Amyloid-β fibrils accumulated in preeclamptic placentas suppress syncytialization of cytotrophoblasts

  1. Kaho Nishioka
  2. Midori Ikezaki
  3. Naoyuki Iwahashi
  4. Miyu Arakawa
  5. Momo Fukushima
  6. Noa Mori
  7. Mika Mizoguchi
  8. Yuko Horiuchi-Tanizaki
  9. Megumi Fujino
  10. Takami Tomiyama
  11. Yoshito Ihara
  12. Kenji Uchimura
  13. Kazuhiko Ino
  14. Kazuchika Nishitsuji

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Abstract

Cerebral deposition of fibrillar amyloid-β (Aβ) is a pathological hallmark of Alzheimer’s disease. While Aβ is present in human placentas and accumulates in preeclamptic placentas characterized by poor placentation, the production and role of Aβ in the human placenta remain unclear. Because hypoxia in mid-to-late pregnancy is a risk for preeclampsia, we found that levels of hypoxia-inducible factor 1-α and β-secretase (BACE-1) increased concurrently with placental Aβ deposition in late stage preeclamptic placentas. We also found that a human cytotrophoblast (CTB) model, BeWo cells, actually produced Aβ species, and that hypoxia increased Aβ production and BACE-1 protein levels. Aβ42 fibrils inhibited CTB syncytialization, a critical step in maintaining pregnancy, by inducing loss of membrane localization of cell-cell adhesion molecules. Primary human CTBs confirmed these observations. Taken together, our results suggest that increased Aβ production in CTBs by hypoxia may lead to the formation of Aβ fibrils, which inhibit syncytiotrophoblast formation and are detrimental to pregnancy. Thus, our results reveal the novel role of Aβ fibrils in the pathogenesis of preeclampsia.

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    Reply to the reviewers

    Reviewer #1 (Significance (Required)):

    This study aims to bridge a gap between the mechanisms of preeclampsia and neurodegenerative disorders, and this through the existence of misfolded proteins in the preeclamptic placenta which has been reported before, in particular the beta amyloid protein, known as operative in Alzheimer's disease (AD) in particular.

    Our response: We sincerely appreciated the reviewer’s constructive comments.

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

    Minor remarks

    1. It is classical now to present in extenso the WB as supplementary data for Fig 3, 4 and 5. Our response: We will include the full blots in Supplemental information.

    It seems that the beta amyloid signal is not stronger for the early onset and the late onset PE samples. Have the authors an interpretation?

    Our response: The current manuscript includes both early-onset and late-onset cases. Thus, we are certain that amyloid beta deposition is involved in both early- and late-onset PE. We will discuss this matter.

    The figure 4b does not show the BeWo labeling in forskolin with or without beta amyloid peptides, why? It would be illustrative to show a decrease in the fusion processes

    Our response: In Fig. 4A, we pretreated BeWo cells with Aβ fibrils and after that, cell fusion was induced by Fsk. On the other hand, in Fig. 4b, we treated BeWo with Aβ fibrils and investigated the protein levels and subcellular localization of ZO-1 and E-cadherin. Fig.4b shows that expressions of proteins involved in cell-cell interaction were reduced by Aβ fibril treatment without Fsk. Cell-cell interaction before syncytialization is required for cell fusion, and these proteins disappear after cell fusion. Thus, our results demonstrate that elimination of cell-cell interaction by Aβ fibrils resulted in reduced cell fusion induced by Fsk. This is why we treated BeWo cells with Aβ fibrils before the induction of cell fusion by Fsk, and BeWo labeling in forskolin with or without Aβ fibrils will result in a loss of ZO-1 and E-cadherin regardless of the occurrence of cell fusion. We will discuss this matter in more detail.

    How do the authors explain that exposure to fibrils did not seem to slow down significantly the fusion process, even though markers are decreased?

    Our response: Since we previously demonstrated that loss of membrane E-cadherin slows the fusion (Iwahashi et al., Endocrinology, 2019, PMID: 30551188), we believe that reduction of membrane localization of E-cadherin also slows the fusion process. We will discuss this matter further.

