Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

  1. Daniel V Oliveira
  2. Kirsten G Coupland
  3. Wenchao Shao
  4. Shaobo Jin
  5. Francesca Del Gaudio
  6. Sailan Wang
  7. Rhys Fox
  8. Julie W Rutten
  9. Johan Sandin
  10. Henrik Zetterberg
  11. Johan Lundkvist
  12. Saskia AJ Lesnik Oberstein
  13. Urban Lendahl
  14. Helena Karlström

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Abstract

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  1. Version published to 10.15252/emmm.202216556
  2. Review Commons

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

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    Reply to the reviewers

    1. General Statements [optional]

    We thank the reviewers for fair and constructive comments, and for good suggestions for how to further improve the manuscript. We also appreciate the comments on novelty and potential importance for future clinical approaches for CADASIL. Below we provide our point-by-point responses to the reviewers’ comments. We start with a description of planned revisions, followed by describing changes already carried out in the transferred manuscript.

    2. Description of the planned revisions

    We plan to experimentally address the following points raised by the reviewers.

    First, we will address the ASMA staining in the arterioles by isolating brain microvessels from the TgN3R182C150 mouse and WT mouse and strain for ASMA and perlecan/PDGFRb (stain pericytes and VSMC) to in more detail visualize that ASMA is only expressed in the arterioles and not in the pericytes in the capillaries (reviewer 1, point 4).

    Second, we will assess the safety and toxicity of the active immunotherapy (reviewer 2, point 4). Specifically, we plan to analyse the structure and morphology of the kidneys from sham and vaccinated mice for renal damage by histology, H/E staining as well as creatinine levels in the serum. If the histology shows signs of damage (such as necrosis, apoptosis or granules in the tubules), we will stain the tissue with KIM1 for acute renal damage and caspase 3 for apoptosis. Moreover, we will analyse the inflammation C-Reactive Protein (CRP) marker in the serum by western blot analysis and/or ELISA and finally Neurofilament Light chain protein in the serum by SIMOA analysis to monitor if the vaccination is causing any neurodegeneration.

    Third, we will improve the image quality for Figure 5A (reviewer 1, point 5).

    Reviewer #1 (Evidence, reproducibility and clarity):

    Summary:

    This study have used immunogenic aggregates formed by recombinant Notch3 fragments EGF 1-5 that contain CADASIL NOTCH3 R133C and wild type NOTCH3 to inoculate a CADASIL mouse model TgN3R182C as an active immunisation therapy of CADASIL. The vaccinated mice showed a decreased deposition of NOTCH3 around brain capillaries, reduced blood NOTCH3 ECD and microglia activation, suggesting a potential novel therapy for future treatment of CADASIL. The lack of impact on retinal vasculature, body weight, and general behaviour of the treated mice indicate the safety of the therapy.

    We thank the reviewer for the positive comments and for finding it to be a potential novel and safe therapy for CADASIL.

    Major comments:

    The results are interesting and the manuscript was carefully written and presented. However, the reduction of NOTCH3 in blood samples and the deposition around capillaries were so modest, plus there was no significant change of NOTCH3 deposition in arterioles. This questions the real effectiveness of this immunisation therapy when translating to clinical patients.

    We thank the reviewers for the positive words on the manuscript and understand the concerns over the lack of effects in the arterioles. We still believe that observing NOTCH3 ECD reductions around capillaries and in the blood is a significant step forward and supports further endeavors in the area of active immunization, especially considering that the treatment is well tolerated with no overt NOTCH3-related toxicity effects. With this said, we of course realize that there would still be a long way towards clinical use, but all therapy developments have to start somewhere, and future studies (from us and others) can build on the data presented here. There are a number of possible explanations as to why there was not a significant reduction of NOTCH3 around the arterioles. It might be due to that the TgN3R182C150 model is a mild disease model which has a NOTCH3 accumulation onset around 6-7 months of age that starts in the capillaries and at later ages spreads to the arterioles, and that a different time axis or longer immunization period would also lead to a reduction of NOTCH3 in the arterioles, which can be addressed in future experiments. We however believe it was worthwhile to first explore an early immunization protocol, as it may pave the way to a treatment strategy that can be used early on the disease progress, maybe already at the pre-symptomatic phase.

    Minor comments:

    • Figure EV1 is redundant as Figure 2C has the same information.

    We agree the figures are redundant and have deleted Figure EV1 in our new version and renamed EV2 and EV3 to EV1 and EV2, respectively.

