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Thank you for further correcting your paper. May I ask you, next time, to be very careful when labeling the figures. It was a pity that you made mistakes (fortunately spotted out by the reviewer). I wish you good luck for the successful continuation of your studies.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
Both reviewers were largely but not entirely satisfied with your revision. Please, pay attention to their remarks (one of the reviewer attached an annotated copy of your manuscript) and spend the necessary time and effort to further improve your submission. Also, try to have your text re-read and corrected for language preferably by a native-English speaking colleague knowledgeable in the field of study (if you have to use a commercial translation bureau, be sure they fully grasp the scientific content of your paper in order to avoid ambiguities and scientific misunderstandings or errors). Do not forget to submit a rebuttal where you indicate in details how and where in the paper you have taken the comments of the reviewers into consideration. I look forward to hearing from you in these matters.
[# PeerJ Staff Note: The review process has identified that the English language must be improved. PeerJ can provide language editing services - please contact us at [email protected] for pricing (be sure to provide your manuscript number and title) #]
1. Please confirm Line136-137 about portein lysate procedure. Does really need lysis buffer incubating with cell line for 30min at 4 degree? But not as usual, adding lysis buffer to the cells in the plate wells, a couple of minutes late, scraping them off on ice? Also, usually 12,000rpm, 30 min in centrifuge, should be done at 4 degree but not room temperature?
2. You don't really try many concentration, why emphasize so much " concentration depended"? Suggest don't use these words, 5uM and10uM work, that is it.
This is a original primary research with aims and scope of the journal, and research questions are well defined, relevant and meaningful. Methods described with detail information for replication, and investigation performed with a high technique and ethical standard as well.
All the data provided are relatively sound, and conclusions are well stated and supported by results.
It is much better for writing after edited, but always keep in mind this is a scientific report. Any conclusion you draw based on the data you got, don't say more than what your data could tell. More accurate, better.
The authors have done a great job revising the manuscript which reads much better now.
All questions have been addressed and improvements have been made accordingly.
I have a few minor remarks about the current version of the manuscript:
1. There is a typo in Abeta molecular mass indicated on western blot in Fig.5A. Abeta40 should be around 4.3kDa (not 32kDa) and Abeta42 around 4.5kDa (not 35kDa).
2. Raw data for Abeta42 is missing from supplementary data (uncropped blots from Fig.5).
3. From the raw data on uncropped blots (panel PS1-G) it looks like upon icaritin treatment PS1 expression goes up in 2 experiments out of 3, which probably warrants correction.
4. Raw western blot data for Fig.5E: the label should read "ADAM10" not "AMDM10".
As you can see, your submission triggered quite extensive but also very constructive reports. This should encourage you to revise your submission and come with what would probably be a much stronger and convincing paper. If you are ready to do the required work, please submit a detailed rebuttal in which you explain how and where the modifications have been made. If you disagree with some of the comments and/or suggestions made, explain why and provide convincing supporting evidence. This rebuttal will be an essential element for me to propose a definitive decision. Please, note that your revised version will be subjected to a new round of review by the same or by different reviewers. I cannot, therefore, make any commitment at this stage about the final acceptance of your submission.
1. title editing from "Icaritin, a beta-secretase 1 inhibitor, impedes amyloidogenic pathway of amyloid precursor protein secretion in APP-PS1-HEK293 cell" to " Icaritin, a beta-secretase 1 inhibitor, inhibits amyloid precursor protein secretion in APP-PS1-HEK293 cels by impeding amyloidogenic pathway ";
2. Does APP-PS1-HEK293 cells purchased? then from where? What is the nature of this cell line? Is APP-PS1 overexpressed in HEK293 cels? Please give some background information about the cells;
3. Line 168, "the Aβ1-40 content is decreased", please specify where? the Aβ1-40 both intracellualr and extracelluar or ...?
Please see all other suggestions as following:
Line 20, BACE1 ---à beta-secretase 1 (BACE1);
Line 28, delet “And”;
Line 29, change “when” to “ at”; add “and “ between 5um and 1o um.
Line 30, add “and “ between 5um and 1o um.
Line 30, “ lower”, please specify intracellular or extracellular or both?