    • Could the authors attempt a labeling with the Di-8, an interesting quantitative marker of cell fusion (see ref PMID: 38019394).*

    Our response: We have shown that pretreatment of BeWo cells and human primary cytotrophoblasts (CTBs) inhibited induction of syncytin-1 and β-hCG. Syncytin-1 is a critical driver of syncytialization and formation of the syncytiotrophoblast layer, and β-hCG is one of the major products of syncytiotrophoblasts. Thus, induction of these proteins is widely used as syncytialization markers of trophoblasts. On the other hand, Di-8-ANEPPS is a potentiometric fluorescent dye that may be used assess cell fusion simply and economically. Although we understand the robustness of this method, we believe that the current data are sufficient to demonstrate that Aβ fibril pretreatment inhibited syncytialization of BeWo cells and CTBs.

    Reviewer #2 (Significance (Required)):

    Investigating the deposition of Aβ in the placenta could enhance our understanding of pregnancy complications such as PE, fetal growth restriction, and neurodevelopmental risks. However, further research on this topic is necessary.

    Our response: We sincerely appreciate the critical reading and constructive comments of the reviewer. We agree that further research on protein aggregation and the pathogenesis of preeclampsia is necessary. We will discuss this matter in the discussion.

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

    Major comments

      • If CTBs are treated with Aβ, and if it affects STB, what happens with EVT? Why didn't they check with EVT if the authors wanted to link with PE?*

    Our response: We thank the reviewer for the critical comment. We investigated Aβ generation by an EVT model cell, HTR8/SVneo cells. We found that HTR8/SVneo cells produced much less amount of Aβ compared to BeWo cells (unpublished). Gao et al. reported that Aβ aggregates induced autophagy in HTR8/SVneo cells and suggested that an excessive autophagy may be detrimental and be involved in the development of preeclampsia (Gao et al., J Mol Histol, 2024, PMID: 38777993). We will discuss this matter in the discussion.

    On the other hand, we have already investigated the effects of Aβ monomers in EVTs, and discovered that even low levels of Aβ produced by EVTs promote EVT invasiveness and have a physiological function. Please see below. We will add these new data in the revised manuscript.

      • Did the authors look for pathologies related to Aβ deposition on PE placentas?*

    Our response: We did not observe any pathologies near the Aβ deposition.

      • Line# 103, the IF images don't show that BACE1 is around HIF1. There are no merged images, and the results are over- or underestimated.*

    Our response: We agree with the reviewer. There is a time discrepancy between HIF activation and BACE1 induction. Our immunohistochemical analysis showed that PE placentas are in a chronic hypoxia condition and that BACE1 was increased in PE placentas. Our cell-based assay supports that HIF1α stabilization by Roxadustat increased BACE1 levels in BeWo cells. We will tone down the results section of the immunohistochemical analysis.

      • What is the intended purpose of using Roxadustat? If it inhibits HIF1α, could you explain the reason behind the increased expression of HIF1α? Furthermore, is there evidence to support the efficacy of this compound?*

    Our response: Roxadustat inhibits the proline hydroxylation of HIF1α and thereby inhibits the ubiquitination and degradation of HIF1α via the ubiquitin proteasomal system. In this study, we used Roxadustat as a HIF1α stabilizer to investigate whether BACE1 levels are increased with hypoxia and HIF1. Our data showed that treatment of BeWo cells with Roxadustat increased HIF1α levels, supporting the efficacy of Roxadustat. We will include this information in the result section for clarity.

      • Is Aβ deposition very specific to PE, or can it also occur for other reasons during pregnancy?*

    Our response: To date, no report has been found showing Aβ deposition in placentas other than PE. The deposition of protein aggregates, including those of Aβ and transthyretin, has previously been reported in PE. However, the presence and role of these protein deposits in placentas under pathological conditions, in addition to PE, remains to be elucidated. Several stresses such as hypoxia and ER stress may lead to deposition of protein aggregates in the placenta. These points will be discussed in the discussion.

      • BACE1 is expressed in Normal#2 and #3 but not in #1, #4, and #5. Why is this expressed in #2 and #3? Is there anything wrong with these samples? If patients had gestational hypertension or some other complications?*

    Our response: We did not find any other complications in the normal placentas. In the brain, Aβ is constitutively generated and thus, thought to play physiological roles. The amount of Aβ is determined by the balance between the production and the clearance. A sustained imbalance of Aβ production and Aβ clearance will lead Aβ aggregation and deposition. We found that BeWo cells expressed BACE1 in a normoxic condition and thus, normal placentas may express BACE1 and generate small amounts of Aβ. Our results suggested that chronic hypoxia in PE placentas resulted in increased BACE1 expression and increased Aβ production, which may eventually result in Aβ aggregation and deposition, because the aggregation process of Aβ is concentration-dependent. We will include this point in the revised manuscript.

