Attenuated CSF‐1R signalling drives cerebrovascular pathology

Abstract Cerebrovascular pathologies occur in up to 80% of cases of Alzheimer's disease; however, the underlying mechanisms that lead to perivascular pathology and accompanying blood–brain barrier (BBB) disruption are still not fully understood. We have identified previously unreported mutations in colony stimulating factor‐1 receptor (CSF‐1R) in an ultra‐rare autosomal dominant condition termed adult‐onset leucoencephalopathy with axonal spheroids and pigmented glia (ALSP). Cerebrovascular pathologies such as cerebral amyloid angiopathy (CAA) and perivascular p‐Tau were some of the primary neuropathological features of this condition. We have identified two families with different dominant acting alleles with variants located in the kinase region of the CSF‐1R gene, which confer a lack of kinase activity and signalling. The protein product of this gene acts as the receptor for 2 cognate ligands, namely colony stimulating factor‐1 (CSF‐1) and interleukin‐34 (IL‐34). Here, we show that depletion in CSF‐1R signalling induces BBB disruption and decreases the phagocytic capacity of peripheral macrophages but not microglia. CSF‐1R signalling appears to be critical for macrophage and microglial activation, and macrophage localisation to amyloid appears reduced following the induction of Csf‐1r heterozygosity in macrophages. Finally, we show that endothelial/microglial crosstalk and concomitant attenuation of CSF‐1R signalling causes re‐modelling of BBB‐associated tight junctions and suggest that regulating BBB integrity and systemic macrophage recruitment to the brain may be therapeutically relevant in ALSP and other Alzheimer’s‐like dementias.

Thank you for the submission of your manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you.We have now received feedback from two of the three reviewers who agreed to evaluate your manuscript.As the referee #1 will unfortunately not be able to return his/her report in a timely manner, we prefer to make a decision now in order to avoid further delay in the process.Should referee #1 provide a report, we will send it to you, with the understanding that we will not ask you extensive experiments in addition to the ones required in the enclosed reports from referee #2 and #3.
As you will see from the reports below, while the referee #3 is overall supportive of the study, referee #2 raises serious criticism that should be addressed in a major revision of the current manuscript.The main focus of the revision should be on 1) providing evidence that endothelial cells express CSF1R by performing direct immunohistochemistry on brain sections or as suggested by the referee #2 you should "de-emphasize the results from CSF1R interventions in endothelial cells" and 2) addressing the difference/similarity between the effects of mutant CSF1R expression versus CSF1R haploinsufficiency in vitro and in regard to the mutation phenotypes observed in CSF1R haploinsufficient mouse model.
Addressing the reviewers' concerns in full will be necessary for further considering the manuscript in our journal.Acceptance of the manuscript will entail a second round of review.Please note that EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript.For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.
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I look forward to receiving your revised manuscript .***** Reviewer's comment s ***** Referee #2 (Comments on Novelty/Model System for Author): In our perspective, the authors make two critical conceptual errors with the use of inadequate model systems and problematic technical quality (including missing quantifications for claimed effects).These issues largely question the strength of the evidence and conclusions drawn.1st error lies in use of endothelial cells to study the CSF1R receptor signalling.These cells do not express the CSF1 receptor in vivo as supported by numerous public resources of cerebral single cell sequencing.
2nd error is to explain the effects of CSF1R mutations with haploinsufficiency models.Said mutations in heterozygous context lead to degradation of nonmutated allele protein resulting in ablation of signalling.The authors instead of introducing these mutations, use heterozygous knockouts that express naive allele which is not targeted for proteolytic degradation.
For details please refer to the comments for the author's section.

