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Peer-Review Record

Liraglutide Treatment Ameliorates Neurotoxicity Induced by Stable Silencing of Pin1

Int. J. Mol. Sci. 2019, 20(20), 5064; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205064
by Marzia Bianchi 1, Valentina D’Oria 2, Maria Rita Braghini 3, Stefania Petrini 2 and Melania Manco 1,*
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Int. J. Mol. Sci. 2019, 20(20), 5064; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205064
Submission received: 30 September 2019 / Accepted: 11 October 2019 / Published: 12 October 2019

Round 1

Reviewer 1 Report

Overall this is an excellent paper, it is on a highly relevant topic and asks a very interesting question and arrives at a meaningful conclusion.  I have some specific concerns related to experimental methodology that are presented in comments on the results section and would need to be addressed before I can recommend for publication. 

 

Detailed comments are below:

 

 

Abstract

 

1. What does “improve mitochondrial network” mean?

 

2. It would be preferable if the abstract more clearly portrayed what the most important results are and presented key data points and made the methods and experimental system more clear to the reader.

 

Introduction

 

1. It would be helpful if the paragraph on 2DG and MG gave a clearer picture of what is already know about the effect of these stressors in the mouse model.

 

Results

 

1. In figure 1B it is difficult to see the representative images.  Consider using black and white to show the channels separately and then show combined color, ideally with red and green and perhaps at higher overall brightness.

 

2. How was 100 nM dose of Liraglutide chosen?  This needs to be discussed and if translational relevance is the goal then it must be somehow related to clinical dosing.  I would suggest that some concentration response experiments would be appropriate for key findings.

 

3. The methodology to determine survival in Figure 4A and Figure 4B is unclear both in the figure legend and the text.  Two concerns about the kit used: 1. How is a difference between cell death and changes in proliferation being distinguished?  2. It is based on NADPH, but in later work there is a suggestion of an effect on mitochondria—is it possible that the observed change in NADPH is a result of this and does not actually prove a change in cell survival?

 

4. In my judgement it is difficult to make a clear conclusion about the mitochondrial network from the group of images in Figure 5.  At a minimum quantification of some kind must be done to describe this phenomenon and prove it with adequate scientific rigor.  Additionally, the authors must cite references showing clearly that this type of approach is an accepted way to analyze mitochondria that correlates to meaningful changes in their function.

 

5. In figure 6, I have the same concerns as in #3 above about how cell survival is measured.  Additionally, while it is interesting to see pcr changes in pro-apoptotic mrna, it is well understood that these findings do not necessarily equate to changes in apoptosis.  The authors must actually demonstrate apoptosis (and a change in apoptosis) using a well recognized technique like TUNEL.

 

Discussion

 

1. Something is clearly missing on line 300.

 

2. It would be helpful if the authors can describe any clinical evidence that bolsters translational relevance of their findings.

 

3. A limitations section should be included, particularly given that this work is done entirely in cell culture.

 

Authors' response:

 

We meant fission and fusion. We recognized this definition was misleading and therefore we deleted.  Accordingly we rephrased as it follows: “did not revert mitochondrial dysfunction in Pin1 KD model” (lines 31-32).

 

Done. … “We tested the hypotheses in Pin1 silenced cells (SH-SY5Y) treated with 2-deoxy-D-glucose (2DG) and methylglyoxal (MG), stressors causing altered glucose trafficking, glucotoxicity and protein glycation. Rescue by liraglutide was investigated. Pin1 silencing caused increased levels of reactive oxygen species, upregulated energy metabolism as suggested by raised levels of total ATP content and mRNA of SIRT1, PGC1α, NRF1;  enhanced mitochondrial fission events as supported by raised protein expression of FIS1 and DRP1. 2DG and MG reduced significantly cell viability in all the cell lines. In Pin1 KD clones, 2DG exacerbated altered mitochondrial dynamics causing higher rate of Fission events. Liraglutide influenced insulin signaling pathway (GSK3b/Akt); improved cell viability also in cells treated with 2DG; but it did not revert mitochondrial dysfunction in Pin1 KD model. In cells treated with MG, liraglutide enhanced cell viability, reduced ROS levels and cell death (AnnexinV/PI); and trended to reduce anti-apoptotic signals (BAX, BCL2, CASP3)…”

 

We provided detail on 2DG and MG in the introduction. See lines 82 to 106.

 

We edited the figure. The representative images were acquired with confocal at 60X magnification.

 

The 100 nM dose of liraglutide was chosen based on the response of parental cell SH-SY5Y (cell viability) to different concentration of liraglutide (Supplementary figure). We reported that liraglutide 100 nM is equal to 0.37 mg while the minimum dose in T2D patients is 0.6 mg up to 3 mg/d in patients with obesity. A recent study in people with obesity (ref 68 in the manuscript) demonstrated that the concentration of liraglutide in the cerebrospinal fluid is very low suggesting reduced passage.  Therefore, there is need to quantify exactly the passage of the GLP-1 across the BBB to adjust eventually the dose for clinical purposes.

 

The methodology used to determine cell viability was MTT assay. We specified the methodology (lines 191-192; 256 and legends of Figure 4 and Figure 7). MTT assay reveals metabolically active cells, so the term “survival” was changed in “viability”, because there is no possibility to discriminate between cell death and different proliferation rate.

 

We performed western blotting experiments in Figure 5 A-H to  quantify the increased mitochondrial fission in KD clones by using markers of fusion (OPA1) and Fission (FIS1, DRP1 and pDRP1(S616)). See result, lines 221-242.

We edited the figure 6 (ex 5) adding the p-values for increased number and different distribution of mitochondria among Scramble (control) and P1-C7 KD clone. See lines 243-246.

 

The methodology used to determine cell viability was MTT assay. See reply to your query above.