      • PE placentae were compared with GA matched placentae. What is the expression of BACE1 and RB4CD12 in term control placentae?*

    Our response: We used RB4CD12 as a protein aggregation marker. As shown in Table 1, the current study includes 3 placentas whose gestational ages are over 37 weeks. We did not observe RB4CD12 and Aβ deposition in gestational age-matched control and observed BACE1 expression in one 37 weeks gestational age control. We will include these points in the result section.

      • If AB fibril deposition is hypoxia dependent, what happens at the early gestation, where oxygen conc is 1-2%?*

    Our response: At the early gestation, physiological hypoxia promotes the EVT invasion and helps the remodeling of spiral arteries for oxygen supply. Please see our response above. Severe hypoxia on the CTB side in early gestation may result in a miscarriage before PE develops.

    Minor comments

      • The authors only performed IF and IHC. Please confirm and correct the methods accordingly.*

    Our response: We thank the reviewer for pointing this out. We will correct the methods.

      • Was the BeWo-b21 clone cell line used for all the experiments in this paper? This is the only clone that can be used for BeWo-STB models.*

    Our response: We do not have information about the clone number of BeWo cells used in this study. We purchased them from the American Type Culture Collection (Manassas, VA) and they were authenticated by JCRB Cell Bank (National Institute of Biomedical Innovation Japan, report no. KBN0410). By using the same cells, we published three articles in which we successfully analyzed syncytialization of BeWo cells (Yamamoto et al., Endocrinology, 2017, PMID: 28938427; Iwahashi et al., Endocrinology, 2019, PMID: 30551188; Matsukawa et al., Biomolecules, 2022, PMID: 36008943). We would like to apologize for our mistake in the description of BeWo cells in the methods section and thank the reviewer for providing us with an opportunity to correct our mistake. We will note that BeWo cells were purchased from the American Type Culture Collection (Manassas, VA) and authenticated by JCRB Cell Bank (National Institute of Biomedical Innovation Japan, report no. KBN0410) in the methods section, and will upload the authentication report KBN0410 as a review process file.

      • Have all the experiments on BeWo only been performed once?*

    Our response: We repeated 6 experiments (the repetitions are biological, not technical, replicates). The results are shown as means ± SEM (n = 6) as stated in the Figure legends.

    Reviewer #3 (Significance (Required)):

    While Aβ is present in human placentas and accumulates in preeclamptic placentas, the production and role of Aβ in the human placenta remain unclear. The current findings suggest that increased Aβ production in cytotrophoblast by hypoxia may lead to the formation of Aβ fibrils, which inhibit syncytiotrophoblast formation and are detrimental to pregnancy, revealing a novel role of Aβ fibrils in the pathogenesis of preeclampsia.

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

    The authors found that Amyloid β suppressed cytotrophoblasts syncytialization, which is innovative. The authors used human patient samples and human primary CTB culture which are powerful data.

    Our response: We appreciate the reviewer’s thoughtful feedback and support.

    Fig. 3. The authors used Roxadustat to stimulate HIF-1α and showed BACE1 increase. It would be better to have the cells in real hypoxia condition.

    Our response: There is a time discrepancy between the increase in HIF-1α levels by hypoxia and induction of BACE1. Because the purpose of this experiment is to show that increased HIF1-α correlated BACE1 induction, we used Roxadustat as a HIF1-α stabilizer and showed that sustained induction of HIF increased BACE1 levels. However, we do understand the reviewer’s concern. We will include data showing an increase in BACE1 in hypoxic conditions by performing new Western blotting experiments.

    Fig. 4 and 5. The authors used external Amyloid β for stimulation. Would the endogenous Amyloid β levels reach the concentration of external one? It would be better to see the quantitative levels of Amyloid β in Fig. 3b.