Summary:
The manuscript by Delaney C. et al. presents previously undescribed cases of novel mutations in the CSF-1R receptor and attempts to explore the direct effect of these mutations on microglial and vascular endothelial cells with in-vitro and in-vivo models.The strongest novelty angle for the manuscript message comes from clinical evidence of cerebrovascular pathology in carriers of novel CSF-1R mutations.The mutation effects observed in Figure 1 are intriguing and the consequences on vascular phenotypes (presented in Fig 6) are indeed very interesting.The authors eloquently attempt to describe the mutation effects on cellular signalling by the introduction of respective constructs to HEK cells.However, further interpretation of the mechanistic part of the manuscript relies on two critical conceptual assumptions which are either: (major point 1) -unsupported by generally available evidence or (major point 2)inconsistently argued within the manuscript.
Major points: 1).Mechanistic analysis of CSF-1R effects in microglial and endothelial cells is based on the assumption that the CSF1R gene is expressed in both cell types.The assumption that CSF1R is present in endothelial cells in-vivo is critical to interpret results from numerous figures (Fig. 4 c,d; Fig. 5 c,d,f,g,h,i,j,k,l,m,n,o; Fig. 6 k,l,m,n).However, publicly available resources of single-cell RNA sequencing support Csfr1 expression only in microglia but not in endothelial cells.
The notion that CSF-1R protein is expressed in endothelium comes from authors observations and its detection by Western blotting in immortalised Bend3 cell line (deposited by Montesano R, et al. to ATCC in 1990) (Fig. 5 b) and primary mouse endothelial cells (Fig. 5 f,g).However, the purification protocol for primary cells used by the authors uses only centrifugation gradients rather than more selective endothelial cell-specific pull-downs or fluorescent reporter sorting.Even these advanced endothelial-specific purifications methods are prone to microglial contamination (see Vanlandewijck, M. et al.Nature 2018).
As a consequence, a large proportion of the manuscript results that refer to CSF1r role in endothelium lacks rational support in-vivo and risks to propagate serious misinterpretations by the readers of the journal.
The vascular phenotypes present in the CSF1R mutation carriers are distinct and clear but more likely due to interaction between vascular and parenchymal cells in the CNS rather than direct effects of Csfr1 within endothelium.Therefore, I would suggest that the authors: (1) acknowledge in the manuscript the lack of support for Csfr1 expression in endothelial cells in vivo, (2) reconsider the value of their observations and claims based on the Csfr1 presence in endothelium and (3) de-emphasize the results from Csfr1 interventions in endothelial cells e.g. by keeping selected data in the supplementary figures.
2).Interpretation of clinical phenotypes arising from CSF1R mutation with the tools that express one healthy allele relies on the assumption that the effects of haploinsufficiency (e.g.heterozygous Csf1r knockout mice) are the same as the presence of a mutated allele.However, the data presented by the authors does not support this claim and is inconsistently argued in the manuscript.
On one hand, the authors report that the presence of a mutated receptor leads to degradation of both mutated and native allele proteins.E.g. "co-expression of the native CSF-1R gene being attenuated in the presence of the mutant CSF-1R".This standpoint is supported by: -Reduced protein stability for both mutations as argued by authors (Fig. 2 a).
-Empirical evidence from co-expression of native and mutated receptors in HEK cells at 24 and 48 hours (Fig. 2b) -Rescued proteasomal degradation of the mutated receptor by MG132 (Fig. 2j) -The dominant (rather than recessive) phenotype of the CSF1R mutations in ALSP disease.These observations would lead to a conclusion that the presence of a mutated allele gives not only reduced but nullified effect on Csf1r signalling.
On the other hand, the authors use the haploinsufficiency models (Csf1r +/-mice) where the native receptor is still expressed (and should not be targeted for proteasomal degradation) to interpret mutation phenotypes.
This approach is used in Fig. 3  This inconsistency could be solved by introduction of described human CSFR1 mutations to mouse models, but such efforts would be time consuming.Authors could show comparative CSFR1 levels and its activity in both mutated and heterozygosity context at least within the in-vitro setting to clarify these interpretations.This issue should be clearly specified and put in context of data interpretation in the manuscript discussion.

Minor points
In addition, the manuscript often presents data in a (1) subjective, (2) non-quantified and (3) nontransparent way.1).Claims of effects are made without points of reference or controls: e.g.Pathology phenotypes in Fig. 1 c,d,e,f,g,h,i,j.Fig. 6 a,b,c,d,e,f,g,h,i,j. should be shown in reference to tissues without neurodegeneration with similar post-mortem collection interval and age-matched individuals.These figures have potentially highest informative value within the manuscript.Addressing this issue is critical and would allow clearer interpretation.
2).Claims of effects from in-vitro experiments are made without quantifications or statistical analysis: e.g.Fig. 2 d,e,f,g.;Fig. 3 c; Fig. 5 b,c,f,g; Fig. 6 k.Presenting a technical (e.g.reloading of the same lysate e.g. per patient when increasing the nnumber is not available) or, better yet, biological reproduction (repeated in vitro experiment) data would allow for more supported claims.Quantification of data would be necessary to justify the claims of "increased" or "decreased" effects.
3).Whenever the quantifications are indeed present, the figures often conceal the n number and data distribution by using column graphs with standard deviation: e.g.Fig. 3 e,f,g,h,i; Fig. 5 d.This figure format may be justified when presenting data from a standardised experiment like RT-PCR where the n numbers are the same throughout the manuscript, yet even there the n numbers are not mentioned in the figure panels or legends.

