A New Role for RNA G-quadruplexes in Aging and Alzheimer’s Disease

  1. Lena Kallweit
  2. Eric D Hamlett
  3. Hannah Saternos
  4. Anah Gilmore
  5. Ann-Charlotte Granholm
  6. Scott Horowitz

  • Curated by eLife

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    eLife Assessment

    The current human tissue-based study provides convincing evidence correlating hippocampal expressions of RNA guanine-rich G-quadruplexes with aging and with Alzheimer's Disease presence and severity. The results are important and hold promise for deeper understanding of AD's pathogenesis and potential new therapeutic strategies.

    [Editors' note: this paper was reviewed by Review Commons.]

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Abstract

As the world population ages, new molecular targets in aging and Alzheimer’s Disease (AD) are needed to combat the expected influx of new AD cases. Until now, the role of RNA structure in aging and neurodegeneration has largely remained unexplored. METHODS: In this study, we examined human hippocampal postmortem tissue for the formation of RNA G-quadruplexes (rG4s) in aging and AD.We found that rG4 immunostaining strongly increased in the hippocampus with both age and with AD severity. We further found that neurons with accumulation of phospho-tau immunostaining contained rG4s, that rG4 structure can drive tau aggregation, and that rG4 staining density depended on APOE genotype in the human tissue examined.Combined with previous studies showing the dependence of rG4 structure on stress and the extreme power of rG4s at oligomerizing proteins, we propose a model of neurodegeneration in which chronic rG4 formation drives proteostasis collapse. These morphological findings suggest that further investigation of RNA structure in neurodegeneration is a critical avenue for future treatments and diagnoses.

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  1. Version published to 10.7554/elife.105446.1 on eLife
  2. Version published to 10.7554/elife.105446 on eLife
  3. eLife

    eLife Assessment

    The current human tissue-based study provides convincing evidence correlating hippocampal expressions of RNA guanine-rich G-quadruplexes with aging and with Alzheimer's Disease presence and severity. The results are important and hold promise for deeper understanding of AD's pathogenesis and potential new therapeutic strategies.

    [Editors' note: this paper was reviewed by Review Commons.]

  4. eLife

    Reviewer #1 (Public review):

    This is an interesting manuscript where the authors systematically measure rG4 levels in brain samples at different ages of patients affected by AD. To the best of my knowledge this is the first time that BG4 staining is used in this context and the authors provide compelling evidence to show an association with BG4 staining and age or AD progression, which interestingly indicates that such RNA structure might play a role in regulating protein homeostasis as previously speculated. The methods used and the results reported seems robust and reproducible. There were two main things that needed addressing:

    (1) Usually in BG4 staining experiments to ensure that the signal detected is genuinely due to rG4 an RNase treatment experiment is performed. This does not have to be extended to all the samples presented but having a couple of controls where the authors observe loss of staining upon RNase treatment will be key to ensure with confidence that rG4s are detected under the experimental conditions. This is particularly relevant for this brain tissue samples where BG4 staining has never been performed before.

    (2) The authors have an association between rG4-formation and age/disease progression. They also observe distribution dependency of this, which is great. However, this is still an association which does not allow the model to be supported. This is not something that can be fixed with an easy experiment and it is what it is, but my point is that the narrative of the manuscript should be more fair and reflect the fact that, although interesting, what the authors are observing is a simple correlation. They should still go ahead and propose a model for it, but they should be more balanced in the conclusion and do not imply that this evidence is sufficient to demonstrate the proposed model. It is absolutely fine to refer to the literature and comment on the fact that similar observations have been reported and this is in line with those, but still this is not an ultimate demonstration.

    Comments on current version:

    The authors have now addressed my concerns.

  5. eLife

    Reviewer #2 (Public review):

    RNA guanine-rich G-quadruplexes (rG4s) are non-canonical higher order nucleic acid structures that can form under physiological conditions. Interestingly, cellular stress is positively correlated with rG4 induction.

    In this study, the authors examined human hippocampal postmortem tissue for the formation ofrG4s in aging and Alzheimer Disease (AD). rG4 immunostaining strongly increased in the hippocampus with both age and with AD severity. 21 cases were used in this study (age range 30-92).

