Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients

Round 1

Reviewer 1 Report

In this article, the authors have reported results demonstrating observations of alterations in mitochondrial morphology in two mitochondrial disease (MELAS and LS) patient fibroblasts. There is adequate rationale presented for this study, in the context of better understanding the changes that occur within mitochondrial, specifically in an attempt to link mitochondrial structure and function to disease.    

 

The authors have experimental outcomes based on use of use of immunocytochemistry, Western blots, live-cell metabolic analysis, qPCR, siRNA transfection, flow cytometry and additional biochemical assays. These experiments are appropriate to arrive at initial conclusions. Overall, this is an interesting study that demonstrates potential for a better understanding of defects in mitochondrial genome affecting mitochondrial structure and function, in the context of specific diseases.

 

However, there are some major concerns that need to be addressed before this manuscript can be considered for publication.

 

Additional details need to be incorporated in the materials and methods section so that meaningful interpretation of results can be conducted. The following details are needed: What is the constitution of the FBM used to propagate the healthy and diseased fibroblasts? Use of antibiotics is a concern as it can impact mitochondrial function. What is the concentration used? It is useful to note that GDF15 is used as a biomarker to indicate ‘mitochondrial disease’. Although an intriguing biomarker, it is important that the authors demonstrate the presence of the mutation in the samples. If possible, it would be important to also quantitatively evaluate heteroplasmy levels by next-gen sequencing or RFLP in the samples, to make strong correlations with the mitochondrial phenotype. In almost all experiments conducted, more details on the experimental design needs to be included so that significance can be appropriately evaluated. For example: In Figure 4, what is n? Biological or technical replicates? More clarification needed in the experimental design section. In Figure 2, what is n? Biological or technical replicates? More clarification needed in the experimental design section. In Figure 5B, what is n? Biological or technical replicates? More clarification needed in the experimental design section. Data in Figure 1B should be accompanied by a quantitative evaluation of the GDF15 intensity levels. The reference to each of Figure 3A, 3B, 3C in the results section is incorrect. For example, Figure 3A is correctly listed as ATP measurements in the figure caption, while incorrectly referenced as membrane potential measurement in the text. Similar to what is shown in Figure 4B, where is the analysis after siRNA treatment to indicate progressive recovery of mitochondrial morphology?. Comparisons should be done between scrambled and treated samples. What happens in the control fibroblasts when DRP1 is knocked down in very different aged control lines? This data is equally important to better demonstrate the importance of DRP1 in the context of this study. It is useful to note that the authors have measured cellular ROS levels using CellRox. However, in the context of this study, it is important to measure mitochondrial ROS levels using MitoSox, as this will be useful in explaining the ROS levels generated when mitochondrial morphology is altered. Concerns also exist with the minimal samples used in this study. Given the variability that exists in mitochondrial disease, it is important to replicate these findings in at least another cell line for each disease.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The study by Tokuyama and colleagues presents the novel point that DRP1 expression is induced in fibroblasts from MELAS and Leigh syndrome patients. This is an interesting result, that can open a new paradigm in mitochondrial diseases. However, the authors over-generalize their findings to all mitochondrial diseases based only on two samples of patient fibroblasts. This is my first concern: the authors cannot generalize the findings from this study to (all!) "mitochondrial diseases". The spectrum of MDs is vast, and two fibroblast samples are not sufficient to cover it all. The names of the diseases should be mentioned instead. The novelty will not be affected, and the story will immediately be more solid.

Secondly, there are several aspects of this study that require improvement in order to solidify the conclusions.

The introduction is well written, and care was taken by the authors to cite the original papers in a fair way. 

The methods require some improvements, particularly in the methodology that was used to define what "fragmented" mitochondria are (which criteria?), and how the number of cells with fragmented mitochondria was determined (automated? blind? by direct counting?). For all experiments, the number of replicates needs to be shown.

On the results, there is a puzzling start with GDF15 which seems to have no meaning towards the message of the paper and merely verifies someone else's paper in the samples used in this study. Why GDF15? I would suggest that this is removed or sent to supplementary information, to improve the readability of the manuscript.

The sections showing that the respiration is impaired in the MELAS and Leigh samples are technically well done, but are not novel. They are however important to validate the two samples used here. 

Most of my concerns have to do with Figures 4 and 5, which are anyway the core of the message of this manuscript. Figure 4B requires the clarification of the criteria for fragmentation, as noted above. Figure 4C can only be interpreted if the authors show the inhibitory phosphorylation of DRP1 as well, and mostly so because the RATIO of phospho-S616 to total DRP1 is visibly down in the patient fibroblasts. So the ratio of the inhibitory DRP1 phosphorylation to the total is important to be determined. The increase in DRP1 protein levels is very convincing, and also at transcript levels. However, other proteins involved in mitochondrial dynamics should be tested, particularly in fusion, to show that the increase in transcript and protein level is specific for the fission machinery.

Figure 4D is of low quality as such, and needs to be improved to become interpretable (and publishable). Also, quantification of the amount of DRP1 in mitochondria are expected.

In line 240 the authors state that excess cellular ROS cause apoptosis. This may or not be the case, excess ROS can cause different types of cell death. I recommend that the word "apoptosis" is replaced by "cell death". The authors then follow-up Annexin V staining to determine if it is apoptosis. 

In Figure 5A, quantification is required, as discussed for similar experiments above.

Figure 5B - the control cells with DRP1-kd need to be included in this experiment (also 5C), to determine if the effects of silencing DRP1 are a consequence of DRP1 knockdown, or a consequence of blocking fragmentation in cells with damage mitochondria. This is a very important distinction, and crucial to the message of this manuscript.

Figure 5D is fine as is, as long as 5C includes controls+DRP1-kd (and scrambled, of course).

I have a few points on the discussion. The authors measured the total levels of ATP, and found no difference, so it is puzzling that they then claim in line 263-264 that low ATP is a cause of mitochondrial disease. The reference 34 measures ATP synthesis rate, not total levels. And anyway the putative "loss of ATP" in mitochondrial diseases is more a myth of the field, and is not a general observation for mitochondrial diseases (see for example review on PMID 24508276).

In line 272 - dynasore is an inhibitor of dynamin (the endocytic dynamin) not of DRP1. Keep in mind that dynamin was proven by at least two labs NOT to be involved in mitoch fission. Dynasore should be removed from this list.

The experiments with DRP1 knockdown and autophagy are quite interesting, even if not followed-up. I don't see why they are in supplements, I believe they would bring value to the main figures. Keep in mind that perturbation of mitochondrial dynamics was shown to cause lysosomal malfunction and autophagy impairment (PMID 26987902), and that chronic mitochondrial defects trigger a general impairment of autophagy and lysosomes to avoid the complete degradation of the mitochondrial network (PMID 31791731, 30917721).

In sum, this is an interesting study with a novel result and, after addressing some experimental concerns, will be of value to the field of mitochondrial medicine and biology.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed this reviewer concerns. The manuscript is acceptable for publication.