Referee #3 (Remarks for Author):
Study by Delaney describes the role of mutations in colony stimulating factor-1 receptor (CSF-1R) in cerebral vascular pathology in the context of amyloid pathology and Alzheimer's disease.The paper is very well structured and beautifully designed.The identification of the families with ALSP and characterization of their vascular pathology are the real strengths of this study.The results from cell cultures and animal experimentations are very convincing.The findings are novel and very likely to generate a great interest in the field.Minor: It would be nice if the results from Figure 2 could be confirmed, at least in part, in a cell type that is relevant to cerebral vasculature or amyloid pathology.

Editor's Comments
The main focus of the revision should be on 1) providing evidence that endothelial cells express CSF1R by performing direct immunohistochemistry on brain sections or as suggested by the referee #2 you should "de-emphasize the results from CSF1R interventions in endothelial cells" and 2) addressing the difference/similarity between the effects of mutant CSF1R expression versus CSF1R haploinsufficiency in vitro and in regard to the mutation phenotypes observed in CSF1R haploinsufficient mouse model.
As suggested by the editor and Reviewer 2, we have de-emphasized the results from CSF1R in endothelial cells and we concur that the expression of CSF1R in brain endotheial cells is still yet to be fully resolved.Additionally, we have expanded our analysis of CSF1R expression/haploinsufficiency in vitro with quantitative data and believe that our manuscript has been vastly improved.We have included some data in our response related to CSF1R expression in brain endothelial cells simply to highlight that we will continue to explore whether or not CSF1R is actually expressed in these cells.

Referee #2 (Comments on Novelty/Model System for Author): In our perspective, the authors make two critical conceptual errors with the use of inadequate model systems and problematic technical quality (including missing quantifications for claimed effects). These issues largely question the strength of the evidence and conclusions drawn. 1st error lies in use of endothelial cells to study the CSF1R receptor signalling. These cells do not express the CSF1 receptor in vivo as supported by numerous public resources of cerebral single cell sequencing. 2nd error is to explain the effects of CSF1R mutations with haploinsufficiency models. Said mutations in heterozygous context lead to degradation of nonmutated allele protein resulting in ablation of signalling. The authors instead of introducing these mutations, use heterozygous knockouts that express naive allele which is not targeted for proteolytic degradation.
For details please refer to the comments for the author's section.