    This immunostaining co-localized with hyper-phosphorylated tau immunostaining in neurons. The BG4 staining levels were also impacted by APOE status. rG4 structure was previously found to drive tau aggregation. Based on these observations, the authors propose a model of neurodegeneration in which chronic rG4 formation drives proteostasis collapse.

    This model is interesting, and would explain different observations (e.g., RNA is present in AD aggregates and rG4s can enhance protein oligomerization and tau aggregation).

    Main issue from the previous round of review:

    There is indeed a positive correlation between Braak stage severity and BG4 staining, but this correlation is relatively weak and borderline significant ((R = 0.52, p value = 0.028). This is probably the main limitation of this study, which should be clearly acknowledged (together with a reminder that "correlation is not causality"). Related to this, here is no clear justification to exclude the four individuals in Fig 1d (without them R increases to 0.78). Please remove this statement. On the other hand, the difference based on APOE status is more striking.

    Comments on current version:

    The authors have made laudable efforts to address the criticisms I made in my evaluation of the original manuscript.

  6. eLife

    Author response:

    Reviewer #1 (Evidence, reproducibility and clarity):

    This is an interesting manuscript where the authors systematically measure rG4 levels in brain samples at different ages of patients affected by AD. To the best of my knowledge this is the first time that BG4 staining is used in this context and the authors provide compelling evidence to show an association with BG4 staining and age or AD progression, which interestingly indicates that such RNA structure might play a role in regulating protein homeostasis as previously speculated. The methods used and the results reported seem robust and reproducible.

    In terms of the conclusions, however, I think that there are 2 main things that need addressing prior to publication:

    (1) Usually in BG4 staining experiments to ensure that the signal detected is genuinely due to rG4 an RNase treatment experiment is performed. This does not have to be extended to all the samples presented but having a couple of controls where the authors observe loss of staining upon RNase treatment will be key to ensure with confidence that rG4s are detected under the experimental conditions. This is particularly relevant for this brain tissue samples where BG4 staining has never been performed before.

    With what is now known about RNA rG4s and the recent reconciliation of the controversy on rG4 formation (Kharel, Nature Communications 2023), this experiment is no longer strictly required for demonstration of rG4 formation. Despite this change, we did attempt this experiment at the reviewer’s suggestion, but the controls were not successful, suggesting it may not be feasible with our fixing and staining conditions. That said, we agree that despite the G4 staining appearing primarily outside the nucleus, it would be helpful to have some direct indication of whether we were observing primarily RNA or DNA G4s, and so we performed an alternate experiment to determine this.

    In our previous submission, we had performed ribosomal RNA staining (Figure S7), and the staining patterns were similar to that of BG4, especially the punctate pattern near the nuclei. Therefore, we directly asked whether the BG4 was largely binding to rRNA and have now shown the resulting co-stain in Figure 3b. These results show that at least a large amount of the BG4 staining does arise from rG4s in ribosomes. At high magnification, we observe that the BG4 stains a subset of the ribosomes, consistent with previous observations of high rG4 levels in ribosomes both in vitro and in cells (Mestre-Fos, 2019 J Mol Biol, Mestre-Fos 2019 PLoS One, Mestre-Fos 2020 J Biol Chem), but this had never been demonstrated in tissue. This experiment has therefore both answered the primary question of whether we are primarily observing rG4s, as well as provided more detailed information on the cellular sublocalization of rG4 formation, and provided the first evidence of rG4 formation on ribosomes in tissue.

    (2) The authors have an association between rG4-formation and age/disease progression. They also observe distribution dependency of this, which is great. However, this is still an association which does not allow the model to be supported. This is not something that can be fixed with an easy experiment and it is what it is, but my point is that the narrative of the manuscript should be more fair and reflect the fact that, although interesting, what the authors are observing is a simple correlation. They should still go ahead and propose a model for it, but they should be more balanced in the conclusion and do not imply that this evidence is sufficient to demonstrate the proposed model. It is absolutely fine to refer to the literature and comment on the fact that similar observations have been reported and this is in line with those, but still this is not an ultimate demonstration.

    We agree that these are correlative studies (of necessity when studying human tissue), but recent experiments have shown that rG4s affect the aggregation of Tau in vitro – and we have now better clarified this in the text itself. We have now also been more careful in drawing causative conclusions as shown in the revised text.

