TDP-43 Pathology and Prionic Behavior in Human Cellular Models of Alzheimer’s Disease Patients

Round 1

Reviewer 1 Report

In a general way, the article is well-written, original, innovative, clear and objective. It fits in the aims and scopes of the Journal. Excellent work!

Even so, I recommend to the authors a few things:

Abstract

“that seem to have prion-like characteristic”

“pathogenesis and its cell-to-cell propagation”

Put the keywords by alphabetic order

“suitable experimental models for mechanistic”

I think that it is important to mentioned that Alzheimer’s disease is also frequently associated with type 3 diabetes (please, read, for example: doi:10.3390/ijms21093165)

Materials and Reagents

Please, mention the reference of the Ethic committee approval.

“The establishment of lymphoblastoid”

Please, mention the company and origin of the reagents, antibodies and of the main equipaments used.

Is not possible to increase the number of participants?

“an SDS polyacrylamide gel and”

“chemiluminescent substrate”

“followed by a”

“anti-TDP-43 (1:100, Proteintech 67345-1-Ig) or anti-phosphor-TDP-43 (1:100, Proteintech 22309-1-AP).”

“manufacturers' recommendations.”

“p-value < 0.05”

 “Leica Application Suite X and Image J software” : please, mention the versions.

“of each group of four control”

“in the legend of Fig,1, (A)”

“To this end, we analysed”

“… selection were performed using Image J software.”

“exosomes in different neurological diseases. For this reason, we first performed Western”

“in most AD cases, regarding disease severity,”

“Of note, a band of approximately 35”

“4%”

“The % of cells with the presence of 25 KDa-TDP-43 extracellular fragments was”

“osteosarcoma”

“there was an apparent increase in particle size mean and concentration in severe AD cases.”

“markers since they are not”

“suggests that they can participate in the dissemination of”

“it is worth highlighting that fragmented”

“in the communication”

“TDP-43, were detected in EVs isolated from AD lymphoblasts.”

Merge the citations

Remove the italic in some “Figure” titles, legends and of “Increased”

Is it possible that over factors related to the lifestyle influence the results verified, like alcohol, tobacco, among others? Please, clarify this detail.

Author Response

RESPONSE TO REVIEWER #1

Ms ID: biomedicines-1580246

We thank Reviewer 1 for his/her kind comments

Following the Reviewer advise, we have corrected all typo errors.

The corresponding version # for Leica Application Suite X and Image J software are now included in the revised version of our Ms.

A mention to the association between AD and diabetes type 3 has been included in the introduction section.

Is it possible that over factors related to the lifestyle influence the results verified, like No alcohol, tobacco, among others? Please, clarify this detail.

We have observed the same feature (TDP-43 mis-function) in four independent patients with the same results and, although we do not know anything about their life style, the results are the same. One possibility may be that life style influence in starting the pathology but once it is produced, pathological features are the same.

Author Response File: Author Response.pdf

Reviewer 2 Report

Cuevas and colleagues summarize in the present study, entitled ‘TDP-43 pathology and prionic behavior in human cellular models of Alzheimer`s disease patients’, the current status of knowledge about molecular pathology characterizing Alzheimer’s disease (AD), focusing on of role of TDP-43 binding protein. For this purpose, lymphocytes samples from patients diagnosed with different severity of AD were used and the TDP-43 pathology was analyzed against controls using Western blot and immunocytochemical techniques. Results showed an increase in TDP-43 fragmentation, as well as increased phosphorylation and aberrant localization of TDP-43 in patients diagnosed with severe AD.

The main strength of this paper is that it addresses an interesting and timely question, finds a novel solution based on carefully selected methods for protein detection and provides a clear answer to the possible role of TDP-43 in AD pathogenesis and in its cell to cell propagation. In general, I think the idea of this article is really interesting and the authors’ fascinating observations on this timely topic may be of interest to the readers of Biomedicines. However, some comments, as well as some crucial evidence that should be included to support the authors’ argumentation, need to be addressed to improve the quality of the article, its adequacy, and its readability prior to the publication in the present form. My overall judgment is to publish this article after the authors have carefully considered my suggestions below, in particular reshaping the Introduction and Discussion sections.