    Our response: Because the aggregation of Aβ requires a high concentration of a micromolar order, we used synthetic Aβ fibrils for stimulation. We propose that chronic hypoxia in preeclampsia leads to an elevated local concentration of Aβ through a sustained increase in Aβ production, which eventually results in Aβ fibrillogenesis and deposition of Aβ fibrils. Therefore, it will be difficult for the Aβ concentrations generated by BeWo cells to reach a level sufficient for fibrillogenesis. We will discuss this point in the revised manuscript. In addition, we have already performed ELISA assays to quantitatively analyze Aβ generation by BeWo cells. We will include these ELISA data in the revised manuscript.

    Reviewer #4 (Significance (Required)):

    The manuscript addresses an important theme recently identified to address the heterogeneous etiology of preeclampsia. Although the authors have used in vitro approaches, the study could have been a solid if not for some major concerns.

    The authors have focused on an already demonstrated phenomenon but have tried to validate the findings using their in vitro approaches. The manuscript is well written but some lapses for correct references.

    Our response: We thank the reviewer for the critical reading of our manuscript and his/her constructive comments. As the reviewer pointed out, recent studies suggest that preeclampsia is a proteinopathy. However, the mechanisms by which protein aggregate plays detrimental roles in placentation has not been well-understood. In the present study, we discovered a detrimental role of Aβ fibrils in syncytiotrophoblast formation.

    Reviewer #4 (Evidence, reproducibility and clarity (Required)):

    Major comments:

      • In lines 50 and 51 of Introduction, the authors provide references to two publications. However, several other articles have appeared before or after these publications that demonstrated evidence for proteinopathy in the placenta and circulation of preeclampsia patients. The reviewer has gone through the literature and found several publications. For example, Kalkunte et al were the first ones to demonstrate the etiology of proteinopathy in preeclampsia placenta and focused on a protein called transthyretin (Am J Pathol. 2013, 183(5):1425-1436). Similarly, Cheng et al demonstrated using a novel blood test that serum from early onset and late onset preeclampsia manifestations contained Aβ and transthyretin (Nature Sci Rep. 2021;11:15934). Jash et al showed the presence of cis P-tau in the placenta and serum of early and late onset preeclampsia patients (Nat Commun. 2023;14:5414). This article and another article by Cheng et al (Hypertension 79(8):1738-1754) revealed that aggregated cis P-tau and transthyretin are etiologically critical for the onset of preeclampsia. There have been several other review and original articles that have talked about Alzheimer's like etiology in preeclampsia (Olie et al, JAMA Netw Open, 2024; e2412870; Basit et al, BMJ 2018; 363:k4109; Schliep et al, Hypertension 2023; 80:257-267, Cheng et al, Am J Reprod Immunol. 2016;75:372-381).*

    Our response: We sincerely appreciate the reviewer for his/her helpful comment. We will revise the introduction by citing the references recommended by the reviewer.

    • Following up on the comments made above, the authors talk about induction of Aβ in hypoxia-treated human trophoblasts represented by an established cell line, BeWo, and primary human trophoblasts. However, it is not clear whether Aβ42 as stated in the manuscript was detected as an aggregated structure or a protein coupled with RB4CD12 aggregate marker. It would have been helpful if the authors could provide direct evidence for Aβ aggregation.*

    Our response: Based on our previous findings showing that highly sulfated domains of heparan sulfate are common components of protein aggregate deposits, we used RB4CD12, which recognizes these domains, as a marker of protein aggregate deposition. These include aggregates of Aβ in Alzheimer’s disease, transthyretin in ATTR, and p53 aggregates in p53-mutated cancers (Hoshono-fukao et al., Am J Pathol, 2012, PMID: 22429964; Kameyama et al., Am J Pathol, 2019, PMID: 30414409; Iwahashi, PNAS, 2020, PMID: 33318190). Please also see our reply to Comment 5 below. We will perform additional immunohistochemical analysis with the β0001 anti-Aβ antibody and the ProteoStat dye that recognizes protein aggregates.