    Minor point:

    (3) rG4s themselves have been shown to generate aggregates in ALS models in the absence of any protein (Ragueso et al. Nat Commun 2023). I think this is also important in the light of my comment on the model, could well be that these rG4s are causing aggregates themselves that act as nucleation point for the proteins as reported in the paper I mentioned. Providing a broader and more unbiased view of the current literature on the topic would be fair, rather than focusing on reports more in line with the model proposed.

    We agree and have modified the discussion and added a broader context, including the Ragueso report described above.

    Reviewer #1 (Significance):

    This is a significant novel study, as per my comments above. I believe that such a study will be of impact in the G4 and neurodegenerative fields. Providing that the authors can address the criticisms above, I strongly believe that this manuscript would be of value to the scientific community. The main strength is the novelty of the study (never done before) the main weakness is the lack of the RNase control at the moment and the slightly over interpretation of the findings (see comments above).

    Reviewer #2 (Evidence, reproducibility and clarity):

    RNA guanine-rich G-quadruplexes (rG4s) are non-canonical higher order nucleic acid structures that can form under physiological conditions. Interestingly, cellular stress is positively correlated with rG4 induction. In this study, the authors examined human hippocampal postmortem tissue for the formation ofrG4s in aging and Alzheimer Disease (AD). rG4 immunostaining strongly increased in the hippocampus with both age and with AD severity. 21 cases were used in this study (age range 30-92). This immunostaining co-localized with hyper-phosphorylated tau immunostaining in neurons. The BG4 staining levels were also impacted by APOE status. rG4 structure was previously found to drive tau aggregation. Based on these observations, the authors propose a model of neurodegeneration in which chronic rG4 formation drives proteostasis collapse.

    This model is interesting, and would explain different observations (e.g., RNA is present in AD aggregates and rG4s can enhance protein oligomerization and tau aggregation).

    Main issue:

    There is indeed a positive correlation between Braak stage severity and BG4 staining, but this correlation is relatively weak and borderline significant ((R = 0.52, p value = 0.028). This is probably the main limitation of this study, which should be clearly acknowledged (together with a reminder that "correlation is not causality”.

    We believe that we had not explained this clearly enough in the text (based on the reviewer’s comment), as the correlation mentioned by the Reviewer was for the CA4 region only, and not the OML, which was substantially more correlated and statistically significant (Spearman R= 0.72, p = 0.00086). As a result, we believe this was a miscommunication that is rectified by the revised text:

    “In the OML, plotting BG4 percent area versus Braak stage demonstrated a strong correlation (Spearman R= 0.72) with highly significantly increased BG4 staining with higher Braak stages (p = 0.00086) (Fig. 2b).”

    Related to this, here is no clear justification to exclude the four individuals in Fig 1d (without them R increases to 0.78). Please remove this statement. On the other hand, the difference based on APOE status is more striking.

    We did not mean to imply that deleting these outliers was correct, but merely were demonstrating that they were in fact outliers. To avoid this misinterpretation, we have now deleted the sentence in the Figure 1d caption mentioning the outliers.

    Minor suggestions

    - "BG4 immunostaining was in many cases localized in the cytoplasm near the nucleus in a punctate pattern". Define "many"

    This is seen in nearly every cells and this is now altered in the text and is now identified as ribosomes containing rG4s using the rRNA antibody (Fig. 3b).

    - Specify that MABE917 corresponds to the specific single-chain version of the BG4 antibody

    Yes, this is correct, and this clarification has been added to the manuscript

    - Define PMI, Braak, CERAD (add a list of acronyms or insert these definitions in Fig 1b legend)

    These definitions have all been added when they first appear.

    - Fig 3: scale bar legend missing (50 micrometers?)

    This has been added, and the reviewer was correct that it was 50 micrometers.

    - Supplementary data Table 1: indicate target for all antibodies

    The target for each antibody has been added to supplementary Table 1.

    - Supplementary data Table 2: why give ages with different levels of precision? (e.g. 90.15 vs 63)

    We apologize for this oversight and have altered the ages to the same (whole years) in the figure.

    - Supplementary data Fig 1 X-axis legend: add "(nm)" after wavelength. Sequence can also be added in the legend. Why this one? Max/Min Wavelengths in the figure do not match indications in the experimental part. Not sure if that part is actually relevant for this study.