Major points: 1). Mechanistic analysis of CSF-1R effects in microglial and endothelial cells is based on
the assumption that the CSF1R gene is expressed in both cell types.The assumption that CSF1R is present in endothelial cells in-vivo is critical to interpret results from numerous figures (Fig. 4 c,d; Fig. 5 c,d,f,g,h,i,j,k,l,m,n,o; Fig. 6 k,l,m,n We thank Reviewer #2 for these comments and we fully acknowledge the need for further validation of CSF-1R expression within the mouse brain endothelium in vivo.In that regard and as suggested, we have moved data from Figure 6, PLX3397 treatment of endothelial cells and the effects of direct CSF-1R inhibition on endothelial tight junction expression, to Extended View Figure 7 to de-emphasise the direct effects of CSF-1R on cerebrolvascular endothelial TJ maintenance.We have also alluded to this in the introduction. While not part of the revised manuscript, we will continue to explore the expression pattern of CSF-1R in BBB associated endothelial cells.As alluded to by Reviewer#2, it has been previously reported in primary endothelial cells and mouse CNS endothelium ( 1 ), in addition to murine neurons ( 2 ), however we acknowledge the problems associated with the various protocols as outlined.
Added to this, we have directly enquired through personal communication with the Betsholtz lab regarding CSF-1R expression in their datasets.We have included below some scRNAseq data indicating a subset of Iba1 -endothelial cells (Figure 1) from microvascular samples that do express Csf1r in the mouse brain (Figure 2).Clearly however, microglia express huge amounts of CSF-1R when compared to EC's.Furthermore, we have also isolated mouse microvessels and performed direct IHC for CSF-1R (Figure 3).While we do show a degree of signal overlap between claudin-5 positive EC's and CSF1R, we are showing this data simply to suggest that future work will be needed to explore whether CSF-1R is definitively expressed or not in the cerebrovascular endothelium.This standpoint is supported by: -Reduced protein stability for both mutations as argued by authors (Fig. 2 a).
-Empirical evidence from co-expression of native and mutated receptors in HEK cells at 24 and 48 hours (Fig. 2b) -Rescued proteasomal degradation of the mutated receptor by MG132 (Fig. 2j) -The dominant (rather than recessive) phenotype of the CSF1R mutations in ALSP disease.These observations would lead to a conclusion that the presence of a mutated allele gives not only reduced but nullified effect on Csf1r signalling.
We fully agree with Reviewer #2.We are showing that the mutated allele is one involving loss of function.The data suggests that the variant CSF-1R isoforms found in ALSP are true loss of function, lacking signalling capacity (Figure 2, b -g) while additionally being actively targeted for degradation via the UPS (Figure 2, j).Furthermore, we demonstrate that this increased UPS activity has no impact on cellular stress or toxicity (Supplementary Figure 3).Taken together, this indicates that in the context of ALSP, individuals are functionally heterozygous for CSF-1R, therefore allowing us to utilise a CSF-1R heterozygous mouse model to further investigate the disease.
We demonstrate that MG132 can rescue variant CSF-1R expression, without restoring signalling capacity (supplementary figure 4).MG132 targeted degradation is specific to the variant isoform, indicating that native CSF-1R would not be degraded through this pathway as shown by protein levels being unaffected by MG-132 treatment (Figure 2, j).We similarly directly demonstrate that in the heterozygous state, reduced signalling capacity persists (Figure 2, d, e, f, g) supporting the dominant nature of ALSP as arising from haploinsufficiency, rather than a dominant-negative effect.Again, this supports our use of heterozygous mice.
On the other hand, the authors use the haploinsufficiency models (Csf1r +/-mice) where the native receptor is still expressed (and should not be targeted for proteasomal degradation) to interpret mutation phenotypes.This approach is used in Fig. 3 h,i; Fig. 4 a,b,c,d; Fig. 5 e-o

; These observations may reflect partially reduced receptor expression rather than mutation effects (lack of signalling).
We have established the ALSP context to be one of haploinsufficiency.There is no cell stress effect from the variant receptor being targeted for degradation.Additionally, CSF-1R signalling capacity is effectively in the heterozygous state, therefore a heterozygous mouse model is the ideal molecular model of CSF-1R bioactivity in ALSP.

This inconsistency could be solved by introduction of described human CSFR1 mutations to mouse models, but such efforts would be time consuming. Authors could show comparative CSFR1 levels and its activity in both mutated and heterozygosity context at least within the in-vitro setting to clarify these interpretations.
This issue should be clearly specified and put in context of data interpretation in the manuscript discussion.

Minor points
In addition, the manuscript often presents data in a (1) subjective, (2) non-quantified and (3) non-transparent way.1).Claims of effects are made without points of reference or controls: e.g.Pathology phenotypes in Fig. 1 c,d,e,f,g,h,i,j.Fig. 6 a,b,c,d,e,f,g,h,i,j

. should be shown in reference to tissues without neurodegeneration with similar post-mortem collection interval and age-matched individuals. These figures have potentially highest informative value within the manuscript. Addressing this issue is critical and would allow clearer interpretation.
The neuropathological features displayed in Figure 1 were detected during autopsy by our colleague and co-author Prof Michael Farrell and the processing of sections used pathognomonic markers for diagnostic pruposes.In that regard it is challenging to obtain FFPE non-diseased human cortical tissue, dissected and identically processed for a side-byside comparison.We fully concur however that a non-diseased comparision is important and we have now provided tight junction, CD68 and GFAP immunohistochemistry from agematched non-diseased, non-demented flash-frozen human tissues (See Figure EV6).The absence of cerebrovascular and neurodegenerative pathologies in the healthy brain is well established in the literature.The presence of the hallmark pathology of ALSP within the white matter itself is criteria for diagnosis, being a qualitative assessment rather than quantitative.Testing for the pathognomic markers of axonal spheroids, demyelination and amyloid is not routinely performed in tissue obtained during non-demented or cognitively healthy human autopsy.
2).Claims of effects from in-vitro experiments are made without quantifications or statistical analysis: e.g.Fig. 2 d,e,f,g.;Fig. 3 c; Fig. 5 b,c,f,g; Fig. 6 k.Presenting a technical (e.g.reloading of the same lysate e.g. per patient when increasing the nnumber is not available) or, better yet, biological reproduction (repeated in vitro experiment) data would allow for more supported claims.Quantification of data would be necessary to justify the claims of "increased" or "decreased" effects.3).Whenever the quantifications are indeed present, the figures often conceal the n number and data distribution by using column graphs with standard deviation: e.g.Fig. 3 e,f,g,h,i; Fig. 5 d.This figure format may be justified when presenting data from a standardised experiment like RT-PCR where the n numbers are the same throughout the manuscript, yet even there the n numbers are not mentioned in the figure panels or legends.
We acknowledge this oversight on our part and we now included fully quatified figures to represent our replicates, representation of data and statistical analyses in full.This is now reflected throughout all figures.Highest novelt y value wit hin the manuscript lies in the discovery of new CSFR1 mut at ions and mechanist ic descript ions of how they affect the downst ream signalling of this recept or.