    • P5 last line "there was a prominent loss of monomeric NOTCH3 EGF1-5 when WT and R133C fragments were mixed as compared to incubating them separately (Fig 2C)". Why the aggregation is more obvious when mutant protein fragment mixed with wild type comparing mutant fragment alone?

    The reviewer raises an interesting point. While we do not strictly know why a mixing of mutated and wildtype fragments promote aggregation, our observations are in line with previous reports. Duering et al. Hum. Mol. Genet, 2011 used a single-particle approach SIFT (scanning for intensely fluorescent targets) to study the co-aggregation of WT and mutant NOTCH3 EGF1-5 proteins and reported that mixtures of WT and mutant proteins showed dual colour high-intensity signals, representing de novo aggregates. They also observed WT/R133C multimer formation over three days as compared to five days used in our study. It is also of note that most CADASIL cases are heterozygous for the NOTCH3 mutation, which suggests that wildtypeand mutant NOTCH3 proteins co-exist in vivo, which also have been confirmed by co-immunoprecipitation experiments from cell lysates by Opherk et al. Hum. Mol. Genet. 2009. In the light of these previous reports, we performed a variety of ratio between WT and R133C NOTCH3 EGF1-5 proteins and opted for a 1:1 mixture since it showed the highest amount of multimers in relation to monomers after incubation.

    • How was the dosage or concentration of aggregated protein (0.5 mg/ml) used for immunisation determined?

    The dosage concentration was calculated by measuring the purified protein with a BCA absorbance assay. The protein was then mixed with the adjuvant or PBS to the desired concentration. This is now more clearly described in the Materials and Methods.

    • P7 line 1-2, while using ASMA as a marker for arteries/arterioles, didn't the author see any expression of ASMA in pericytes (capillaries)?

    We have used a direct fluorescent conjugated ASMA antibody for this mouse strain and another mouse strain with same mutation but higher NOTCH3 expression (described in Rutten et al, Acta Neuropathol Commun, 2015) as well as for the wildtype mice. In all cases, we find that pericytes have no or very low ASMA expression. This is in line with a report from Ghezali et al. Ann Neurol 2018, in which they used perlecan as a marker for pericytes in the rat Notch3 R169C mutation model, since ASMA did not stain the capillaries. The lack of ASMA staining in pericytes is in agreement with our previous large-scale single cell RNA-seq study of the mouse brain vasculature, where we find very little ASMA (ACTA2) gene expression in pericytes, while it is abundant in the vascular smooth muscle cells (Vanlandewijck et al., Nature 2018).

    With this said, we will in the revised version include images on isolated brain microvessels from the N3TgR182C150 mouse model and WT that are stained with ASMA and perlecan/PDGFRb to clearly show that ASMA only stains the arterioles and not the capillaries, see Point 2 “Description of planned revisions” part above.

    • In Figure 5A, the NOTCH3 ECD signal looks like similar between Shame and Vaccinated, although the quantifications seem significant in Figure 5B. The author may discuss the robustness of the quantitation method used and therefore the conclusion (i.e., active immunization with NOTCH3 EGF1-5 WT/R133C aggregates specifically reduces the amount of NOTCH3 aggregates around cerebral capillaries.).

    We agree that Figure 5A is not clearly showing the differences between vaccinated and sham although highly significant. We have included more representative images and we have also improved the description of the quantification in the method part (page 18-19).

    • In Figure 8B, what does the "% of microglia area" mean and how was it calculated?

    The % of microglia area represents the percentage that the CD68 staining occupies per microglia area (stained by Iba1), meaning that the vaccinated mice contain more activated microglia compared to sham. This was calculated by measuring the area of the CD68 staining that colocalizes with the microglia. In the transferred version, we have improved the description of this in the text (page 18-19).

    • Figure 9A doesn't seem to support the conclusion "There was also a trend towards more NOTCH3 ECD deposits inside or in close vicinity to microglia in vaccinated TgN3R182C150 mice compared to control or sham-vaccinated TgN3R182C150 mice, although the difference did not reach statistical significance", as it is not so convincing that the signals of the NOTCH3 ECD staining co-localise with microglia in the vaccinated sample. Besides, what's the meaning or functional significance about "% of microglia area", "number/1000 um2 microglia", and "average size per microglia"?