    • What appeared to be more surprising is the statement on lines 162 and 163 that cultured CTBs produced Aβ40/42. Again, it is not clear whether the authors are talking about aggregated Aβ or just induction of Aβ. Why should normal CTBs produce Aβ? It is not clear whether this is a transient expression or a long-term phenomenon. The issue is distinction between normal and adverse pregnancy conditions, and the latter associated with protein aggregation as suggested in the literature.*

    Our response: BeWo cells and cultured CTBs produce Aβ peptides in a normoxic condition. In the brain, neurons constitutively produce Aβ peptides, which have physiological roles such as controlling neuronal hyperexcitability, enhancing of synaptic plasticity, and improving memory (reviewed in Kent et al., Acta Neuropathol, 2020, PMID: 32728795). The amount of Aβ in the brain is regulated by the balance between Aβ production and Aβ clearance, and the imbalance of the production and the clearance may result in an increase in Aβ local concentration and Aβ aggregation. Our results showing that hypoxia increased Aβ production in BeWo cells suggest that chronic hypoxia, which is a risk of preeclampsia, may lead to a sustained increase in Aβ production and an elevated local concentration of Aβ at or near the site of Aβ production. We will discuss these points in the discussion.

    In the present study, we showed that aggregated form of Aβ (i.e., Aβ fibrils) was detrimental to the CTB differentiation. On the other hand, we already found that Aβ monomers promoted EVT invasion (please see the below). We believe that promotion of EVT invasion by Aβ monomers represent a physiological function of Aβ in the placenta. We will include these new data in the revised manuscript and we will also perform experiments with BeWo cells and Aβ monomers in order to investigate whether Aβ monomers have some roles in CTB differentiation.

    • The authors have adequately pointed to importance of hypoxia in the onset of preeclampsia-like features. As a matter of fact, Lai et al demonstrated in a mouse pre-clinical model that hypoxia could induce severe features of preeclampsia (Hypertension. 2011;57:505-514). The use of hypoxia as driver of Aβ induction is appreciated.*

    Our response: We agree with the reviewer that studies using preclinical animal models are an important topic for the future. __We will discuss this point in the discussion. __

    • In Fig. 1, although the authors have used DIC approach, it would have been helpful if they presented individual Aβ and RB4CD12 green and red channels, and a merged profile. For example, PE #4 sample does not appear to have much RB4CD12. Again, there is a question of aggregated or native protein structures. It is difficult to have a satisfactory statistical analysis. Did the authors look for Aβ in the anchoring villi region of the placenta?*

    Our response: We will show the green and red channel images individually. We have noticed that we detected Aβ deposition without RB4CD12 signals. Aβ is small peptides of 40 to 42 amino acid residues and is extracellularly released after the production. Non-deposited Aβ monomers are not detected by immunohistochemical analysis, because these soluble Aβ peptides are spread out in the tissue fluid. Thus, in our statistical analysis, we calculated only merged signals of Aβ and RB4CD12, which suggests that our data show the aggregated and deposited Aβ. We will note this point in the results. In addition, we will perform immunohistochemical analysis with the anti-Aβ antibody and the ProteoStat dye. Please also see our response to Comment 2 above. We did not observe Aβ deposition at the anchoring villi.

    • Fig. 2 does not show significant staining for HIF1-α in PE placental tissue.*

    Our response: In a normoxic condition, HIF1-α is constitutively expressed but degraded via the proline-hydroxylation and the subsequent ubiquitination and degradation in the proteasome. Because the proline-hydroxylation is oxygen-dependent, hypoxia induce HIF1-α accumulation. Thus, our data suggest a hypoxic environment in the preeclamptic placentas. We will note this point in the results section.

    • Fig. 3B, why should there be Aβ40/42 under normoxic conditions? This is the most pertinent concern and the authors are validating significant expression of Aβ40/42 under normal conditions. In normal pregnancy placenta, this protein has not been detected.*

    Our response: Aβ peptides are constitutively produced in BeWo cells, and the production was enhanced by hypoxia. Aβ is small peptides of 40 to 42 amino acid residues. We did not observe Aβ signals in the immunohistochemical analysis of the normal pregnancy placentas, because Aβ peptides that do not aggregate and deposit in the placenta were distributed in the tissue fluid and lost before and during the processing of the placentas for the paraffin-embedding and immunostaining. Our immunohistochemical analysis detects only Aβ deposition. Thus, the absence of Aβ signals in the immunohistochemical analysis of normal placentas does not mean that normal placenta does not produce any Aβ peptides.

