F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.138354.1 Brief Report Articles Validation and in vivo characterization of research antibodies for Moesin, CD44, Midkine, and sFRP-1.

[version 1; peer review: 2 approved with reservations]

 Doolen Suzanne Conceptualization Data Curation Formal Analysis Investigation Methodology Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-1843-7510 1 Ayoubi Riham Investigation Methodology Visualization Writing – Original Draft Preparation Writing – Review & Editing 2 Laflamme Carl Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Resources Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-5906-025X 2 Betarbet Ranjita Conceptualization Methodology Project Administration Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4792-8952 3 4 Zoeller Elizabeth Project Administration Writing – Review & Editing https://orcid.org/0000-0001-9588-3114 3 Williams Sean-Paul G. Investigation Methodology Project Administration Writing – Original Draft Preparation Writing – Review & Editing 1 Fu Haian Conceptualization Funding Acquisition Methodology Project Administration Resources Supervision Visualization Writing – Original Draft Preparation Writing – Review & Editing 3 5 6 Levey Allan I. Conceptualization Funding Acquisition Methodology Project Administration Resources Supervision Visualization Writing – Original Draft Preparation Writing – Review & Editing 7 8 Sukoff Rizzo S. J. Conceptualization Funding Acquisition Methodology Project Administration Resources Supervision Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-1460-8127 a 1 Aging Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, 15219, USA Neurology and Neurosurgery, McGill University, Montreal, Québec, H3A 2B4, Canada Department of Pharmacology, Emory University, Atlanta, Georgia, 30322, USA Center for Neurodegenerative Disease, Emory University, Atlanta, Georgia, 30322, USA Winship Cancer Institute, Emory University, Atlanta, Georgia, 30322, USA Chemical Biology Discovery Center, Emory University, Atlanta, Georgia, 30322, USA Goizueta Alzheimer's Disease Research Center, Emory University, Atlanta, Georgia, USA Neurology, Emory University, Atlanta, Georgia, 30322, USA rizzos@pitt.edu

No competing interests were disclosed.

 31 8 2023 2023 12 1070 31 7 2023 Copyright: © 2023 Doolen S et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer’s disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified.

Methods: We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as “sFRP-1” (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice.

Results: Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT.

Conclusions: These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins.

  Alzheimer’s disease Moesin sFRP-1 Midkine antibody characterization Western blot mice 5xFAD National Institutes of Aging RF1AG057443 Government of Canada OGI-210 National Institutes of Aging U54AG065187 This work was supported by the Emory-Sage-SGC TREAT-AD center established by the National Institutes of Aging (NIA) U54AG065187 grant and additional support by RF1AG057443 and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (OGI-210). RA is supported by a Mitacs postdoctoral followship. Introduction

Alzheimer’s Disease (AD) is a debilitating neurodegenerative disorder affecting an estimated 6.7 million Americans age 65 years and older with no clear understanding of the disease mechanism ( ‘2023 Alzheimer’s disease facts and figures’ 2023). Leading mechanistic hypotheses, such as the amyloid and tau hypotheses, have yet to generate fully efficacious therapies that can prevent or stop AD. As part of a national effort to develop therapeutics and biomarkers for AD, the Accelerated Medicines Partnership for Alzheimer’s Disease (AMP-AD) Consortium has been leveraging unbiased molecular profiling data at the genomic, transcriptomic, proteomic and metabolomic levels to further understanding of AD pathogenesis. Recent studies as part of AMP-AD have used a tandem mass tag mass spectrometry (TMT-MS) approach of postmortem brain tissues to reveal new AD-related protein co-expression modules ( Seyfried et al. 2017; Johnson et al. 2020). These protein modules were strongly correlated with higher neuropathological burden and worse cognitive outcomes ( Seyfried et al. 2017; Johnson et al. 2020). One such module referred to as Module 4 or “M4” included Moesin and CD44 and had the strongest correlation with higher neuropathological burden and worse cognitive outcomes ( Seyfried et al. 2017; Johnson et al. 2020). M4 was enriched in microglial and astrocytic proteins and contains proteins involved in response to inflammation. Another novel AD-associated module, M42 or the matrisome module, had a significant correlation with global pathology and is a source of promising therapeutic targets and biomarkers for AD. M42, which was not present in RNA networks, contained several proteins that have previously been identified by TMT-MS and shown to be correlated with amyloid-beta deposition in brain as well as with Midkine and sFRP-1 ( Higginbotham et al. 2020; Ping et al. 2018; Bai et al. 2020; Johnson et al. 2018, 2022).