    The CD spectrum in Sup Fig 1 is the sequence that had previously been shown to aid in tau aggregation seeding, but had not been suspected by those authors to be a quadruplex. So we tested that here and showed it is a quadruplex, as described at the end of the introduction. We have added wording to the figure legend to clarify where its corresponding description in the main text can be found. We have also checked and corrected the wavelength and units.

    - Supplementary data Fig 7: Which ribosomal antibody was used?

    The details of this antibody have now been added to Supplementary Table 2 which lists all the antibodies used.

    Reviewer #2 (Significance):

    Provide a link between Alzheimer disease and RNA G-quadruplexes.

    Reviewer #3 (Evidence, reproducibility and clarity):

    This study investigated the formation of RNA G quadruplexes (rG4) in aging and AD in human hippocampal postmortem tissue. The rG4 immunostaining in the hippocampus increases strongly with age and with the severity of AD. Furthermore, rG4 is present in neurons with an accumulation of phosphorylated tau immunostaining.

    Major comments

    (1) The method used in this study is primarily immunostaining of BG4, and the results cannot be considered correct without additional data from more multifaceted analyses (biochemical analysis, RNA expression analysis, etc.).

    We respectfully disagree with the Reviewer’s assessment of the value of these experiments. The most relevant biochemical experiments at the cellular and molecular level showing the role of G4s in aggregation in general and Tau in particular have been done and are referenced in the text. The results here stand on their own and are highly novel and significant, as evaluated by both of the other reviewers. There has been no previous work demonstrating the presence of rG4s in human brain – either in controls or in patients with AD. AD is a complex condition that only occurs spontaneously in the human brain and no other species; because of this complexity, novel aspects are best first studied in human brain tissue using the methods employed here.

    (2) Overall, the quality of the stained images is poor, and detailed quantitative analysis using further high quality data is essential to conclude the authors' conclusions.

    We have again looked at our images and they are not poor quality -they are confocal images taken at recommended resolution of the confocal microscope. It is possible the poor quality came from pdf compression by the manuscript submission portal, which is beyond our control as they were uploaded at high resolution. These data were quantified by scientists who were blinded to the diagnosis of each case. The level of description on the detailed quantification is higher than we have observed in similar studies. We therefore disagree with the reviewer’s conclusion.

    Reviewer #3 (Significance):

    Overall, this study is not a deeply analyzed study. In addition, the authors of this study need further understanding regarding G4.

    It is also unclear why the reviewer believes that we do not have sufficient understanding of G4s, and would request that the reviewer instead provides specific comments regarding what is lacking in terms of knowledge on G4s, as we respectfully disagree with this judgement of our knowledge-base (see other G4 papers from the Horowitz lab, Begeman, 2020, Litberg 2023, Son, 2023 referenced below).

    Litberg TJ, Sannapureddi RKR, Huang Z, Son A, Sathyamoorthy B, Horowitz S. Why are G-quadruplexes good at preventing protein aggregation? Jan;20(1):495-509. doi: 10.1080/15476286.2023.2228572. RNA Biol. (2023)

    Son A, Huizar Cabral V, Huang Z, Litberg TJ, Horowitz S. G-quadruplexes rescuing protein folding. May 16;120(20):e2216308120. doi: 10.1073/pnas.2216308120. Proc Natl Acad Sci U S A (2023)

    Guzman BB, Son A, Litberg TJ, Huang Z, Dominguez , Horowitz S. Emerging Roles for G-Quadruplexes in Proteostasis FEBS J.doi: 10.1111/febs.16608. (2022)

    Begeman A, Son A, Litberg TJ, Wroblewski TH, Gehring T, Huizar Cabral V, Bourne J, Xuan Z, Horowitz S. G-Quadruplexes Act as Sequence Dependent Protein Chaperones. EMBO Reports Sep 18;e49735. doi: 10.15252/embr.201949735. (2020)

  7. Version published to 10.1101/2023.10.02.560545v4 on bioRxiv
  8. Version published to 10.1101/2023.10.02.560545v3 on bioRxiv
  9. Review Commons

    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

    We thank the reviewers for their thorough review of our manuscript and believe it has been much improved based on their comments.

    A detailed response to each comment is itemized below.