    • Figs. 4 and 5 present the crux of the conclusions that the authors are trying to draw from their study. Aβ peptide solution was incubated for 5 days at 370C to prepare so called Aβ fibril-like structures. What is the purity of fibril structures? Does this preparation show toxic effects on cell viability? Human trophoblasts expressing E-cadherin fail to participate in endovascular cross-talk with endothelial cells, a process required for spiral arteries. It appears that either BeWo cells or primary trophoblasts used in this study represent trophoblasts from third trimester. It is not clear why should Aβ fibril like structures should inhibit ZO-1 and E-cadherin or β-hCG (Fig. 5) for that matter. In Fig. 5C, there does not seem to be a major effect of Aβ fibrils. Did the authors try synthetic Aβ as a control. These experiments could have been meaningful but for proper controls.*

    Our response: Synthetic Aβ was purchased from Peptide Institute (Osak, Japan). The purity is >95%. We will include the data sheet as a review process file. In case that the reviewer wants to know the fibril content of the preparation, we will calculate the fibril content by using Native PAGE followed by Western blotting. We did not observe any cytotoxicity of the preparation as shown in Supplemental Fig. S3.

    We previously showed that membrane localization of cell-cell interaction proteins such as ZO-1 and E-cadherin in cytotrophoblasts is required for syncytialization (Iwahashi et al., Endocrinology, 2019, PMID: 30551188; Matsukawa et al., Biomolecules, 2022, PMID: 36008943). Because Aβ aggregates disrupt membrane localization of tight junction proteins partly by inducing excess autophagy (Marco et al., Neurosci Lett, 2006, PMID: 16644119; Chan et al., Exp Cell Res, 2012, PMID: 29856989), we hypothesized that Aβ fibrils may also disrupt membrane localization of ZO-1 and E-cadherin in BeWo cells. We are focusing on the effect of Aβ fibrils on cytotrophoblasts at the late stage of pregnancy when the remodeling of spiral arteries is completed. We understand the importance of investigating the effects of Aβ and Aβ fibrils on early pregnancy. We will cite an article showing the effects of Aβ aggregates on EVTs (Gao et al., J Mol Histol, 2024, PMID: 38777993) and include our data showing the Aβ monomer functions on EVT invasion. Please also see our reply to Comment 3 above. As for Fig. 5C, we will improve the quality of images. We will also perform experiments to investigate whether Aβ monomers alone affect syncytialization of BeWo cells.

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    Referee #4

    Evidence, reproducibility and clarity

    This manuscript focuses on the role of amyloid β (Aβ) in hypoxia-exposed human trophoblasts. Recent reports in the literature have confirmed the presence Aβ and other proteins, including Tau, transthyretin, and TDP-43, in placental tissue derived from preeclampsia deliveries. These proteins are recognized as hallmark causative factors for Alzheimer's disease related dementias. Hypoxia has also been shown to induce expression of these proteins, including Aβ, in human trophoblasts. In this regard, detection of Aβ hypoxia-exposed human trophoblast may not be a novel finding. This said, the manuscript presents some solid information and could have been very informative study. However, several conceptual, technical and literature concerns remain unaddressed and dampen the reviewer's enthusiasm for this study.

    Major comments:

    1. In lines 50 and 51 of Introduction, the authors provide references to two publications. However, several other articles have appeared before or after these publications that demonstrated evidence for proteinopathy in the placenta and circulation of preeclampsia patients. The reviewer has gone through the literature and found several publications. For example, Kalkunte et al were the first ones to demonstrate the etiology of proteinopathy in preeclampsia placenta and focused on a protein called transthyretin (Am J Pathol. 2013, 183(5):1425-1436). Similarly, Cheng et al demonstrated using a novel blood test that serum from early onset and late onset preeclampsia manifestations contained Aβ and transthyretin (Nature Sci Rep. 2021;11:15934). Jash et al showed the presence of cis P-tau in the placenta and serum of early and late onset preeclampsia patients (Nat Commun. 2023;14:5414). This article and another article by Cheng et al (Hypertension 79(8):1738-1754) revealed that aggregated cis P-tau and transthyretin are etiologically critical for the onset of preeclampsia. There have been several other review and original articles that have talked about Alzheimer's like etiology in preeclampsia (Olie et al, JAMA Netw Open, 2024; e2412870; Basit et al, BMJ 2018; 363:k4109; Schliep et al, Hypertension 2023; 80:257-267, Cheng et al, Am J Reprod Immunol. 2016;75:372-381).
    2. Following up on the comments made above, the authors talk about induction of Aβ in hypoxia-treated human trophoblasts represented by an established cell line, BeWo, and primary human trophoblasts. However, it is not clear whether Aβ42 as stated in the manuscript was detected as an aggregated structure or a protein coupled with RB4CD12 aggregate marker. It would have been helpful if the authors could provide direct evidence for Aβ aggregation.
    3. What appeared to be more surprising is the statement on lines 162 and 163 that cultured CTBs produced Aβ40/42. Again, it is not clear whether the authors are talking about aggregated Aβ or just induction of Aβ. Why should normal CTBs produce Aβ? It is not clear whether this is a transient expression or a long-term phenomenon. The issue is distinction between normal and adverse pregnancy conditions, and the latter associated with protein aggregation as suggested in the literature.
    4. The authors have adequately pointed to importance of hypoxia in the onset of preeclampsia-like features. As a matter of fact, Lai et al demonstrated in a mouse pre-clinical model that hypoxia could induce severe features of preeclampsia (Hypertension. 2011;57:505-514). The use of hypoxia as driver of Aβ induction is appreciated.
    5. In Fig. 1, although the authors have used DIC approach, it would have been helpful if they presented individual Aβ and RB4CD12 green and red channels, and a merged profile. For example, PE #4 sample does not appear to have much RB4CD12. Again, there is a question of aggregated or native protein structures. It is difficult to have a satisfactory statistical analysis. Did the authors look for Aβ in the anchoring villi region of the placenta?
    6. Fig. 2 does not show significant staining for HIF1-α in PE placental tissue.
    7. Fig. 3B, why should there be Aβ40/42 under normoxic conditions? This is the most pertinent concern and the authors are validating significant expression of Aβ40/42 under normal conditions. In normal pregnancy placenta, this protein has not been detected.
    8. Figs. 4 and 5 present the crux of the conclusions that the authors are trying to draw from their study. Aβ peptide solution was incubated for 5 days at 370C to prepare so called Aβ fibril-like structures. What is the purity of fibril structures? Does this preparation show toxic effects on cell viability? Human trophoblasts expressing E-cadherin fail to participate in endovascular cross-talk with endothelial cells, a process required for spiral arteries. It appears that either BeWo cells or primary trophoblasts used in this study represent trophoblasts from third trimester. It is not clear why should Aβ fibril like structures should inhibit ZO-1 and E-cadherin or β-hCG (Fig. 5) for that matter. In Fig. 5C, there does not seem to be a major effect of Aβ fibrils. Did the authors try synthetic Aβ as a control. These experiments could have been meaningful but for proper controls.

    Significance

    The manuscript addresses an important theme recently identified to address the heterogeneous etiology of preeclampsia. Although the authors have used in vitro approaches, the study could have been a solid if not for some major concerns.

    The authors have focused on an already demonstrated phenomenon but have tried to validate the findings using their in vitro approaches. The manuscript is well written but some lapses for correct references.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors found that Amyloid β suppressed cytotrophoblasts syncytialization, which is innovative. The authors used human patient samples and human primary CTB culture which are powerful data.

    Fig. 3. The authors used Roxadustat to stimulate HIF-1α and showed BACE1 increase. It would be better to have the cells in real hypoxia condition.

    Fig. 4 and 5. The authors used external Amyloid β for stimulation. Would the endogenous Amyloid β levels reach the concentration of external one? It would be better to see the quantitative levels of Amyloid β in Fig. 3b.

    Significance

    While Aβ is present in human placentas and accumulates in preeclamptic placentas, the production and role of Aβ in the human placenta remain unclear. The current findings suggest that increased Aβ production in cytotrophoblast by hypoxia may lead to the formation of Aβ fibrils, which inhibit syncytiotrophoblast formation and are detrimental to pregnancy, revealing a novel role of Aβ fibrils in the pathogenesis of preeclampsia.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors examine the deposition of amyloid-β (A β) peptides that accumulate in the brains of patients with Alzheimer's disease (AD). The authors demonstrated the expression of HIF-1 in the pre-eclamptic (PE) placental tissue using immunofluorescence (which is not novel), alongside the expression of BACE1. These experiments were also validated using BeWo and primary trophoblast cells cultured under hypoxia to mimic one of the characteristics of PE. However, this manuscript is quite preliminary, and many additional experiments are necessary to confirm the deposition of Aβ fibrils in PE. The authors treated CTB and observed the effects on STB, but in PE, the main cell lineage affected is extravillous trophoblast (EVT) cells, which invade the spiral artery. The defect in this invasion is one of the major causes of PE. Therefore, the authors should investigate the effect of hypoxia and Aβ deposition on EVT invasion. Overall, this work appears very incomplete, and further experiments are warranted.