Although the quality of research antibodies has been long been an issue ( Weller 2018), technical, economic and sociological challenges obstacles impede proper antibody characterization. The recent democratization of the CRISPR-Cas9 technology allows the generation of ideal isogenic controls for proper antibody characterization ( Laflamme et al. 2019, 2021). We have characterized commercial antibodies against Moesin ( Alshafie et al. 2023), Midkine ( Ayoubi et al. 2023a), sFRP-1 ( Ayoubi et al. 2023b). As commercial research antibodies become available and validated in commercially available human knock-out cell lines, it is essential to extend the characterization of these antibodies in murine tissues to enable their use in AD-mouse models.

The 5xFAD mouse is a well-characterized transgenic model that manifests Aβ plaque deposition as early as 4-6 months of age ( Jawhar et al. 2012; Oakley et al. 2006). Here we have characterized the expression of the TREAT-AD target proteins Moesin, CD44, Midkine and sFRP-1 in the 5xFAD mouse model in comparison to age-matched non-transgenic C57BL/6J controls. This allows not only for confirmation of the presence and/or changes in the protein level of target proteins but these antibodies also serve as tools for future in vivo target engagement studies as we evaluate the potential of novel therapeutic agents to modulate disease.

 Methods

In vivo studies were in line with NIH Guide for The Care and Use of Laboratory Animals ( Council 2011) and followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines ( Kilkenny et al. 2010; Doolen & Rizzo 2023b). Prior to study initiation (April 2023) all in vivo procedures were reviewed approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC). All efforts were made to ameliorate harm to the animals by adherence to the 3 Rs alternatives; replacement, reduction and refinement ( Tannenbaum and Bennett 2015).

Adult male and female 5xFAD (JAX MMRRC Stock #034840; B6.Cg-Tg (APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax) and C57BL/6J (JAX stock #000664) were used for these studies. These 5XFAD mice are congenic on the C57BL/6J substrain and were received directly from the Jackson laboratory which maintains a genetic stability and specific pathogen-free biosecurity program, and provides appropriate assurances and genotyping protocols ( www.jax.org). Subjects were 7-12 weeks upon arrival to the University of Pittsburgh animal facility and were group-housed within sex (up to 4 per cage) in a dedicated mouse housing room with a 12hr:12hr light:dark cycle (lights on at 7:00am). The University of Pittsburgh animal facility is a specific pathogen-free facility that is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). An Animal Welfare Assurance is on file with OPRR-NIH. Subjects were maintained in the dedicated mouse housing facility with ad libitum food and water until the intended age of the experiments which was pre-determined based on our previous published characterization of the 5XFAD mouse model ( Oblak et al. 2021). Specifically, 5XFAD mice and controls were aged to 6-9 months which is an age in which 5XFAD mice demonstrate significant AD relevant pathophysiology including amyloid plaque deposition in brain and neuroinflammation, relative to C57BL/6J controls ( Oblak et al. 2021) Tissue samples were collected during the light cycle. (All efforts were undertaken to ameliorate animal suffering including appropriate methods of anaesthesia for terminal tissue collection.

The total number of animals used for all experiments were n= 4 5XFAD and n=4 C57BL/6J. These studies were initially conducted with minimal sample sizes (n=3-4) in line with historical published data for 5XFAD mice showing robust phenotypes of AD-related pathologies ( Oblak et al. 2021) and in line with protocols from the MODEL-AD consortium ( ADKnowledgeportal.synapse.org).

Western blots were reproduced and confirmed from initial pilot data, under blinded conditions. The experimenter was not aware of the group allocation during the experiment and data analysis. A staff member who serves the role of a colony manager that was not involved directly with the experiment coded the samples as A and B. The staff member provided the unblind code after the data were analyzed by the experimenter and reviewed for quality control by the supervisor. No samples were excluded from this study.