    Thank you for the comments and we understand the concern, given the highly complex morphology and dynamics of microglia, which makes it difficult to describe potential differences in a straightforward manner. In the first graph in Figure 9B we show that the vaccinated mice had a higher percentage of microglia with NOTCH3 ECD deposits. However, these deposits did not occupy more area of the microglia (% microglia area, second graph), or showed a significant increase in the number of deposits per microglia area (number/1000 µm2 microglia, third graph). The average size of these deposits did not increase in a significant way per microglia area (average size per microglia µm2, fourth graph) in comparison to sham and non-vaccinated mice. We have rephrased the text to better indicate that the proportion of microglia with NOTCH3 deposits increased, while not the area or number of deposits within a microglial cell (Results, page 9).

    Reviewer #1 (Significance):

    CADASIL is the most common genetic small vessel disease that leads to cognitive defect and eventual vascular dementia. Current, there is no specific treatment available. Similar to a number of neurodegenerative conditions caused by protein accumulations like Alzheimer's disease, Parkinson disease and Huntington disease etc, NOTCH3 protein accumulation represents a key pathological change in CADASIL and therefore a drug target. The approach of active immunisation therapy described in this paper demonstrated a novel method for the treatment of this condition. Although the effectiveness of the therapy in the transgenic mouse model of CADASIL was yet highly impressive, this paper provides a proof-of-principle that the active immunisation is more or less functional and, most importantly, tolerable. One other advantage of this approach is that the immunisation therapy is not restricted to a specific NOTCH3 mutation. After further development this strategy could potentially benefit patients in the future.

    This paper may be interested by researchers working on diseases that are caused by specific protein accumulation or aggregations.

    My expertise is in the area of studying the molecular mechanisms of CADASIL and other genetic small vessel diseases.

    We thank the reviewer for these comments and that the manuscript provides proof-of-principle for a novel strategy towards a safe and tolerable therapy for future treatment of CADASIL patients. As such, we believe the study will be a useful platform and resource for future studies from us and others.

    Reviewer #2 (Evidence, reproducibility and clarity):

    The article submitted by Daniel V. Oliveira et al to Review Commons and titled "NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model" describes a novel active immunization therapy against the aggregated NOTCH3 mutant protein associated to CADASIL pathology. This strategy induces a significant reduction in NOTCH3 deposition around brain capillaries, increase of microglia activation and lowering of serum levels of NOTCH3, that demonstrates the potential clinical value of the therapy. In addition, the authors report that the therapy is safe and tolerable.
    The study is well written, very clear and the results are very promising.

    We thank the reviewer for positive comments on our work and pleased that you find the manuscript well and clearly written and containing promising data.

    Same minor comments need to be clarified before publication.

    1. In the introduction is indicated that ".....Aducanumab recently being approved for treatment of AD by the Food and Drug Association (Budd Haeberlein et al, 2017; Mintun et al, 2021; Tolar et al, 2020)." In the discussion, Page 10, "In the quest for AD therapies, emerging encouraging results suggest a clinically meaningful effect from immunotherapies aimed at clearing Ab-amyloid which recently also have led to the first FDA approval of a passive vaccine for treatment of AD (Demattos et al, 2012; Golde et al, 2009; Sevigny et al, 2016).
      References should be updated with recent clinical studies about the efficacy of Aducanumab.

    This is a valid point. We have updated the manuscript accordingly, and included a recent study on the effect of Aducanumab (Knopman et al. Alzheimers Dement. 2021).

    1. Schedule used to induce active immunization should be justified or referenced.

    We appreciate this comment and realize that we did not explain the rationale for the immunization scheme in the original version. We used a similar immunization schedule as used by Kontsekova et al. Alzheimer's Research & Therapy 2014 in their study of active immunization of a tau peptide in a transgenic rat Alzheimer disease model. This is now mentioned in the text on page 6. The slight modifications of the Kontsekova et al., scheme are also described in the Materials and Methods, page 15 (we made some slight modifications in order to be compatible to mouse and also immunized with an aggregated protein of 25kD instead of a 2 kD tau peptide).

    1. CADASIL is associated with high risk of stroke, dementia and migraine. Did the authors check the brain of TgN3R182C150 mice by MRI, for instance? This analysis could be interesting in order to increase the clinical impact and the translational value of the therapy.

    Thank you for pointing this out. We have previously reported MRI data on a more severe CADASIL mouse model (TgN3R182C350), which harbors the same transgene but expressed at a 2.3 times higher level compared to the mouse model used in this study (Rutten et al. Acta Neuropathol. Commun. 2015 and Gravesteijn et al. Transl. Stroke Res. 2020). We did not observe any consistent differences on MRI or behavior between the TgN3R182C350 model and controls at 20 months of age. From this data we do not expect to see any differences on MRI in our milder model at 7 months of age. We have however included a short description of the previous MRI data in the transferred version on page 12.