Comments

• In general, I recommend authors to use more evidence to back their claims, especially in the Introduction of the article, which I believe is currently lacking. Thus, I recommend the authors to attempt to deepen the subject of their manuscript, as the bibliography is too concise: nonetheless, in my opinion, less than 50 articles for a research paper are really insufficient. Currently, authors cite only 26 papers, and they are dramatically few. Therefore, I suggest the authors to focus their efforts on researching relevant literature: I believe that adding more studies will help to provide better and more accurate background to this study. In this review, I will try to help the authors by suggesting relevant literature that suits their manuscript.
• Page 1, Introduction: I suggest the authors to reshape the Introduction section, which seems inhomogeneous and dispersive. I think that more information about pathophysiology and neurological changes of Alzheimer’s disease would provide welcome background here. Thus, I suggest the authors to make such an effort to provide a brief overview of the pertinent published literature that offers a perspective on the definition, causes, and symptoms of AD, because as it stands, this information is not highlighted in the text. In this regard, I believe that the statement ‘Alzheimer’s Disease (AD), the most common form of dementia in people over 65 years of age is characterized by two main neuropathological hallmarks…’ needs some other necessary citations. In particular, according with this sentence, I would recommend citing a review that examined pathophysiological basis and biomarkers of AD pathology (https://doi.org/3390/ijms21249338) and a study in which authors investigated age-related impairments in the ability to process contextual information and in the regulation of responses to threat, addressing that structural and physiological alterations in the prefrontal cortex and medial temporal lobe determine cognitive changes in advanced aging, that can eventually cause patterns of cognitive dysfunctions observed in patients with AD/MCI (https://doi.org/10.1038/s41598-018-31000-9). I firmly believe that these improvements will help to provide a more coherent and defined background.
• Page 1, Introduction: Authors stated that ‘TDP-43 was originally found to associate with frontal temporal lobar degeneration with ubiquitin inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS)’. In my opinion, adding some references that address how functional alterations of the frontal lobe impact memory and error-driven learning in humans may improve the theoretical background of the present article and its argumentation: evidence from an electrophysiological study suggested that medio-frontal ERP signals of prediction error tracks the timing of salient events, and highlighted how alterations in medial prefrontal cortex could impact on the patients’ capacity of signal errors in the prediction of outcomes (https://doi.org/10.1162/jocn_a_01074). Additionally, a recent review on vmPFC subregional contributions addressed the role of vmPFC in processing safety-threat information and their relative value, and how this region is fundamental for the evaluation and representation of stimulus-outcome’s value needed to produce sustained physiological responses (https://doi.org/10.1038/s41380-021-01326-4). Secondary, authors can also see studies that have focused on this topic (https://doi.org/10.1162/NECO_a_00779; https://doi.org/10.1111/cns.12835; https://doi.org/10.1038/s41386-021-01101-7).
• Page 2, Subjects: Can the authors provide the specific number of patients and healthy controls that were recruited for this study?
• Page 4, Immunological assays: I suggest rewriting/reorganizing this paragraph for clarity. In my opinion, authors should present information about protein sequences in a more clear and structured way, such as using a table or an explanatory figure, because as it stands, it is quite difficult to readers to understand data.
• Page 5, Results: Please provide full statistical details to ensure in-depth understanding and replicability of the findings.
• Page 13, Discussion: This manuscript focuses specifically on TDP-43 expression levels in AD patients, analyzing the role of this protein in AD pathogenesis. In this regard, I believe that it may be useful adding some references that have described multi-target techniques used to assess pathways implicated in the progression of neurodegeneration and to identify effective therapy for alleviating cognitive impairment in AD, in order to improve the theoretical background of the present article: interestingly a recent review investigated the contribution of emotional stimuli on action control and proposed that executive functions and emotions are thought to compete for the same resources, examining how frontal cortex-dependent resources, that are normally involved in motor control, are also involved in emotion detection, thus pointing out a relation between action control and emotion processing that is impaired in patients with AD due to atrophy of prefrontal cortex or limbic system (https://doi.org/10.1016/j.brat.2021.103963). Authors can also see additional studies that have focused on this topic (https://doi.org/10.1016/j.cortex.2019.04.008; https://doi.org/10.1053/j.semnuclmed.2020.12.002).
• Also, I believe that the ‘Conclusion’ paragraph would benefit from some thoughts as well as in-depth considerations by the authors because as it stands, it is very descriptive but not enough theoretical. Authors should make an effort, trying to explain the theoretical implication as well as the translational application of their research, to adequately convey what they believe is the take-home message of their study, and therefore discuss theoretical and methodological avenues in need of refinement, suggesting a path forward in AD treatment. In this regard, recent evidence suggests that the application of new methods in Alzheimer’s treatment, such as the Non-invasive brain stimulation techniques (NIBS), have shown promising results in humans ( https://doi.org/1097/WCO.0000000000000669). Importantly, I recommend citing recent studies that revealed that the application of NIBS induces long-lasting effects, noninvasively modulating the cortical excitability, and modulating a variety of cognitive functions: for example, a recent review acknowledged the implementation of NIBS to modulate in general fear memories (https://doi.org/10.1016/j.neubiorev.2021.04.036). I suggest also a review on the efficacy of NIBS and IBS in AD (https://doi.org/10.3389/fpsyt.2018.00201).
• In according to the previous comment, I would ask the authors to also include a ‘Limitations and future directions’ section before the end of the manuscript, in which authors can describe in detail and report all the technical issues brought to the surface.
• Regarding the Figures: I suggest to modify all figures for clarity and providing higher-quality images because, as it stands, the readers may have difficulty comprehending them. In my opinion, data settings are overcrowded and written with a very small font. Also, please change the scale of the vertical axis and use the same minimum/maximum scale value in all the graphs in all the figures and reorganize the graphs’ space, to provide a better understanding and a direct interpretation of the
• The reference list is incorrect: authors should check the Journal’s guidelines again and provide the abbreviated journal name in italics, the year of publication in bold, the volume number in italics.