Mouse brain collection

For terminal tissue collection, mice were anesthetized with isoflurane to the surgical plane of anesthesia, and brains were collected following decapitation. The brain was extracted and rinsed in ice-cold PBS, cerebellum was removed, and the cortex was bisected into left and right hemispheres then snap frozen and stored at -80 °C until use. Each hemibrain was weighed and then immersed in 1 ml/100 mg tissue homogenization buffer (THB; 2 mM Tris (pH 7.4), 250 mM sucrose 0.5 mM EDTA 0.5 mM EGTA) supplemented with 1X Pierce Protease Inhibitor (Thermo Scientific #A32953) and 1x Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich #P5726, St. Louis, MO). The tissue was homogenized for 20 sec on ice with a Benchmark D1000 hand-held homogenizer beginning at medium and increasing to high speed. Total protein concentration of the resulting homogenate was measured using a Bradford assay ( Bradford 1976). Briefly, 5 μl of homogenate diluted 10× in PBS was added to a 96-well plate. Pierce™ Coomassie Plus (Bradford) Assay Reagent (Life Technologies, Chicago, IL, USA; 250 μl) was added to each well and the plate was read at 595 nm using a SprectraMax i3x (Molecular devices) with SoftMax Pro V7.0.2. The protein concentration was calculated by comparing to a standard curve generated by adding varying known concentrations of Albumin Standard (0, 125, 250, 500, 750, 1000, 1500 and 2000 μl/ml; (Thermo Scientific #23210, Rockford, IL, USA) in duplicate to wells.

 Western blots

Homogenate from N=4 individual 5XFAD subjects and homogenate from N=4 individual C57BL/6J subjects, which served as WT controls, were included on each gel. Equal amounts of protein (25 μg) samples were prepared in 1x loading buffer (4x: 4 ml 100% glycerol, 2.4 ml 1M Tris/HCl (pH 6.8), 0.8 g SDS, 4 mg bromophenol blue, 3.1 ml H2O, 0.5 ml beta-mercaptoethanol) and incubated at 95 °C for 5 min. Samples were separated with SDS-polyacrylamide gel electrophoresis (4–15% Mini-PROTEAN ® TGX™ Precast Protein Gels, 10-well, 30 μl, Bio-rad #4561083) using 120 V for 60 min, then transferred onto a nitrocellulose membrane using a Trans-Blot SD Semi-Dry Electrophoretic transfer cell (Bio-rad #170-3940) at 25 V for 30 min. Non-specific binding was blocked using EveryBlot Blocking Buffer (Bio-rad #12010020) for 5 min at room temperature with gentle rocking. Primary antibodies were prepared in EveryBlot Blocking buffer at the dilutions indicated in Table 1. Blots were immersed in primary antibody solutions overnight at 4 °C with gentle rocking. Membranes were then washed with 1X TBST (3 × 5 min) and immersed in 1:1000 fluorescent secondary antibody (Moesin, CD44 and sFRP-1, StarBright blue 700 goat anti-rabbit IgG, Bio-rad #12004161; Midkine, IRDye 800CW Donkey anti-Goat IgG, Licor, Lincoln, NE, USA) and 1:2000 hFAB™ Rhodamine Anti-GAPDH Antibody (Bio-rad #12004167) for 1 h at room temperature. Membranes were then washed (2 × 5 min in 1X TBST; 2 × 5 min in 1X TBS) then scanned with a Bio-rad ChemiDoc MP Imaging System. Pixel intensity was quantified using ImageJ, the public domain NIH Image program (available at http://rsb.info.nih.gov/nih-image/). Statistical analyses and graphing were performed using GraphPad Prism Version 9.3.1 (San Diego, CA).

Summary of commercial antibodies use in western blot assays.
Target protein Product # Company Source Dilution used RRID (Antibody Registry) Clonality Clone # Conc. (μg/μL) Fold change in 5xFAD brain v C57BL/6J
Moesin ab52490 Abcam Rabbit 1:1000 AB_881245 recombinant-mono EP1863Y 0.19 2.8
CD44 ab189524 Abcam Rabbit 1:500 AB_2885107 recombinant-mono EPR18668 0.47 1.9
Midkine AF7769 R&D Systems Sheep 1:200 AB_291796 polyclonal - 1.0 17.8
sFRP-1 ab267466 Abcam Rabbit 1:1000 AB_2904616 recombinant-mono EPR23092-253 0.46 11.9
Results Selection of antibodies