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

    This is an interesting manuscript where the authors systematically measure rG4 levels in brain samples at different ages of patients affected by AD. To the best of my knowledge this is the first time that BG4 staining is used in this context and the authors provide compelling evidence to show an association with BG4 staining and age or AD progression, which interestingly indicates that such RNA structure might play a role in regulating protein homeostasis as previously speculated. The methods used and the results reported seem robust and reproducible.

    In terms of the conclusions, however, I think that there are 2 main things that need addressing prior to publication:

    1. Usually in BG4 staining experiments to ensure that the signal detected is genuinely due to rG4 an RNase treatment experiment is performed. This does not have to be extended to all the samples presented but having a couple of controls where the authors observe loss of staining upon RNase treatment will be key to ensure with confidence that rG4s are detected under the experimental conditions. This is particularly relevant for this brain tissue samples where BG4 staining has never been performed before.

    Response____: With what is now known about RNA rG4s and the recent reconciliation of the controversy on rG4 formation (Kharel, Nature Communications 2023), this experiment is no longer strictly required for demonstration of rG4 formation. Despite this change, we did attempt this experiment at the reviewer's suggestion, but the controls were not successful, suggesting it may not be feasible with our fixing and staining conditions. That said, we agree that despite the G4 staining appearing primarily outside the nucleus, it would be helpful to have some direct indication of whether we were observing primarily RNA or DNA G4s, and so we performed an alternate experiment to determine this.

    In our previous submission, we had performed ribosomal RNA staining (Figure S7), and the staining patterns were similar to that of BG4, especially the punctate pattern near the nuclei. Therefore, we directly asked whether the BG4 was largely binding to rRNA and have now shown the resulting co-stain in Figure 3b. These results show that at least a large amount of the BG4 staining does arise from rG4s in ribosomes. At high magnification, we observe that the BG4 stains a subset of the ribosomes, consistent with previous observations of high rG4 levels in ribosomes both in vitro and in cells (Mestre-Fos, 2019 J Mol Biol, Mestre-Fos 2019 PLoS One, Mestre-Fos 2020 J Biol Chem), but this had never been demonstrated in tissue. This experiment has therefore both answered the primary question of whether we are primarily observing rG4s, as well as provided more detailed information on the cellular sublocalization of rG4 formation, and provided the first evidence of rG4 formation on ribosomes in tissue.

    1. The authors have an association between rG4-formation and age/disease progression. They also observe distribution dependency of this, which is great. However, this is still an association which does not allow the model to be supported. This is not something that can be fixed with an easy experiment and it is what it is, but my point is that the narrative of the manuscript should be more fair and reflect the fact that, although interesting, what the authors are observing is a simple correlation. They should still go ahead and propose a model for it, but they should be more balanced in the conclusion and do not imply that this evidence is sufficient to demonstrate the proposed model. It is absolutely fine to refer to the literature and comment on the fact that similar observations have been reported and this is in line with those, but still this is not an ultimate demonstration.

    * *

    Response: ____We agree that these are correlative studies (of necessity when studying human tissue), but recent experiments have shown that rG4s affect the aggregation of Tau in vitro - and we have now better clarified this in the text itself. We have now also been more careful in drawing causative conclusions as shown in the revised text (see yellow highlighted portions of the text).

    Minor point:

    1. rG4s themselves have been shown to generate aggregates in ALS models in the absence of any protein (Ragueso et al. Nat Commun 2023). I think this is also important in the light of my comment on the model, could well be that these rG4s are causing aggregates themselves that act as nucleation point for the proteins as reported in the paper I mentioned. Providing a broader and more unbiased view of the current literature on the topic would be fair, rather than focusing on reports more in line with the model proposed.

    * *

    __Response: ____ We agree and have modified the discussion and added a broader context, including the Ragueso report described above. __

    __Reviewer #1 (Significance (Required)): __ This is a significant novel study, as per my comments above. I believe that such a study will be of impact in the G4 and neurodegenerative fields. Providing that the authors can address the criticisms above, I strongly believe that this manuscript would be of value to the scientific community. The main strength is the novelty of the study (never done before) the main weakness is the lack of the RNase control at the moment and the slightly over interpretation of the findings (see comments above).