    1. It is indicated that the therapy did not affect to the vascular structure of the retina, suggesting that endogenous Notch signaling was not affected by vaccination and therefore the therapy is safe; however, additional tox analysis should be included to confirm the biocompatibility, such as blood pressure analysis, inflammation markers, renal and hepatic damage marker.

    Thank you for pointing out this important issue regarding the safety aspects for our active immunotherapy. We will address this by additional experiments of the renal damage by histology and inflammation markers and neuronal damage markers in the serum with western blotting/ELISA and SIMOA, which we have written in more detail in Point 2 “Description of planned revisions” part above.

    The suggestions for blood pressure and hepatic damage analysis are valid but would require that we restart novel series of experiments from scratch, as we currently do not have immunized mice up and running. Such an experiment would take several months, and with the subsequent analysis, most likely more than a year. We believe this time axis is too long, and we therefore respectfully suggest waiving these experiments. Also, we unfortunately did not collect livers from the first round of animals, so analysis of livers would have a similarly long time axis.

    Reviewer #2 (Significance):

    The study is well written, very clear and the results are very promising. Some references related with the effect of Aducanumab in AD should ne updated. Safety and Tox analysis of the therapy should be complemented with additional assays.

    We thank the reviewer for these positive comments. We have updated the manuscript with a new reference regarding the effect of Aducanumab. For the safety and tox analysis we will do additional experimental analysis which we have addressed above in “Description of planned revisions”.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    We deleted Figure EV1 in our new version and renamed EV2 and EV3 to EV1 and EV2, respectively. (Reviewer 1, point 1).

    We provide an improved description of how the dosage concentration was calculated by measuring the purified protein with a BCA absorbance assay (Reviewer 1, point 3) (Materials and Methods, page 15).

    We have improved the description of the quantification of the immunohistochemistry in the method part (Reviewer 1, point 5) (Materials and Methods, page 18-19).

    We have improved the description of microglia in the quantification in the method part (Reviewer 1, point 6 and 7) (Materials and Methods, page 18-19).

    We have rephrased the text to better indicate that the proportion of microglia with NOTCH3 deposits increased, while not the area or number of deposits within a microglial cell (Results, page 9).

    We have updated the manuscript accordingly, and included a recent study on the effect of Aducanumab (Knopman et al. Alzheimers Dement. 2021). (Reviewer 2, point 1).

    We have provided a rationale and a more detailed description of the immunization protocol. (Reviewer 2, point 2) (Materials and Methods, page 15).

    We have included a short description of the previous MRI data in the transferred version. (Reviewer 2, point 3), (Discussion, page 12)

    We have included the following new references (Vanlandewijck et al., Nature 2018, Knopman et al. Alzheimers Dement. 2021, Kontsekova et al. Alzheimer's Research & Therapy 2014, Gravesteijn et al. Transl. Stroke Res. 2020).

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    The suggestions for blood pressure and hepatic damage analysis are valid but would require that we restart novel series of experiments from scratch, as we currently do not have immunized mice up and running. Such an experiment would take several months, and with the subsequent analysis, most likely more than a year. We believe this time axis is too long, and we therefore respectfully suggest waiving these experiments. Also, we unfortunately did not collect livers from the first round of animals, so analysis of livers would have a similar time axis.

  3. 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

    The article submitted by Daniel V. Oliveira et al to Review Commons and titled "NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model" describes a novel active immunization therapy against the aggregated NOTCH3 mutant protein associated to CADASIL pathology. This strategy induces a significant reduction in NOTCH3 deposition around brain capillaries, increase of microglia activation and lowering of serum levels of NOTCH3, that demonstrates the potential clinical value of the therapy. In addition, the authors report that the therapy is safe and tolerable.
    The study is well written, very clear and the results are very promising.

    Same minor comments need to be clarified before publication.