Line 47, “problem facing China” ---à “problem China facing”
Line 38-39: same change as the title
Line 52: first letter should be in capital for “nobel prize”
Line 53, “his” ---à “her”
Line 57: delet “ and it”
Line 60: “besides” replaced by “furthermore “
Line 63: “model rats through” replaced by “ rats model by”
Line 64: abeta generation? May abeta accumulation better?
Line 65: “its neuroprotective effection” replaced by “ possibly neuroprotective effects in vitro”
Line 102: “.” Replaced by “,”, add”then” in front of “ cells”
Line 103: replace “and” with a “,”
Line 110: add “ content” before“ following”
Line 114: change “cells” to “cell “
Line 124: add a subtitle “ Western Blots”;
“ describe ahead” replaced by “ following procedures”; add” Briefly” in front of “After”
Line 133: “during” replaced by “ with”; “ with” replaced by “ by”
Line 134: “antibody” replaced by “ secondary antibodies”
Line 137: add a subtitle “ Immunofluroscence”
Line 138: “ -20 degree “ or “4 degree “? Please confirm
Line 141 and 142: please confirm “ 37 degree” or “ room temperature “? After fixation, why need to be done at 37 degree? It is not live cells anymore, correct?
Line 155: replace “.” By “ ;” ; then add “ Just” before “like”; add “ are” before “both”
Line 160 and 161: “ when” replaced by “at”
Line 161 and 162: replace “,” with “and” between “ 5uM and 10uM”
Line162: add “ that” after “ suggest”; delet”,””
Line 163: replace “ and” by “ at”
Line 166 and 167: move “ culture media obtained from APP-PS1-HEK293 cells” to the “,”, in front of “ the contents”
Line 168: please specify the “decreased” both intracellular and extracellular or ...?
Line 176 ,177 and 180: “with control” replaced by “ with the control group”
Line183: “with control” replaced by “ with that in control group”
Line 186: “ with” replaced by” from”; also please specify what is the difference? Components or functions?
Line 191: where are the data of increased expression of a-secretase?
Line 192: “ this is “ replaced by “ which are”; “ has” replaced by “ have”
Line 196: delet”we”
Line197: “didn’t expect” replaced by “ unexpected “; delet” role”; replaced “ when” with”at”
Line 198: add “ and” in front of “ maybe”
Line200: “problems” replaced with” points”?
Line 201: “patient” replaced by “ patients”
Line204: “ do “ replaced by “ does”
Line 216: “ rat” replaced by “ rats”; add “ and” before“ these”
Line 220 and 221: the same change as title
Q1: Some background information of APP-PS1-HEK293 cells needed; for APP-PS1-HEK293 cells, non Aβ-42 could be available or produced? If yes, why measure Aβ-40 but not Aβ-42 when exposed APP-PS1-HEK293 cells to ICAT? Since the amyloid plaques in Alzheimer’s brains consist of mostly Aβ42 and some plaques contain only Aβ42, even though Aβ40 concentration is several-fold more than Aβ42.
ICAT could decrease Aβ-40 both intracellular and extracellular, but doesn’t mean Aβ-42 (amyloid plaques consisting mostly or only) accordingly decreased as well, since the concentration of Aβ-40 in CSF has been found to be several fold more than that of Aβ-42. Of course, high concentration of Aβ-40 doesn’t mean Aβ-42 must be high either, but depends how much (or the ratio) Aβ-40 transformed to Aβ-42 or even the degradation ratio of Aβ-42 to Aβ-40.
Q2: Did authors by any chance test α-secretase pathway about APPsα or AICD ( APP intracellular domain)changing?
In many instances, an increase in non-amyloidogenic APP metabolism is coupled to a reciprocal decrease in the amyloidogenic processing pathway, and vice-versa, as the α- and β-secretase moieties compete for APP substrate. As a result, if tested a-secretase pathway, even if it a non-amyloidogenic pathway, but could support amyloidogeic BACE1 pathway; if tested AICD, which play a role in transcriptional transactivation, may provide a clue for mRNA and protein expression changing.
Some expressions are not accurate, and rewording needed.
Please see the edited file uploaded.
