A Link between Mitochondrial Dysregulation and Idiopathic Autism Spectrum Disorder (ASD): Alterations in Mitochondrial Respiratory Capacity and Membrane Potential

Round 1

Reviewer 1 Report

Authors fulfilled my suggestions

Author Response

Dear reviewer 1,

Thank you for your comments and suggestions.

Reviewer 2 Report

Any new data (sound data) in the field of autism research is welcome. If must hoeveer be taken into account that it is not easy at all do molecular studies relaed to autism research.

The main issue is with cells used in the study. In google or pubmned search I am unable to find what they are. Actually for ALCL I find in Pubmed anaplastic large cell lymphoma (nothing related to autism). Those cells with this autism-linked meaning are used only in labs from Malaysia and they are not (apparetnly) aware of the Rose et al PLOS One 2004 paper that already proves (well proved) that

"We demonstrate, for the first time, that LCLs derived from children with AD have an abnormal mitochondrial reserve capacity before and after exposure to increasingly higher concentrations of 2,3-dimethoxy-1,4-napthoquinone (DMNQ), an agent that increases intracellular reactive oxygen species (ROS)."

Notice that "LCL derived from ..." is not an ALCL, i.e. I have still no clue on what is the ACLC cell line .

There are quite a number of other main issues: I wonder what 

ROUTINE respirationis but I am sure that any two differnt cell lines will provide different values. So what this results tells?. Overall to compare data obtained in two different celll lines is tricky. We may comapre  B and T lymphcites from healthy individuals and find differences. In summary to find differences in two different cell lines do not give any information on what autism is causing to mitochodria, not to mention that information on what is the gene(s) that are mnutated in ALCL versus NALCL should be known.

FIgure 1 must be deleted. I find it confusing (I teach the biochemistry in mitochodria every year). Apart from confusing, a Figure on how mitochondria works is not needed.

Author Response

Dear Editor in Chief,

Thank you for your comments, insights and suggestions on our manuscript.

Comments: Any new data (sound data) in the field of autism research is welcome. If must hoeveer be taken into account that it is not easy at all do molecular studies relaed to autism research.

Response: Thank you for your comment.

Comments: The main issue is with cells used in the study. In google or pubmned search I am unable to find what they are. Actually for ALCL I find in Pubmed anaplastic large cell lymphoma (nothing related to autism). Those cells with this autism-linked meaning are used only in labs from Malaysia and they are not (apparetnly) aware of the Rose et al PLOS One 2004 paper that already proves (well proved) that

"We demonstrate, for the first time, that LCLs derived from children with AD have an abnormal mitochondrial reserve capacity before and after exposure to increasingly higher concentrations of 2,3-dimethoxy-1,4-napthoquinone (DMNQ), an agent that increases intracellular reactive oxygen species (ROS)."

Notice that "LCL derived from ..." is not an ALCL, i.e. I have still no clue on what is the ACLC cell line .

Response: Thank you for your comment. Apologies for the confusion caused. Yes, they are indeed lymphoblastoid cell lines (LCLs) derived from two siblings whereby one sibling is autistic (hence termed as ALCL in our manuscript), and the healthy control is from an apparently healthy sibling with no observation of behavioral and neurological disorders (LCL derived from non-autistic sibling, hence termed as NALCL in our manuscript). Both of the cell lines were purchased from Autism Genetic Resource Exchange (AGRE; Los Angeles, CA, USA). The terms used have been described in section 4 (Materials and methods) under subsection 4.2 (Cell culture).

“Both lymphoblastoid cell lines (LCLs) were purchased from Autism Genetic Resource Exchange (AGRE; Los Angeles, CA, USA). The cells are derived from male siblings in which one sibling has been diagnosed with ASD (LCL derived from autistic child, ALCL; Cell ID: 065604, aged 11) and the healthy control is from an apparently healthy sibling with no observation of behavioral and neurological disorders (LCL derived from non-autistic sibling, NALCL; Cell ID: 065603, aged 12).”

Comment: There are quite a number of other main issues: I wonder what ROUTINE respirationis but I am sure that any two differnt cell lines will provide different values. So what this results tells?. Overall to compare data obtained in two different celll lines is tricky. We may comapre  B and T lymphcites from healthy individuals and find differences. In summary to find differences in two different cell lines do not give any information on what autism is causing to mitochodria, not to mention that information on what is the gene(s) that are mnutated in ALCL versus NALCL should be known.