We have characterized most commercially available antibodies against Moesin ( Alshafie et al. 2023), Midkine ( Ayoubi et al. 2023a), sFRP-1 ( Ayoubi et al. 2023b) and CD44 ( Ayoubi et al. 2021) using human WT and knockout cell lines for three research applications, including Western blot. For this study, we selected antibodies based on the following criteria: i) high antibody selectivity by WB based on our previous antibody characterization studies, ii) predicted reactivity with mouse based on the internal validation from the manufacturer, iii) band at the expected size by Western blot using mouse brain lysate ( Figure 2) and iv) antibody clonality as we prioritize renewable reagents such as mouse monoclonal or rabbit recombinant antibodies. We selected the recombinant anti-Moesin ab52490, the recombinant anti-CD44 ab189524 and the recombinant anti-sFRP-1 ab267466 antibodies as they met our criteria. However, none of the previously characterized Midkine antibodies was expected to react with mouse Midkine. By searching on antibody manufacturers’ website, we identified the polyclonal antibody AF7769 from Bio-techne generated to specifically detect mouse Midkine. We validated AF7769 as we have done previously ( Ayoubi et al. 2023a) ( Figure 1). AF7769 met our selection criteria and was further selected for the downstream experiments. The underlying data for this study has been uploaded to Zenodo ( Doolen & Rizzo 2023a).

Midkine antibody screening by Western Blot on culture media.

Lysates of HAP1 (WT and MDK KO) were prepared, and ~30 μg of protein was processed for Western blot with the Midkine antibody. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO media and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane.

 Protein expression is increased in mouse models of AD

We next used the selected antibodies on protein extracts from cortex of 9-month aged 5xFAD mouse brain homogenates and compared to age-matched C57BL/6J controls. Our immunoblotting results depicting their reactivity in mouse brain homogenate are shown in Figure 2. The antibody source information and the fold change in protein levels in 5xFAD are in Table 1. Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa and its expression was 2.8 times higher in 5xFAD compared to WT mouse brain ( Figure 2A). Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa and its expression was 1.9 times higher in 5xFAD compared to WT mouse brain ( Figure 2B). Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT mouse brain ( Figure 2C). Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted and its expression was 11.9 times greater in 5xFAD compared to WT mouse brain ( Figure 2D). Interestingly, both Midkine and sFRP-1 are secreted proteins and could still be identified in crude protein extracts from homogenates, prepared as described above.

Analysis of Moesin, CD44, Midkine and sFRP-1 protein level in 5XFAD AD mouse model.

Representative Western blots (left) and quantification (right). (A) Anti-Moesin; (Abcam #ab52490) reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa and 2.8 times greater expression in 5XFAD relative to WT. (B) Anti-CD44 (Abcam #ab189524) reacted with a band at the predicted size of 82 kDa and 1.9 times greater expression in 5XFAD relative to WT. (C) Anti-Midkine (R&D Systems #AF7769) reacted with a band ~16 kDa with 17.8 times greater expression in 5XFAD relative to WT. (D) Anti-sFRP-1 (Abcam #ab267466) reacted with a band at 35 kDa as predicted with 11.9 times greater expression in 5xFAD relative to WT. Brightness and contrast were adjusted in figure for visualization purposes and were applied equally to entire blot. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by t-test.

 Discussion

Validated reagents are important tools for studying underlying biological mechanisms that may contribute to disease and are a critical component of rigorously interrogating potential novel therapeutic targets for AD. Contrastingly, reagents such as antibodies that are not rigorously characterized could lead to spurious, irreproducible data and significant waste of resources. The present studies extend the characterization of antibodies that have been validated in knock-out cell lines to western blots to confirm their utility for in vivo studies. Given the evidence reporting that postmortem brain tissue from AD patients have elevated levels of Moesin, CD44, Midkine, and sFRP-1 relative to case controls, we selected an AD mouse model to evaluate these antibodies. As predicted, 5XFAD mice at an age at which significant amyloid plaque deposition is present ( Oblak et al. 2021, 2022), demonstrated increases in Moesin, CD44, Midkine, and sFRPR1, relative to age- and sex- matched non-transgenic controls. These data confirm the utility of these specific antibodies as validated reagents for western blotting of brain tissue, and also highlight the presence of these target proteins in a mouse model of amyloidosis. Taken together, the present studies provide an approach for confirming and extending the validation of antibodies from in vitro tissue preparations to in vivo assays and also provide insight into the utility of the 5XFAD mouse model for studying Moesin, CD44, Midkine, and sFRP-1.

 Data availability Underlying data

Zenodo: in vivo characterization of research antibodies for Moesin, CD44, Midkine, and sFRP-1. https://doi.org/10.5281/zenodo.8043971 ( Doolen & Rizzo 2023a).

 Reporting guidelines

Zenodo: ARRIVE checklist for ‘Validation and in vivo characterization of research antibodies for Moesin, CD44, Midkine, and sFRP-1.’ https://doi.org/10.5281/zenodo.8160965 ( Doolen & Rizzo 2023b).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgements

An earlier version of this article can be found on Open Lab Notebooks ( https://openlabnotebooks.org/in-vivo-characterization-of-antibodies-directed-against-treat-ad-target-proteins-in-mouse-model-of-ad-pathology/).

