
Residuals vs fitter values for model shown in Figure 3B: 2. The authors interpret their 217phosphotau data within a mainstream model that neocortical amyloidosis is a trigger for inducing the spread of medial temporal tangles to the neocortex. Here is an alternative explanation: Obviously the total amount of aggregated tau is much lower when tangles are limited to the medial temporal cortex than when tangles are also spreading into neocortex. Hence, if 217phosphotau reflects aggregated tau, it is more likely that a relationship will be found with neocortical than with medial temporal tau. Under this hypothesis the difference in the relationship of phosphotau to medial temporal versus neocortical tau is due to 1. a quantitative difference in total brain tangle load 2. the fact that neocortical spread of tau is rarely seen in amyloid negative individuals.
We agree with this, and we have added a section in the discussion about this: (page 10, line 8) "(and since the total amount of aggregated tau is low in subjects with tau tangles limited to the medial temporal cortex, it is more difficult to detect an association between tangle load and plasma P-tau217 in those individuals compared to when tangles have also spread into neocortex)".
3. Likewise, on p 6 the interpretation of the observation that "Higher tau PET was associated with higher plasma P-tau217 in those with high (but not in those with low) Aβ PET", must take into account that those with a high Abeta PET have a much wider range in tau PET levels. When the range of the predictor variable is wide, the likelihood to find a significant association if there is one is much higher than when the range of the predictor variable is narrow (as in the amyloid negative cases). The authors' interpretation that 'plasma P-tau217 is independently related to both Aβ and tau build-up' is interesting but an alternative, more trivial explanation should also be entertained.
We agree, and we have added a section about this in the results (page 7, line 14): "(but we acknowledge that the narrow range of tau PET in the individuals with low Aβ PET makes it more difficult to detect a significant association with plasma P-tau217, compared to in those with high Aβ PET, where the range of tau PET is wider)".
Very minor comments: 1. Figure 3: when the linear regression does not reach significance there should be no regression line drawn in the plot We have removed the regression lines from Figure 3B and Figure 3C (and from relevant panels in Figure 6).

Referee #3 (Comments on Novelty/Model System for Author):
This is a well done study using state of the art biomarkers in humans it is sufficiently powered.
We are grateful to the reviewer for this overall comment.
Referee #3 (Remarks for Author): I think the data speaks for itself that pTau217 in blood is strongly associated with amyloid and tau pathology. This extends previous studies of this and other pTau epitopes.
However, association does not prove causality and the manuscript at times goes a litel to far or is not precise enough with inference. One example is the statement: "We also found that plasma P-tau217 levels mediated the effect of Aβ load on tau load." We have clarified that this was a statistical mediation, and changed it to "We also found that plasma P-tau217 levels statistically mediated the effect of Aβ load on tau load." (page 3, line 18).
This and a number of other similar interpretative statements should be modified to reflect that it is an association and as a they did in the abstract say they potentially support a hypothesis.
We have made a number of changes throughout the text to modify statements that may be interpreted as too strong. We now point out in the introduction that this is an observational study ("the data from this observational study", page 3, line 21). We mention at several places that the mediation effect represents "statistical mediation". On page 5 (line 13) we have changed the sentence "These results showed that plasma P-tau217 may partly explain the link between build-up of Aβ and tau pathology" to "These results suggest that plasma P-tau217 may partly explain the link between build-up of Aβ and tau pathology". We have also changed the sentence (page 5, line 25) "These results show that Aβ pathology is linked to increased phosphorylation and/or release of tau" to "These results suggest that Aβ pathology is linked to increased phosphorylation and/or release of tau".
A missing link here is data on a pure tauopathy (PSP, FTDP-17)it would strengthen the manuscript to include data on this group. Figure 4. These results showed that PSP/CBD patients (who did not have amyloid pathology) did not have increased plasma P-tau217 compared to individuals without significant primary pathologies. This supports that plasma P-tau217 is increased primarily in subjects with both amyloid and tau pathology. We have also added a sentence about this in the abstract: "P-tau217 was not elevated in patients with primary non-Alzheimer's disease tauopathies (N=9)."