Response: Thank you for the comment. Apologies for the lack of clarification and explanation on what the respiration states are. The definitions of the respiration states have been simplified in Table 2 under section 4 (Materials and methods) under subsection 4.3 (High–resolution respirometry).

The discussion section (section 3) has been refined to elaborate more on what ROUTINE respiration is and what are the differences in the respiration rates between ALCL and NALCL observed means in relation to mitochondria function.

“ROUTINE respiration is the respiration controlled by intrinsic energy demand. It represents energy demand under steady-state conditions. During ROUTINE respiration, cell respiration relies on endogenous substrates in the cells only..........”

We appreciate your insight for future direction of the study regarding the gene mutation analysis in ALCL versus in NALCL. We have added this in discussion section in line number 276:

“However, clarification of this hypothesis needs to be done via sequencing of the mtDNA of both cell lines”

However, the main focus of this current study is on mitochondria function measuring respiration rates at different respiratory states (ie ROUTINE, LEAK, ETS and OXPHOS), mitochondrial membrane potential and cytochrome coxidase (CIV) activity using high-resolution respirometry, O2k from Oroboros.

With regards to the issue of the information validity from two different cell lines, we do agree that different normal individual also may provide different findings on the mitochondrial activity. However, in this study we use two different cell lines, from siblings, ie sibling as a control to provide a better confined “environment” in terms of genetics and epigenetics and the controls could provide more valid comparison as it can amplify confounding by factors not shared by siblings. Interestingly, we found the significant findings for several parameters measured at least for this subset (ALCL-NALCL pair). This data may provide preliminary findings and ideas for future research on mitochondria and autism.

Comment: FIgure 1 must be deleted. I find it confusing (I teach the biochemistry in mitochodria every year). Apart from confusing, a Figure on how mitochondria works is not needed.

Response: Thank you for your suggestion. The figure has been deleted as suggested.

Dear Guest editor,

Thank you for your comments, insights and suggestions on our manuscript.

Comments: The main issue of this paper is the experimental design that compares only two lymphoblastoid cell lines from male brothers, with or without autism. The data shown are the average of various replicates performed in the same lymphoblastoid cell line. In this regard, the authors do not consider the relevant individual variability in autism. These data are preliminary and do not allow to state the mechanisms of mitochondrial] dysfunction recorded. Therefore, they should use more lymphoblastoid cell lines from different subjects. At least three lymphoblastoid cell lines for both autistic and healthy subjects.

Response: Thank you for your comment and yes, we agree with the comment. However, we are not able to add more lymphoblastoid cell lines from different subjects as the study has been completed. To clarify on the issue, we have added a new section “Limitations & future direction” where we have elaborated further our limitations and we included your constructive suggestion for future directions.

However, eventhough only one pair of LCL was used, the results showed significant and interesting findings on the mitochondrial functions specifically in the respiration rates at different respiratory states (ie ROUTINE, LEAK, ETS and OXPHOS), mitochondrial membrane potential and cytochrome c oxidase (CIV) activity. This shows that there are differences in mitochondrial bioenergetics or perhaps an alteration on mitochondrial physiology that contributes to the pathophysiology of autism.

Unlike other studies that use age- and gender-matched controls, this study uses sibling as a control to provide a better confined “environment” and the controls could provide more valid comparison as it can amplify confounding by factors not shared by siblings.

However, we admit that there is variability in ASD and therefore general conclusion should be made with caution “This finding might provide an insight on only a subset of ASD but not on all ASD.” Eventhough our findings are prelimary but these data might offer a better ideas on future direction of mitochondrial study and autism.

Comment: In addition, the authors should report in method section or new table all the lymphoblastoid cell line characteristics including cell ID, source and age of the subject. They can find an example in Rose et al. Oxidative stress induces mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines in a well-matched case control cohort. PLoS One. 2014 Jan 8;9(1):e85436. In this form the manuscript is not suitable for the publication. 

Response: Thank you for your comment. The source of the cells are mentioned in the method section;

“Both lymphoblastoid cell lines (LCLs) were purchased from Autism Genetic Resource Exchange (AGRE; Los Angeles, CA, USA).“

The cells ID and the age of subjects have been added to the method section as well.

“The cells are derived from male siblings in which one sibling has been diagnosed with ASD (LCL derived from autistic child, ALCL; Cell ID: 065604, aged 11) and the healthy control is from an apparently healthy sibling with no observation of behavioral and neurological disorders (LCL derived from non-autistic sibling, NALCL; Cell ID: 065603, aged 12).”

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Please revise for Style improvement.