Amyloid plaques, extracellular deposits primarily composed of the 4 kDa, 40–42 amino acid Aβ peptide, a product of amyloid precursor protein (APP) proteolysis, and neurofibrillary tangles (NFTs), intracellular aggregates of the microtubule associated protein tau are two characteristic brain lesions at the microscopic level to define Alzheimers’ Disease (AD). Aβ peptide is generated following the sequential cleavage of APP by β- and γ-secretase in the amyloidogenic pathway. A beta-secrtase1 (BACE1) exhibits all the properties of the β-secretase, and as the key rate-limiting enzyme that initiates the formation of Aβ, BACE1 is an attractive drug target for AD. This study explore the Icaritin, (BACE1) inhibitor, inhibits the amyloid precursor protein secretion by impeding the amyloidogenic pathway.
1. The ms needs to be run by a native speaker to improve the structure and clarity of the sentences.
2. Not all relevant literature is cited (please see below). A similar compound has been investigated recently in an AD model, this needs to be mentioned in the discussion.
Choice of a cell model not explained.
3. Article structure is OK, figures are of acceptable quality. More details about experimental design need to be provided in figure legends which are currently too short to be informative.
4. Data don't fully support the conclusions. Other APP fragments (sAPPbeta, sAPP alpha, CTF) need to be analysed as per an extended comment below.
1. Research is original.
2. Research question is not very clearly defined (please see comment below).
3. Data are preliminary and don't fully support the conclusion.
4. Methods need more detail, e.g. how intracellular beta was measured? what BACE1 substrate was used? (please see comment below)
The data present in this report do not fully support the conclusion that icaritin impedes amyloidogenic pathway. More proteolytic fragments of APP processing need to be measured.
Here, Feng et al describe anti-amyloidogenic effects of icaritin, a flavonoid compound from a Chinese medical herb. The work is mostly done in APP-PS1-HEK293 cells and the only data that report direct effect of icaritin on BACE1 activity is coming from a BACE1 activity kit (substrate used in this assay was not indicated).
Icaritin has some documented neuroprotective properties in vitro and the authors hypothesize that it should also have an effect on BACE1 activity based on its chemistry. Their previous work show neuroprotective effects on icaritin on rats memory in vitro. It would be good to change the word “secretion” to “processing” in the title because here the authors don’t investigate APP protein secretion.
To analyse the effect of icaritin on amyloidogenic processing, the authors looked at mRNA levels and protein levels of PS1, ADAM10 and BACE1 and abeta40 and saw that these amyloidogenic components were downregulated while ADAM10 was upregulated upon treatment (Fig.5). In order to know whether proteolytic activities of these enzymes in cells also change upon treatment with icaritin, sAPPalpha, sAPPbeta and CTF need to be measured by Western blotting or ELISA.
The compound was tested on cells at 0.5, 5 and 10uM (Fig.5). In terms of LDH leakage rate (Fig. 3 B), it is not clear why there was low level of leakage (=good viability) at 5 and 10uM but not at 0.1-1uM of icaritin. The LDH data make it difficult to interpret western blot data on Fig.5 and cell viability data (Fig.3 A).
Fig.5A: what is the molecular weight of the fragments shown on Western blots? Did the authors detect other long and short abeta species? Why abeta42 was not measured alongside abeta40 to see if ratio of 42:40 is not shifted towards generation of longer beta species?
Fig.4: how was the intracellular Abeta extracted and measured? More details on cell lysate processing need to be provided.
In the introduction, the authors state that genetic evidence demonstrated that abeta plays a pivotal role in AD pathogenesis (lines 47-50). It would be more cautious to say that genetic evidence indicates that dysregulated gamma-secretase-mediated processing of APP is involved in familial AD. Abeta is just one of the proteolytic fragments that happens to accumulate in AD brain. There is also accumulation of misfolded tau which may have a significant role in AD pathogenesis, too.
There is a recent study (doi:10.7150/ijbs.6232) exploring anti-amyloidogenic effects of “icariin” which is a compound obtained from the same plant (Epimedium) as icaritin but with a higher molecular weight. They observed a reduction in APP and BACE1 expression in APPV717I Tg mouse model of AD. It would be helpful to discuss structural similarities/ differences between icaritin and icariin.
Overall, the data present in this report are preliminary and do not fully support the conclusion that icaritin impedes amyloidogenic pathway. More proteolytic fragments of APP processing need to be measured.
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