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

    RNA guanine-rich G-quadruplexes (rG4s) are non-canonical higher order nucleic acid structures that can form under physiological conditions. Interestingly, cellular stress is positively correlated with rG4 induction. In this study, the authors examined human hippocampal postmortem tissue for the formation ofrG4s in aging and Alzheimer Disease (AD). rG4 immunostaining strongly increased in the hippocampus with both age and with AD severity. 21 cases were used in this study (age range 30-92). This immunostaining co-localized with hyper-phosphorylated tau immunostaining in neurons. The BG4 staining levels were also impacted by APOE status. rG4 structure was previously found to drive tau aggregation. Based on these observations, the authors propose a model of neurodegeneration in which chronic rG4 formation drives proteostasis collapse. This model is interesting, and would explain different observations (e.g., RNA is present in AD aggregates and rG4s can enhance protein oligomerization and tau aggregation).

    Main issue: There is indeed a positive correlation between Braak stage severity and BG4 staining, but this correlation is relatively weak and borderline significant ((R = 0.52, p value = 0.028). This is probably the main limitation of this study, which should be clearly acknowledged (together with a reminder that "correlation is not causality".

    __Response: ____ We believe that we had not explained this clearly enough in the text (based on the reviewer's comment), as the correlation mentioned by the Reviewer was for the CA4 region only, and not the OML, which was substantially more correlated and statistically significant (____Spearman R= 0.72, p = 0.00086). As a result, we believe this was a miscommunication that is rectified by the revised text: __

    "In the OML, plotting BG4 percent area versus Braak stage demonstrated a strong correlation (Spearman R= 0.72) with highly significantly increased BG4 staining with higher Braak stages (p = 0.00086) (Fig. 2b)."

    * *

    Related to this, here is no clear justification to exclude the four individuals in Fig 1d (without them R increases to 0.78). Please remove this statement. On the other hand, the difference based on APOE status is more striking.

    * *

    Response: We did not mean to imply that deleting these outliers was correct, but merely were demonstrating that they were in fact outliers. To avoid this misinterpretation, we have now deleted the sentence in the Figure 1d caption mentioning the outliers.

    Minor suggestions

    • "BG4 immunostaining was in many cases localized in the cytoplasm near the nucleus in a punctate pattern". Define "many"

    Response: This is seen in nearly every cells and this is now altered in the text and is now identified as ribosomes containing rG4s using the rRNA antibody (Fig. 3b).

    • Specify that MABE917 corresponds to the specific single-chain version of the BG4 antibody

    __Response:____ Yes, this is correct, and this clarification has been added to the manuscript __

    • Define PMI, Braak, CERAD (add a list of acronyms or insert these definitions in Fig 1b legend)

    Response: ____These definitions have all been added when they first appear.

    • Fig 3: scale bar legend missing (50 micrometers?)

    Response:____ This has been added, and the reviewer was correct that it was 50 micrometers.

    • Supplementary data Table 1: indicate target for all antibodies

    Response: ____The target for each antibody has been added to supplementary Table 1.

    • Supplementary data Table 2: why give ages with different levels of precision? (e.g. 90.15 vs 63)

    Response:____ We apologize for this oversight and have altered the ages to the same (whole years) in the figure.

    • Supplementary data Fig 1 X-axis legend: add "(nm)" after wavelength. Sequence can also be added in the legend. Why this one? Max/Min Wavelengths in the figure do not match indications in the experimental part. Not sure if that part is actually relevant for this study.

    Response: The CD spectrum in Sup Fig 1 is the sequence that had previously been shown to aid in tau aggregation seeding, but had not been suspected by those authors to be a quadruplex. So we tested that here and showed it is a quadruplex, as described at the end of the introduction. We have added wording to the figure legend to clarify where its corresponding description in the main text can be found. We have also checked and corrected the wavelength and units.

    • Supplementary data Fig 7: Which ribosomal antibody was used?

    Response: The details of this antibody have now been added to Supplementary Table 2 which lists all the antibodies used.

    Reviewer #2 (Significance (Required)):

    Provide a link between Alzheimer disease and RNA G-quadruplexes.

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

    This study investigated the formation of RNA G quadruplexes (rG4) in aging and AD in human hippocampal postmortem tissue. The rG4 immunostaining in the hippocampus increases strongly with age and with the severity of AD. Furthermore, rG4 is present in neurons with an accumulation of phosphorylated tau immunostaining.

