PONE-D-20-34809

The effects of CSTB duplication on APP/amyloid-β pathology and cathepsin activity in a mouse model

PLOS ONE

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? 

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: REVIEW FOR: PLoS One No. PONE-D-20-34809 The effects of CSTB duplication on APP/amyloid-β pathology and cathepsin activity in a mouse model

This is a timely paper on a study that addresses a very important issue in the field of Down syndrome as well as dementia science. It is vital to understand why the DS trisomy of chromosome 21 greatly increases the risk of developing Alzheimer's disease, particularly since gene(s) on chromosome 21 other than APP were shown to accelerate amyloid pathology when triplicated. To determine if three copies of CSTB, the endogenous inhibitor cystatin B of lysosomal cysteine cathepsins that is located on chromosome 21, influences Alzheimer-type pathogenesis a tgAPP mouse model of amyloid-β deposition was crossed with a mouse with heterozygous duplication of the Cstb locus. The Cstb duplication increased transcriptional and translational levels of CSTB in the brain, while having no detected effect on plaque deposition or Aβ levels. With the following additional analyses of immunoblots and enzyme assays, along with minor improvements to the text and references, this will be an important study to help understand the DS-AD connection.

1. A clear statement of the hypothesis being tested needs to be included in the abstract and/or introduction, and reflected by the title. This point is made since there exists several (~11) cysteine cathepsin enzymes, and the abstract states that duplication of the gene Cstb in a transgenic APP mouse model resulted in an increase in transcriptional and translational levels of Cstb but had no effect on Aβ or plaques… and the increased CSTB did not alter the activity of cathepsin B enzyme. Thus, it should be clearly stated the selectivity of the hypothesis, i.e. we determined if three copies of the mouse gene Cstb is sufficient to modulate Aβ accumulation and cathepsin B activity in a transgenic APP mouse model. Also, as Cstb duplication is noted as increasing transcription levels of Cstb in tg mice, please address why no statistical significance is noted in Figure 1A for tgAPP vs. dupCstb*tgAPP (also no such notation between those two groups in Fig. 1B).

2. With cathepsin B being the focus of the tested hypothesis, and that establishing a negative result requires more extensive analysis than to establish a positive change, additional background information and data are warranted. For instance, Mueller-Steiner et al. (ref. 22) indicate that message, protein, and activity levels of cathepsin B are increased by Aβ-related proteinopathy, but the putative compensatory response failed to occur in older tg mice which may be related to the aging risk factor for AD. Such should be mentioned in the context of whether CSTB plays a role in cathepsin B’s compensatory response and related regulation of the enzyme’s maturation process. And, since the ages of tg mice used in the Wu et al. study cannot make such comparison as in the Mueller-Steiner study, measures of cathepsin B maturation (active CatB or actCatB:proCatB ratio) can be assessed in immunoblot samples to enhance the study, and/or the specific assessment of lysosomal cathepsin B activity in isolated lysosomes via Percoll step as previously shown (Butler et al. 2011 PLoS ONE 6:e20501). Such analyses would provide improved coverage of whether Cstb regulation influences key features of cathepsin B maturation, trafficking, and localized activity that have been shown linked to AD-type pathology and may be critical for the DS-AD connection.

3. The anti-APP immunoblot in Figure 1D should be shown in full for purpose of assessing CTFs (or lack thereof if a positive control that the antibody recognizes them). Relevance on this matter from “Lysosomal Dysfunction in Down Syndrome is Mediated by APP-βCTF” by Nixon’s lab (Jiang et al. 2019 J Neurosci 39:5255) and others indicating APP-CTFs-derived endosomal dysfunction is early hallmark of both AD and DS (Pérez-González et al. 2019 Neurobiol Aging 84:26). Also, enhancing cathepsin B reduced APP-CTFs in AD mice (Hwang et al. 2019 Internatl J Mol Sci 20(18):4432). Thus up-regulated CSTB would be expected to increase APP-CTFs.

4. Discussion could include brief mention of the crosstalk between the lysosomal cathepsin network and the proteasome pathway as another potential component of the complex DS-AD relationship, as the two protein clearance systems may both contribute to the proteostatic stress in Down syndrome. See Aivazidis et al. 2017 Plos ONE 12(4):e0176307 and Farizatto et al. 2017 PLoS ONE 12(8): e0182895.

Lastly, minor but important reference coverage and corrections are warranted in the Introduction. Ref. 24 is noted as showing that knocking out Cstb lowered cathepsin B activity (typo?). This is a problem with logic for the Wu study and misrepresents Yang et al. that clearly says Cystatin B deletion in TgCRND8 elevating cathepsin activities, results of enzymatic activity for cathepsin B.

Several cathepsin B studies noted above would also add for a more appropriate coverage of the important topic. As for Ref. 21 regarding “cathepsin B inhibitors CA074Me or E64d lead to reduced Aβ levels”, please note that E64d is well-known for blocking the protease calpain more potently than cathepsin B, and that E64d is reported to produce complete neuroprotection in cathepsin B knockout mice (Hook et al. 2014). As for CA074me, it has been shown to inhibit cathepsin B without having inhibitory action on human b-secretase (Butler et al. 2011).

Reviewer #2: Cystatin B (CSTB) is an endogenous inhibitor of cathepsin proteases, the current study aimed to evaluate the effects of duplication of CSTB on amyloid pathology using dupCstBXtgAPP mice. The authors found CSTB duplication resulted in an increase in the expression of CSTB in the cortex from 3-month old mice, but had no effects on the enzymatic activity on cathepsin B, the endogenous substrate of CSTB. Furthermore, CSTB duplication had no effects on neither the soluble nor insoluble Aβ in 6-month old tgAPP mice. The study is relatively simple and clear; however, revisions are indicated.

Major comments:

1) The effects of CSTB duplication on APP processing were examined using 6-month old mice. It will be reasonable to evaluate the expression of CSTB and enzymatic activity of cathepsin B using the same sample, but not use the sample from 3-month old mice.

2) According to figure1 D and E, the expression of APP was obvious changed in dupCstB*tgAPP mice compared to tgAPP mice in figure. 1D, but no difference was shown in figure.1E.

3) If the inhibition of cathepsins has been saturated by endogenous CSTB in 6-month old tgAPP mice, duplication of CSTB was destined to have no effects on relative pathologies. Consideration and discussion on this point is lacking.

4) A positive control using human recombinant cathepsin B in the CatB activity assay is absolutely needed.

Again, the authors should make an effort for exploring the effects of Cystatin B on AD pathologies, not limited to Amyloid beta.

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Reviewer #1: No

Reviewer #2: No

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The effects of CSTB duplication on APP/amyloid-β pathology and cathepsin B activity in a mouse model

PONE-D-20-34809R1

Dear Dr. Wiseman,

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Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have addressed all my concerns regarding CatB-related importance for involvement in neuropathogenic insults.

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Reviewer #1: No