    Major comments

    • If CTBs are treated with Aβ, and if it affects STB, what happens with EVT? Why didn't they check with EVT if the authors wanted to link with PE?
    • Did the authors look for pathologies related to Aβ deposition on PE placentas?
    • Line# 103, the IF images don't show that BACE1 is around HIF1. There are no merged images, and the results are over- or underestimated.
    • What is the intended purpose of using Roxadustat? If it inhibits HIF1, could you explain the reason behind the increased expression of HIF1? Furthermore, is there evidence to support the efficacy of this compound?
    • Is Aβ deposition very specific to PE, or can it also occur for other reasons during pregnancy?
    • BACE1 is expressed in Normal#2 and #3 but not in #1, #4, and #5. Why is this expressed in #2 and #3? Is there anything wrong with these samples? If patients had gestational hypertension or some other complications?
    • PE placentae were compared with GA matched placentae. What is the expression of BACE1 and RB4CD12 in term control placentae?
    • If AB fibril deposition is hypoxia dependent, what happens at the early gestation, where oxygen conc is 1-2%?

    Minor comments

    • The authors only performed IF and IHC. Please confirm and correct the methods accordingly.
    • Was the BeWo-b21 clone cell line used for all the experiments in this paper? This is the only clone that can be used for BeWo-STB models.
    • Have all the experiments on BeWo only been performed once?

    Significance

    Investigating the deposition of Aβ in the placenta could enhance our understanding of pregnancy complications such as PE, fetal growth restriction, and neurodevelopmental risks. However, further research on this topic is necessary.

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Proving that more Beta-amyloid are produced in preeclampsia, and that impacts negatively trophoblast cell fusion is interesting and provides a potential mechanism for interpreting some specific cases of preeclampsia. The authors analyzed the placenta from five control and five preeclamptic pregnancies (4 early onset et 1 late onset).

    The authors show first by IHC that amyloid beta and aggregate markers are apparently exclusively detected in the PE samples, the same observation is done for detection of HIF1alpha and BACE1, the enzyme that is responsible for the generation of amyloid peptides from digestion of the APP membrane neuron protein. After having used placental samples, the authors moved to the BeWo cell model, where they could analyze specifically cell biology in the context of syncytialization. The authors inhibited HIF1a prolylation (thus stabilizing it even in normoxia), and this leaded to the increase of BACE1, of beta-amyloid molecules, as shown by WB analyses; the same result was obtained by exposure to hypoxia, while a BACE1 inhibitor had the opposite effect.

    An interesting issue is the demonstration provided by the authors that in this model, syncytialization is decreased by Beta-amyloid fibrils, together with decreased hCG expression and decreased Syncytin-1. The authors also validate these results on primary human CTB from the third trimester.

    Minor remarks

    1. It is classical now to present in extenso the WB as supplementary data for Fig 3, 4 and 5.
    2. It seems that the beta amyloid signal is not stronger for the early onset and the late onset PE samples. Have the authors an interpretation?
    3. The figure 4b does not show the BeWo labeling in forskolin with or without beta amyloid peptides, why? It would be illustrative to show a decrease in the fusion processes
    4. How do the authors explain that exposure to fibrils did not seem to slow down significantly the fusion process, even though markers are decreased?
    5. Could the authors attempt a labeling with the Di-8, an interesting quantitative marker of cell fusion (see ref PMID: 38019394).

    Significance

    This study aims to bridge a gap between the mechanisms of preeclampsia and neurodegenerative disorders, and this through the existence of misfolded proteins in the preeclamptic placenta which has been reported before, in particular the beta amyloid protein, known as operative in Alzheimer's disease (AD) in particular.

  6. Version published to 10.1101/2025.03.06.641957v1 on bioRxiv