The manuscript potential carries important value presenting the possible mechanisms of Alzheimer’s disease dissemination and the possible relation between DNA binding proteins and AD-associated molecules. I hope that, after these careful revisions, the manuscript can meet the Journal’s high standards for publication.

Best regards

Author Response

RESPONSE TO REVIEWER #2

Ms ID: biomedicines-1580246

We are grateful to the reviewer for his/her insightful comments.  We think that by addressing the issues raised, the manuscript has been greatly improved.

In general, I recommend authors to use more evidence to back their claims, especially in the Introduction of the article, which I believe is currently lacking…

Following the Reviewer’s advise, we have re-written the Introduction section to provide a more extensive and deeper backgroung for thi study. The revised version of the Ms contains now more than 60 references.

Page 2, Subjects: Can the authors provide the specific number of patients and healthy controls that were recruited for this study-

The specific number of patients together with their main characteristics are listed in Table 2

Page 4, Immunological assays: I suggest rewriting/reorganizing this paragraph for clarity

We have edited the corresponding paragraph, and include a new table (Table1) in the revised version of our Ms, containing the list of antibodies used in this sudy

Page 5, Results: Please provide full statistical details to ensure in-depth understanding and replicability of the findings.

 Thanks for your suggestion. The full statistical parameters are now provided in the legends to the figures

Page 13, Discussion: This manuscript focuses specifically on TDP-43 expression levels in AD patients, analyzing the role of this protein in AD pathogenesis. In this regard, I believe that it may be useful adding some references that have described multi-target techniques used to assess pathways implicated in the progression of neurodegeneration and to identify effective therapy for alleviating cognitive impairment in AD, in order to improve the theoretical background of the present article…

We appreciate your constructive criticism and we would like to thank you for your recommendations. The discussion section has been re-written, to include pertinent information regarding the pathophysiological mechanisms through which TDP-43 mediates neurodegeneration in AD. The possible association between TDP-43 deposists in. the limbic region and the coordination between action control and emotion is now acknowledged.  In the revised version of our Ms we have included information about the potenctial of targeting TDP-43 pathology for the developing of new therapeutical strategiesfor AD.

Also, I believe that the ‘Conclusion’ paragraph would benefit from some thoughts as well as in-depth considerations by the authors because as it stands, it is very descriptive but not enough theoretical. Authors should make an effort, trying to explain the theoretical implication as well as the translational application of their research….

We have re-written the Conclusions section and include a paragraph with “limitations and future diections”

Regarding the Figures: I suggest to modify all figures for clarity and providing higher-quality images…

All Figures have been redrawn, with a bigger font size, and increased quality. Nevertheless, the original high quality figures are provided in a Zipp archive

The reference list is incorrect: authors should check the Journal’s guidelines again and provide the abbreviated journal name in italics, the year of publication in bold, the volume number in italics

We apologize, we have now corrected all the references applying the MPDI citation style in the revised version

Author Response File: Author Response.pdf