Referee #3 (Remarks for
The technical qualit y of the pat hological assessment is graded at medium.The aut hors do provide hist ological and mechanist ic assesement s yet do not provide tissue cont rols as a reference to the observed disease-associat ed pat hology.
The medical impact is graded at medium.The aut hors do describe pat hological and mechanist ic consequences of the new mut at ions yet this is largely an observat ory st udy and they do not propose a concept ual or experiment al framework for treat ment st rat egies.
The employed model syst ems are more adequat e in the revised manuscript since the aut hors cont ext ualise the differences in CSFR1 expression bet ween microglia and endot helial cells.Moreover the aut hors convincingly argue for the haploinsuficciency rat her than dominant negat ive phenot ype of the mut at ions.Referee #2 (Remarks for Author): In the revised manuscript the authors adequately contextualize the difference of CSFR1 expression between the microglial and endothelial cells which addressed the main issue nr 1.
The authors also convincingly argue for the haploinsufficiency rather than dominant negative phenotype of the mutations which addressed the main issue nr 2.
The minor issue of adequate quantification analysis and data presentation is largely addressed by new densitometry graphs in Fig. 2 and individual datapoints are now visible on graphs.However the pathological assessments are not referenced to non diseased tissues due to unavailable agematched controls for Fig. 1.
Minor comments for the revised manuscript.1.a.How was the sample size chosen to ensure adequate power to detect a pre-specified effect size?
1.b.For animal studies, include a statement about sample size estimate even if no statistical methods were used.
2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis.Were the criteria preestablished?
3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g.randomization procedure)?If yes, please describe.
For animal studies, include a statement about randomization even if no randomization was used.

Yes
Randomization was used for any experiments using injected material such as amyloid-beta.Littermate controls were used for knockout studies and retroactively genotyped.
Image acquisition (for confocal experiments) and analysis were performed by a blinded investigator.
All animal experiments were conducted in a blinded fashion a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself.Every question should be answered.If the question is not relevant to your research, please write NA (non applicable).We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.
definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).

G-Dual use research of concern F-Data Accessibility
Tallaght/St James's Hospital ethics committee Informed consent was obtained from all subjetcs and the experiments conformed to the principles set out in the WMA Declaration of Helsinkin and Department of Health and Human Services Belmont Report.
Consent to publish was obtained The b.End3 and HEK293T cell lines were purchased from ATCC, the BV-2 cell line was provided by Dr. Marina Lynch, TCIN, Ireland.Primary brain endothelial cells, microglial cells were produced inlab as per methods.Cells were not tested for mycoplasma contimations by kit, however DAPI staining was used to check for smaller cytoplasmic nuclei.