    1. In the introduction is indicated that ".....Aducanumab recently being approved for treatment of AD by the Food and Drug Association (Budd Haeberlein et al, 2017; Mintun et al, 2021; Tolar et al, 2020)." In the discussion, Page 10, "In the quest for AD therapies, emerging encouraging results suggest a clinically meaningful effect from immunotherapies aimed at clearing Ab-amyloid which recently also have led to the first FDA approval of a passive vaccine for treatment of AD (Demattos et al, 2012; Golde et al, 2009; Sevigny et al, 2016).
      References should be updated with recent clinical studies about the efficacy of Aducanumab.
    2. Schedule used to induce active immunization should be justified or referenced.
    3. CADASIL is associated with high risk of stroke, dementia and migraine. Did the authors check the brain of TgN3R182C150 mice by MRI, for instance? This analysis could be interesting in order to increase the clinical impact and the translational value of the therapy.
    4. It is indicated that the therapy did not affect to the vascular structure of the retina, suggesting that endogenous Notch signaling was not affected by vaccination and therefore the therapy is safe; however, additional tox analysis should be included to confirm the biocompatibility, such as blood pressure analysis, inflammation markers, renal and hepatic damage marker, ....

    Significance

    The study is well written, very clear and the results are very promising. Some references related with the effect of Aducanumab in AD should ne updated. Safety and Tox analysis of the therapy should be complemented with additional assays.

  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 #1

    Evidence, reproducibility and clarity

    Summary:

    This study have used immunogenic aggregates formed by recombinant Notch3 fragments EGF 1-5 that contain CADASIL NOTCH3 R133C and wild type NOTCH3 to inoculate a CADASIL mouse model TgN3R182C as an active immunisation therapy of CADASIL. The vaccinated mice showed a decreased deposition of NOTCH3 around brain capillaries, reduced blood NOTCH3 ECD and microglia activation, suggesting a potential novel therapy for future treatment of CADASIL. The lack of impact on retinal vasculature, body weight, and general behaviour of the treated mice indicate the safety of the therapy.

    Major comments:

    The results are interesting and the manuscript was carefully written and presented. However, the reduction of NOTCH3 in blood samples and the deposition around capillaries were so modest, plus there was no significant change of NOTCH3 deposition in arterioles. This questions the real effectiveness of this immunisation therapy when translating to clinical patients.

    Minor comments:

    • Figure EV1 is redundant as Figure 2C has the same information.
    • P5 last line "there was a prominent loss of monomeric NOTCH3 EGF1-5 when WT and R133C fragments were mixed as compared to incubating them separately (Fig 2C)". Why the aggregation is more obvious when mutant protein fragment mixed with wild type comparing mutant fragment alone?
    • How was the dosage or concentration of aggregated protein (0.5 mg/ml) used for immunisation determined?
    • P7 line 1-2, while using ASMA as a marker for arteries/arterioles, didn't the author see any expression of ASMA in pericytes (capillaries)?
    • In Figure 5A, the NOTCH3 ECD signal looks like similar between Shame and Vaccinated, although the quantifications seem significant in Figure 5B. The author may discuss the robustness of the quantitation method used and therefore the conclusion (i.e., active immunization with NOTCH3 EGF1-5 WT/R133C aggregates specifically reduces the amount of NOTCH3 aggregates around cerebral capillaries.).
    • In Figure 8B, what does the "% of microglia area" mean and how was it calculated?
    • Figure 9A doesn't seem to support the conclusion "There was also a trend towards more NOTCH3 ECD deposits inside or in close vicinity to microglia in vaccinated TgN3R182C150 mice compared to control or sham-vaccinated TgN3R182C150 mice, although the difference did not reach statistical significance", as it is not so convincing that the signals of the NOTCH3 ECD staining co-localise with microglia in the vaccinated sample. Besides, what's the meaning or functional significance about "% of microglia area", "number/1000 um2 microglia", and "average size per microglia"?

    Significance

    CADASIL is the most common genetic small vessel disease that leads to cognitive defect and eventual vascular dementia. Current, there is no specific treatment available. Similar to a number of neurodegenerative conditions caused by protein accumulations like Alzheimer's disease, Parkinson disease and Huntington disease etc, NOTCH3 protein accumulation represents a key pathological change in CADASIL and therefore a drug target. The approach of active immunisation therapy described in this paper demonstrated a novel method for the treatment of this condition. Although the effectiveness of the therapy in the transgenic mouse model of CADASIL was yet highly impressive, this paper provides a proof-of-principle that the active immunisation is more or less functional and, most importantly, tolerable. One other advantage of this approach is that the immunisation therapy is not restricted to a specific NOTCH3 mutation. After further development this strategy could potentially benefit patients in the future.

    This paper may be interested by researchers working on diseases that are caused by specific protein accumulation or aggregations.

    My expertise is in the area of studying the molecular mechanisms of CADASIL and other genetic small vessel diseases.

  5. Version published to 10.1101/2022.07.11.499563 on bioRxiv