    Major comments 1.The method used in this study is primarily immunostaining of BG4, and the results cannot be considered correct without additional data from more multifaceted analyses (biochemical analysis, RNA expression analysis, etc.).

    __Response: ____We respectfully disagree with the Reviewer's assessment of the value of these experiments. The most relevant biochemical experiments at the cellular and molecular level showing the role of G4s in aggregation in general and Tau in particular have been done and are referenced in the text. The results here stand on their own and are highly novel and significant, as evaluated by both of the other reviewers. There has been no previous work demonstrating the presence of rG4s in human brain - either in controls or in patients with AD. AD is a complex condition that only occurs spontaneously in the human brain and no other species; because of this complexity, novel aspects are best first studied in human brain tissue using the methods employed here. __

    Overall, the quality of the stained images is poor, and detailed quantitative analysis using further high quality data is essential to conclude the authors' conclusions.

    Response:____ We have again looked at our images and they are not poor quality -they are confocal images taken at recommended resolution of the confocal microscope. It is possible the poor quality came from pdf compression by the manuscript submission portal, which is beyond our control as they were uploaded at high resolution. These data were quantified by scientists who were blinded to the diagnosis of each case.____ The level of description on the detailed quantification is higher than we have observed in similar studies. We therefore disagree with the reviewer's conclusion.

    Reviewer #3 (Significance (Required)):

    Overall, this study is not a deeply analyzed study. In addition, the authors of this study need further understanding regarding G4.

    __Response____: It is also unclear why the reviewer believes that we do not have sufficient understanding of G4s, and would request that the reviewer instead provides specific comments regarding what is lacking in terms of knowledge on G4s, as we respectfully disagree with this judgement of our knowledge-base (see other G4 papers from the Horowitz lab, Begeman, 2020, Litberg 2023, Son, 2023 referenced below). __

    __ ____Litberg TJ, Sannapureddi RKR, Huang Z, Son A, Sathyamoorthy B, Horowitz S. Why are G-quadruplexes good at preventing protein aggregation? Jan;20(1):495-509. doi: 10.1080/15476286.2023.2228572. RNA Biol. (2023)​__

    __ ____Son A*, Huizar Cabral V*, Huang Z, Litberg TJ, Horowitz S. G-quadruplexes rescuing protein folding. May 16;120(20):e2216308120. doi: 10.1073/pnas.2216308120. Proc Natl Acad Sci U S A (2023)__

    ​____Guzman BB*, Son A*, Litberg TJ*, Huang Z*, Dominguez ‡, Horowitz S. Emerging Roles for G-Quadruplexes in Proteostasis FEBS J​.doi: 10.1111/febs.16608. (2022)

    __ ____Begeman A*, Son A*, Litberg TJ, Wroblewski TH, Gehring T, Huizar Cabral V, Bourne J, Xuan Z, Horowitz S‡. G-Quadruplexes Act as Sequence Dependent Protein Chaperones. EMBO Reports Sep 18;e49735. doi: 10.15252/embr.201949735. (2020)__

  10. 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 #3

    Evidence, reproducibility and clarity

    This study investigated the formation of RNA G quadruplexes (rG4) in aging and AD in human hippocampal postmortem tissue. The rG4 immunostaining in the hippocampus increases strongly with age and with the severity of AD. Furthermore, rG4 is present in neurons with an accumulation of phosphorylated tau immunostaining.

    Major comments

    1.The method used in this study is primarily immunostaining of BG4, and the results cannot be considered correct without additional data from more multifaceted analyses (biochemical analysis, RNA expression analysis, etc.).

    1. Overall, the quality of the stained images is poor, and detailed quantitative analysis using further high quality data is essential to conclude the authors' conclusions.

    Significance

    Overall, this study is not a deeply analyzed study. In addition, the authors of this study need further understanding regarding G4.

  11. 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

    RNA guanine-rich G-quadruplexes (rG4s) are non-canonical higher order nucleic acid structures that can form under physiological conditions. Interestingly, cellular stress is positively correlated with rG4 induction. In this study, the authors examined human hippocampal postmortem tissue for the formation ofrG4s in aging and Alzheimer Disease (AD). rG4 immunostaining strongly increased in the hippocampus with both age and with AD severity. 21 cases were used in this study (age range 30-92).