D-Animal Models E-Human Subjects
h,i; Fig. 4 a,b,c,d; Fig. 5 e-o; These observations may reflect partially reduced receptor expression rather than mutation effects (lack of signalling).
Author): Summary: The manuscript by Delaney C. et al. presents previously undescribed cases of novel mutations in the CSF-1R receptor and attempts to explore the direct effect of these mutations on microglial and vascular endothelial cells with in-vitro and in-vivo models.The strongest novelty angle for the manuscript message comes from clinical evidence of cerebrovascular pathology in carriers of novel CSF-1R mutations.The mutation effects observed in Figure 1 are intriguing and the consequences on vascular phenotypes (presented in Fig 6) are indeed very interesting.The authors eloquently attempt to describe the mutation effects on cellular signalling by the introduction of respective constructs to HEK cells.However, further interpretation of the mechanistic part of the manuscript relies on two critical conceptual assumptions which are either: (major point 1) -unsupported by generally available evidence or (major point 2) -inconsistently argued within the manuscript.27th Oct 2020 1st Authors' Response to Reviewers ).However, publicly available resources of single-cell RNA sequencing support Csfr1 expression only in microglia but not in endothelial cells.Allen Atlas: https://celltypes.brain-map.org/rnaseq/mouse_ctx-hip_smart-seqMousebrain.org http://mousebrain.org/genesearch.htmlBetsholtzlab.org(cerebrovascular resource) http://betsholtzlab.org/VascularSingleCells/database.htmlThese resources are the current conceptual reference standard to understand gene expression in brains and would be hard to refute.The notion that CSF-1R protein is expressed in endothelium comes from authors observations and its detection by Western blotting in immortalised Bend3 cell line (deposited by Montesano R, et al. to ATCC in 1990) (Fig.5 b) and primary mouse endothelial cells (Fig.5 f,g).However, the purification protocol for primary cells used by the authors uses only centrifugation gradients rather than more selective endothelial cell-specific pull-downs or fluorescent reporter sorting.Even these advanced endothelial-specific purifications methods are prone to microglial contamination (see Vanlandewijck, M. et al.Nature 2018).As a consequence, a large proportion of the manuscript results that refer to CSF1r role in endothelium lacks rational support invivo and risks to propagate serious misinterpretations by the readers of the journal.The vascular phenotypes present in the CSF1R mutation carriers are distinct and clear but more likely due to interaction between vascular and parenchymal cells in the CNS rather than direct effects of Csfr1 within endothelium.Therefore, I would suggest that the authors: (1) acknowledge in the manuscript the lack of support for Csfr1 expression in endothelial cells in vivo, (2) reconsider the value of their observations and claims based on the Csfr1 presence in endothelium and (3) deemphasize the results from Csfr1 interventions in endothelial cells e.g. by keeping selected data in the supplementary figures.

Figure 2 :
Figure 2: scRNA reads demonstrating Csf1r expression in the Brain Endothelial Cell (BEC) populations of the scRNA dataset

2 ).
Interpretation of clinical phenotypes arising from CSF1R mutation with the tools that express one healthy allele relies on the assumption that the effects of haploinsufficiency (e.g.heterozygous Csf1r knockout mice) are the same as the presence of a mutated allele.However, the data presented by the authors does not support this claim and is inconsistently argued in the manuscript.On one hand, the authors report that the presence of a mutated receptor leads to degradation of both mutated and native allele proteins.E.g. "co-expression of the native CSF-1R gene being attenuated in the presence of the mutant CSF-1R".
Author): Minor: It would be nice if the results from Figure 2 could be confirmed, at least in part, in a cell type that is relevant to cerebral vasculature or amyloid pathology.the submission of your revised manuscript to EMBO Molecular Medicine.I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: Please implement all adjust ment s suggest ed by the referee #2.***** Reviewer's comment s ***** Referee #2 (Comment s on Novelt y/Model Syst em for Aut hor):

4
.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g.blinding of the investigator)?If yes please describe.4.b.For animal studies, include a statement about blinding even if no blinding was done 5.For every figure, are statistical tests justified as appropriate?Do the data meet the assumptions of the tests (e.g., normal distribution)?Describe any methods used to assess it.Is there an estimate of variation within each group of data?Is the variance similar between the groups that are being statistically compared?G* Power software was used for all sample size calculations prior to all experiments No animals were excluded form any experiments All animal experiments were conducted in a blinded fashion, with the analysis similarly performed by an experimenter blinded to the conditions.Manuscript Number: EMM-2020-12889YesThe omnibus K2 (D'Agostino-Pearson) test was used in GraphPad Prism 8.
such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; are tests one-sided or two-sided?are there adjustments for multiple comparisons?exact statistical test results, e.g., P values = x but not P values < x; definition of 'center values' as median or average; definition of error bars as s.d. or s.e.m.

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Source Data should be included to report the data underlying graphs.Please follow the guidelines set out in the author ship guidelines on Data Presentation.Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).G* Power software was used for all sample size calculations prior to all experiments graphs include clearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical replicates.if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range;

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