We have added data on 9 subjects with neuropathology data and pure tauopathies (PSP and CBD). These new data is described in the main text (page 6, line 6) and shown in the new
Overall I think a more conservative discussion is warranted but in general the study will be impactful on the field We sincerely appreciate the positive comments. We have tried to make the discussion more conservative as described above. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from the two referees who were asked to re-assess it. As you will see the referees are now supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following amendments: 1. In the main manuscript file, please do the following: -Provide up to 5 keywords and incorporate them in the main text -Remove the red color font. -In Materials and Methods, include a statement that informed consent was obtained from all human subjects. Please make sure that the information entered in the Checklist regarding human subjects is also included in the Materials and Methods.
2. Funding information is incomplete in the online submission system -Please fix it.
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# Data availability
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Plasma levels of phosphorylated tau, including P-tau217, are elevated in Alzheimer's disease (AD). This study explores the underlying processes associated with the increased levels of plasma P-tau217, using post-mortem data and positron emission tomography (PET) of -amyloid and tau.
• Plasma P-tau217 is independently associated with higher levels of both -amyloid pathology and tau pathology in the brain. • The first changes in plasma P-tau217 may reflect the early accumulation of -amyloid before there is widespread tau aggregation. • Once tau aggregation reaches the neocortex, there is a strong correlation between plasma P-tau217 and the amount of aggregated tau. • Plasma P-tau217 mediates the association between -amyloid accumulation and tau accumulation.
11. As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. a. In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know if you do NOT agree with this.
b. Please note that the Authors checklist will be published at the end of the RPF.
I look forward to seeing a revised version of your manuscript as soon as possible.

Sincerely, Jingyi
Jingyi Hou Editor EMBO Molecular Medicine *** Instructions to submit your revised manuscript *** *** PLEASE NOTE *** As part of the EMBO Publications transparent editorial process initiative (see our Editorial at https://www.embopress.org/doi/pdf/10.1002/emmm.201000094), EMBO Molecular Medicine will publish online a Review Process File to accompany accepted manuscripts.
In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. If you do NOT want this file to be published, please inform the editorial office at contact@embomolmed.org.
To submit your manuscript , please follow this link:

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When submitting your revised manuscript, please include: 1) a .docx formatted version of the manuscript text (including Figure legends and tables) 2) Separate figure files* 3) supplemental information as Expanded View and/or Appendix. Please carefully check the authors guidelines for formatting Expanded view and Appendix figures and tables at https://www.embopress.org/page/journal/17574684/authorguide#expandedview 4) a letter INCLUDING the reviewer's reports and your detailed responses to their comments (as Word file).
5) The paper explained: EMBO Molecular Medicine articles are accompanied by a summary of the articles to emphasize the major findings in the paper and their medical implications for the non-specialist reader. Please provide a draft summary of your article highlighting -the medical issue you are addressing, -the results obtained and -their clinical impact. This may be edited to ensure that readers understand the significance and context of the research. Please refer to any of our published articles for an example. 6) For more information: There is space at the end of each article to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc...

7)
Author contributions: the contribution of every author must be detailed in a separate section. 8) EMBO Molecular Medicine now requires a complete author checklist (https://www.embopress.org/page/journal/17574684/authorguide) to be submitted with all revised manuscripts. Please use the checklist as guideline for the sort of information we need WITHIN the manuscript. The checklist should only be filled with page numbers were the information can be found. This is particularly important for animal reporting, antibody dilutions (missing) and exact values and n that should be indicted instead of a range. 9) Every published paper now includes a 'Synopsis' to further enhance discoverability. Synopses are displayed on the journal webpage and are freely accessible to all readers. They include a short stand first (maximum of 300 characters, including space) as well as 2-5 one sentence bullet points that summarise the paper. Please write the bullet points to summarise the key NEW findings. They should be designed to be complementary to the abstract -i.e. not repeat the same text. We encourage inclusion of key acronyms and quantitative information (maximum of 30 words / bullet point). Please use the passive voice. Please attach these in a separate file or send them by email, we will incorporate them accordingly.
You are also welcome to suggest a striking image or visual abstract to illustrate your article. If you do please provide a jpeg file 550 px-wide x 400-px high.
10) A Conflict of Interest statement should be provided in the main text 11) Please note that we now mandate that all corresponding authors list an ORCID digital identifier. This takes <90 seconds to complete. We encourage all authors to supply an ORCID identifier, which will be linked to their name for unambiguous name identification.
Currently, our records indicate that the ORCID for your account is 0000-0002-8885-7724.
Please click the link below to modify this ORCID: Link Not Available 12) The system will prompt you to fill in your funding and payment information. This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance. This quote takes into account any reduction or fee waivers that you may be eligible for. Authors do not need to pay any fees before their manuscript is accepted and transferred to our publisher. *Additional important information regarding figures and illustrations can be found at https://bit.ly/EMBOPressFigurePreparationGuideline The system will prompt you to fill in your funding and payment information. This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance. This quote takes into account any reduction or fee waivers that you may be eligible for. Authors do not need to pay any fees before their manuscript is accepted and transferred to our publisher.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
Neuropathology cohort: all available subjects with plasma P-tau217 data were included. BioFINDER2 cohort: all available subjects with plasma P-tau217, tau PET and amyloid PET data were included graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

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