Apoptosis response to MG was evaluated by flow cytometry experiments (Figure 8) by using annexin V‐FITC/PI double labelling assay. We used annexin V‐FITC/PI double labelling assay and not TUNEL assay as you suggested in order to discriminate between viable, early and late-apoptotic cells in Scramble (control) and both Pin1 KD clones. Lines 296-314.

 

Checked and rephrased.

 

We stated clearly that findings are far from being transferable at this point from the bench to the bed-side for a number of reasons that we listed in the manuscript conclusion (See lines 611-628 and reply to your query below). Nevertheless, our aim was to better understand mechanisms behind the liaison of T2D and AD and investigate likely target for drug treatment.

 

We enclosed study limitations (lines 618-629). “We are aware that findings of the present investigation are far from being transferable from the bench to the bedside, at least at this time. First, they have been produced in neuroblastoma cells whose metabolism and cell responses might differ from those of primary neuronal cells. Nevertheless, SHSY5Y cells are widely used as an in vitro model of neurons. Pathogenesis and Pin1 mediated mechanisms behind progression of altered brain energy metabolism to neurodegeneration in humans are expected to be more complex involving different cell types (i.e. glial cells) in a network; varying among the different brain areas (i.e. cortical versus non-cortical) and along the history of disease (i.e. prodromal phase, onset and progression). The role of Pin1 in neuronal energy metabolism deserves investigation in robust in vivo models. Secondly, we treated engineered cells with liraglutide 100nM that corresponds to a concentration of 0.37 mg/L, while the dose for clinical use ranges from 0.6 to 1.8 and 3.0 mg per day in patients with T2D and obesity, respectively. An investigation in patients with T2D demonstrated a very low concentration of GLP-1 analog in the cerebrospinal fluid of treated patients suggesting its reduced passage across the brain blood barrier. Nevertheless, the concertation reached in the brain seems sufficient to induce a clinical effect [68]...”

Reviewer 2 Report

Authors studied the role of Pin1 in glucose metabolism and neurodegeneration in the context of type 2 diabetes. Also, they used Liraglutide to rescue brain glucose metabolism affected by Pin1 silencing. I must reject this work for multiple reasons and it is not suitable for publication as it is.

1. Authors used only Neuroblastoma cells in this study and it is vague to come to the conclusions using just one invitro model.

2. The role of GSK-3β in the context of Pin1 was not properly explained. It appears very inappropriate that they linked GSK-3β to Pin1 without proper scientific explanation in figure 1.

3. All the Immunofluorescence images are of poor quality and authors must either redo or replace them with better representatives.

4. There is plenty of literature stating that Sirtuin1 is neuroprotective, especially in neurons. How do the authors explain this compared to the outcome from figure 2 in the current study?

5. Western blots in the figure 3A do not correlate with the bar graphs 3B. Especially GSK-3β and p- GSK-3β bands are of poor quality compared to the figure 1C.

6. The color-coded bar graphs in the figure 4A and B was not properly labeled and it is very confusing to comprehend the outcome.

7. Authors must double check their English as I found so many vague sentences. For example- First line in figure 4A` pre-treated or not` can be replaced to `treated with or without`. Also in the same paragraph `expecially` should be changed to `especially` and so forth.

 

Authors' response

 

We enclosed limitations in the conclusion from line 618-629. “We are aware that findings of the present investigation are far from being transferable from the bench to the bedside, at least at this time. First, they have been produced in neuroblastoma cells whose metabolism and cell responses might differ from those of primary neuronal cells. Nevertheless, SHSY5Y cells are widely used as an in vitro model of neurons. Pathogenesis and Pin1 mediated mechanisms behind progression of altered brain energy metabolism to neurodegeneration in humans are expected to be more complex involving different cell types (i.e. glial cells) in a network; varying among the different brain areas (i.e. cortical versus non-cortical) and along the history of disease (i.e. prodromal phase, onset and progression). The role of Pin1 in neuronal energy metabolism deserves investigation in robust in vivo models. Secondly, we treated engineered cells with liraglutide 100nM that corresponds to a concentration of 0.37 mg/L, while the dose for clinical use ranges from 0.6 to 1.8 and 3.0 mg per day in patients with T2D and obesity, respectively. An investigation in patients with T2D demonstrated a very low concentration of GLP-1 analog in the cerebrospinal fluid of treated patients suggesting its reduced passage across the brain blood barrier. Nevertheless, the concertation reached in the brain seems sufficient to induce a clinical effect [68]…”

 

The relationship between GSK3β and Pin1 was clarified in introduction (lines 59-70) and discussion (lines 331-332 and 364-366).

 

Quality of immunofluorescence images was improved (confocal microscopy with 60x magnification; figures 1 and 6).

 

We hypothesized (lines 351-355) as it follows:  “In both Pin1 KD clones, the reduction of mRNA Pin1 levels was counteracted by the up-regulated expression of SIRT1 and NRF1. The P1-C7 clone showed also the up-regulation of PGC1a, master regulator gene of mitochondria biogenesis and antioxidant gene. In that, it seemed that silencing of Pin1 mimics the effects of starvation in stimulating cell metabolism that, we speculate, could be an attempt of the cell at counteracting neurodegeneration…”

Our speculation is in keeping with growing evidence about a specific fasting-induced regulation of

 

Figure 3A was edited.

 

Figure 4 was edited and we changed the color-coded bar graphs.

 

Edited for English language and typos.

 

Round 1

Reviewer 1 Report

The authors have addressed all of my questions properly. Therefore, I am accepting this to be published in IJMS

Reviewer 2 Report

On re-reading this mansucript and reviewing the response letter, I believe that the authors have substantially improved the manuscript and satisfied the reviews. Thus, I recommend to accept.

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

 

 

 

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