    This immunostaining co-localized with hyper-phosphorylated tau immunostaining in neurons. The BG4 staining levels were also impacted by APOE status. rG4 structure was previously found to drive tau aggregation. Based on these observations, the authors propose a model of neurodegeneration in which chronic rG4 formation drives proteostasis collapse. This model is interesting, and would explain different observations (e.g., RNA is present in AD aggregates and rG4s can enhance protein oligomerization and tau aggregation).

    Main issue

    There is indeed a positive correlation between Braak stage severity and BG4 staining, but this correlation is relatively weak and borderline significant ((R = 0.52, p value = 0.028). This is probably the main limitation of this study, which should be clearly acknowledged (together with a reminder that "correlation is not causality"). Related to this, here is no clear justification to exclude the four individuals in Fig 1d (without them R increases to 0.78). Please remove this statement. On the other hand, the difference based on APOE status is more striking.

    Minor suggestions

    • "BG4 immunostaining was in many cases localized in the cytoplasm near the nucleus in a punctate pattern". Define "many"
    • Specify that MABE917 corresponds to the specific single-chain version of the BG4 antibody
    • Define PMI, Braak, CERAD (add a list of acronyms or insert these definitions in Fig 1b legend)
    • Fig 3: scale bar legend missing (50 micrometers?)
    • Supplementary data Table 1: indicate target for all antibodies
    • Supplementary data Table 2: why give ages with different levels of precision? (e.g. 90.15 vs 63)
    • Supplementary data Fig 1 X-axis legend: add "(nm)" after wavelength. Sequence can also be added in the legend. Why this one? Max/Min Wavelengths in the figure do not match indications in the experimental part. Not sure if that part is actually relevant for this study.
    • Supplementary data Fig 7: Which ribosomal antibody was used?

    Significance

    Provide a link between Alzheimer disease and RNA G-quadruplexes.

  12. 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 #1

    Evidence, reproducibility and clarity

    This is an interesting manuscript where the authors systematically measure rG4 levels in brain samples at different ages of patients affected by AD. To the best of my knowledge this is the first time that BG4 staining is used in this context and the authors provide compelling evidence to show an association with BG4 staining and age or AD progression, which interestingly indicates that such RNA structure might play a role in regulating protein homeostasis as previously speculated. The methods used and the results reported seems robust and reproducible. In terms of the conclusions,. however, I think that there are 2 main things that need addressing prior publication:

    1. Usually in BG4 staining experiments to ensure that the signal detected is genuinely due to rG4 an RNase treatment experiment is performed. This does not have to be extended to all the samples presented but having a couple of controls where the authors observe loss of staining upon RNase treatment will be key to ensure with confidence that rG4s are detected under the experimental conditions. This is particularly relevant for this brain tissue samples where BG4 staining has never been performed before.
    2. The authors have an association between rG4-formation and age/disease progression. They also observe distribution dependency of this, which is great. However, this is still an association which does not allow the model to be supported. This is not something that can be fixed with an easy experiment and it is what it is, but my point is that the narrative of the manuscript should be more fair and reflect the fact that, although interesting, what the authors are observing is a simple correlation. They should still go ahead and propose a model for it, but they should be more balanced in the conclusion and do not imply that this evidence is sufficient to demonstrate the proposed model. It is absolutely fine to refer to the literature and comment on the fact that similar observation have been reported and this is in line with those, but still this is not an ultimate demonstration.

    Minor point:

    1. rG4s themselves have been shown to generate aggregates in ALS models in the absence of any protein (Ragueseo et al. Nat Commun 2023). I think this is also important in the light of my comment on the model, could well be that these rG4s are causing aggregates themselves that act as nucleation point for the proteins as reported in the paper I mentioned. Providing a broader and more unbiased view of the current literature on the topic would be fair, rather than focusing on reports more in line with the model proposed.

    Significance

    This is a significant novel study, as per my comments above. I believe that such a study will be of impact in the G4 and neurodegenerative fields. Providing that the authors can address the criticisms above, I strongly believe that this manuscript would be of value to the scientific community. The main strength is the novelty of the study (never done before) the main weakness is the lack of the RNase control at the moment and the slightly over interpretation of the findings (see comments above).

  13. Version published to 10.1101/2023.10.02.560545v2 on bioRxiv
  14. Version published to 10.1101/2023.10.02.560545v1 on bioRxiv