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Competing interests: No competing interests were disclosed.

 22 3 2024 Copyright: © 2024 Erickson M 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

This article evaluates specific antibodies for proteins upregulated in 5xFAD mouse brain, as found by Western blotting.  Results are straightforward and potentially useful to the reserach community.  Specific comments are below:

1. A human knockout cell line is used to validate antibody specificity.  As this study specifically focuses on mice, it would be important to understand whether specificity is also observed in mouse knockout cells.

2.  5xFAD mice can show sex-specific differences in AD-associated pathological changes.  Group allocation of males and females should be clarified.

3.  Western blotting done here is only semi-quantitative. The increases reflect changes in antibody signal rather than protein expression.

4.  The underlying data in Zenodo aren't appropriately labeled.  It's unclear what blot goes with what antibody or what the lanes are.  Also, the blots appear to have 12 lanes whereas only 8 are shown in the figure.  What are the other 4 lanes? 

5.  Showing the full blots and clearly indicating what is thought to be the specific band is important, and ideally should be included in the main body of the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Blood-brain barrier, Alzheimer's disease, aging, inflammation, neurotoxicology, pharmacology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Doolen Suzanne Aging, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Competing interests: No competing interests were disclosed.

 6 4 2024

This article evaluates specific antibodies for proteins upregulated in 5xFAD mouse brain, as found by Western blotting.  Results are straightforward and potentially useful to the research community.  Specific comments are below:

A human knockout cell line is used to validate antibody specificity.  As this study specifically focuses on mice, it would be important to understand whether specificity is also observed in mouse knockout cells.

 RESPONSE: We acknowledge the importance of ensuring specificity and cross-  reactivity, especially when working with antibodies across species. To address this concern, we conducted Western blot (WB) experiments using human knockout (KO)  cell lines and identified several specific antibodies in WB for all four proteins. The antibodies selected in this study were those predicted by the manufacturers to bind to  the corresponding murine proteins. We further confirmed specificity in WB by  comparing the band sizes between human and murine proteins. It's important to note that for all four proteins, the human and murine proteins are expected to exhibit very  similar molecular weights.

            2.  5xFAD mice can show sex-specific differences in AD-associated pathological changes. Group allocation of males and females should be clarified.

RESPONSE: We have now clarified that both male and female 5XFAD were used (n=2 per sex) for these studies in several places including methods and results.

Western blotting done here is only semi-quantitative. The increases reflect changes in antibody signal rather than protein expression.

RESPONSE: Thank you for this important point. We have added this to the discussion.

 

The underlying data in Zenodo aren't appropriately labeled.  It's unclear what blot goes with what antibody or what the lanes are.  Also, the blots appear to have 12 lanes whereas only 8 are shown in the figure.  What are the other 4 lanes?

RESPONSE: We have now updated the Zenodo notebook and added a file https://zenodo.org/search?q=parent.id%3A8043970&f=allversions%3Atrue&l=list&p=1&s=10&sort=version that shows the additional lanes were other animal models in our lab as we  were surveying other strains in addition to 5XFAD for these experiments and wanted   to maximize all lanes on the gel that were not other 5XFAD mice. Specifically, this was the SAA mouse model (JAX strain# 034711) which had not yet been fully characterized (Lanes 10-13) and Lanes 14 and 15 were n=2 19 month aged WT mice. We chose to only include 5XFAD in the publication given their extensive  characterization and their age-matched WT controls. Since the SAA mouse model did   not have full characterization data yet (e.g. amyloid and/or tau, etc.) and we only had  n=2 19-month aged WT mice and no 19-month aged 5XFAD to compare to we did not  include these in the publication.

 

Showing the full blots and clearly indicating what is thought to be the specific band is important, and ideally should be included in the main body of the manuscript.

RESPONSE: This has now been updated in the Zenodo notebook https://zenodo.org/search?q=parent.id%3A8043970&f=allversions%3Atrue&l=list&p=1&s=10&sort=version” that shows the specific bands which is in addition to what is shown in Figure 2.

10.5256/f1000research.151544.r236011 Reviewer response for version 1 Hopp Sarah C 1 Referee https://orcid.org/0000-0001-5228-0022 Fastenau Caitlyn 2 Co-referee University of Texas Health Science Center San Antonio, San Antonio, Texas, USA University of Texas Health Science Center San Antonio, SAN ANTONIO, TX, USA

Competing interests: No competing interests were disclosed.

 15 2 2024 Copyright: © 2024 Hopp SC and Fastenau C 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The work is clear and concise with consideration of the previous work that highlight the significance of validating the antibodies of interest.

It would be helpful to have more information on the functions of the four different proteins targeted by the different antibodies, and what the changes in expression in the 5XFAD brain lysate might mean biologically. 

The authors should be commended for their clear and reproducible methods with appropriate levels of detail, particularly the methods for selecting the antibodies. 

However, some details from the methods were missing:

Which sex of 5XFAD and WT mice were used for the Western blotting experiment in Figure 2? There are noted sex differences in pathology between sexes, so knowing which mice are males and females could be helpful.

The methods for Figure 1 are missing. Were the cell lysates and Western blots prepared in the same way as the brain lysate? What were the methods for the Ponceau? How was MDK KO confirmed in the HAP1 cells? Where were the HAP1 cells obtained from? How were they grown?

What t-test was run on the data in Figure 2?

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Microglia in Alzheimer's disease

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Doolen Suzanne Aging, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Competing interests: No competing interests were disclosed.

 6 4 2024

The work is clear and concise with consideration of the previous work that highlight the significance of validating the antibodies of interest.

It would be helpful to have more information on the functions of the four different proteins targeted by the different antibodies, and what the changes in expression in the 5XFAD brain lysate might mean biologically. 

The authors should be commended for their clear and reproducible methods with appropriate levels of detail, particularly the methods for selecting the antibodies. 

RESPONSE: Thank you to the reviewers for this acknowledgement.

However, some details from the methods were missing:

 RESPONSE: we appreciate the feedback and have added points for clarity as suggested.

Which sex of 5XFAD and WT mice were used for the Western blotting experiment in Figure 2? There are noted sex differences in pathology between sexes, so knowing which mice are males and females could be helpful.

   RESPONSE: As described in the second paragraph in the methods section both male and female 5XFAD mice were used for these studies. For clarity we have now revised the western blots methods to state: “Homogenate from N=2 male and N=2 female 9-month aged 5XFAD subjects and homogenate from N=4 age-matched individual male C57BL/6J subjects”. We have also added this detail in the results section. There were no differences at this age between male and female 5XFAD mice for these proteins and although only male WT were used as the age-match control (due to females being used as breeders), based on previous studies in 9 month aged WT mice there was no effect of sex in these proteins.

The methods for Figure 1 are missing. Were the cell lysates and Western blots prepared in the same way as the brain lysate? What were the methods for the Ponceau? How was MDK KO confirmed in the HAP1 cells? Where were the HAP1 cells obtained from? How were they grown?

 RESPONSE: The cell lysates and brain homogenates had different preparations for the western blots. The details for the cell culture westerns have now been added as follows:

Wild type (WT) and MDK knockout (KO) HAP1 cells were obtained from Horizon Discovery.  Cell lines were prepared by first washing 3× with PBS then starved for ~18 hrs. Culture media were collected and centrifuged for 10 min at 500 × g to eliminate cells and larger contaminants, then for 10 min at 4500 × g to eliminate smaller contaminants. Culture media were initially concentrated using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma cat. number UFC9010) by centrifuging at 4000 × g for 15 min. The resulting 500 μl of the concentrated media were centrifuged again at 4000 × g for 15 min using Amicon Ultra- 0.5 Centrifugal Filter Units (MilliporeSigma cat. number UFC5010) to 200 μl. ~30μg of protein was loaded and processed for Western blot using the Midkine antibody (AF7769). Western blot was performed using a large 10-20% gradient polyacrylamide gel and transferred on a nitrocellulose membrane. Proteins on the blot were visualized with Ponceau staining which is scanned to show together with the Western blot. The blot was blocked with 5% milk for 1 hr, and the Midkine antibody (AF7769) was incubated overnight at 4°C with 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated on the membrane at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three more washes with TBST. The membrane was incubated with ECL from Pierce (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41). The MDK KO cell line can be confirmed based on the results of the Western blot, as the Midkine signal appears in the lane loaded with the WT cells but disappears in lane loaded with the MDK KO cells. Predicted band size: ~16 kDa.

What t-test was run on the data in Figure 2?

RESPONSE: An unpaired t-test was performed using Graphpad Prism Software Version 9.3.1 for Windows. These details have now been added in the figure legend and the methods.