Biomarkers of Neurodegeneration and Alzheimer’s Disease Neuropathology in Adolescents and Young Adults with Youth-Onset Type 1 or Type 2 Diabetes: A Proof-of-Concept Study

Round 1

Reviewer 1 Report

In this study, the authors have investigated the risk between youth-onset diabetes and neurodegenerative disorders which remains an area that is underexplored and forms the basis of this proof on concept study. As the authors point out, there are numerous studies showing a link between adults (aged over 40) with diabetes and an increased risk of neurodegenerative disorders such as Alzheimer’s disease but research looking at diabetic patients at a much younger age are lacking.  This is an important study that highlights the need for further investigation in this under-researched area.

Overall this manuscript is well written and referenced. The strength and limitations section is well explained highlighting the fact that the authors are aware of the limitations of their study (small sample size) and have highlighted the need for further research.

I recommend some minor edits to the overall text that should be addressed prior to being recommended for publication in this journal.

Point 1- The introduction is very short and I feel non-specialists would benefit from the inclusion of a short paragraph about the plasma and molecular imaging biomarkers. This should include what they are, how they are linked to disease and why you have chosen these particular ones.

Point 2- Well written and explained methods.  The inclusion and exclusion criteria are clear however it should be stated under what conditions and for how long these samples were stored. 

Point 3- What are the significances (if any) in differences in GFAP levels between adolescents and young adults as shown in figure 1 and figures 2A and 2B, lines 25-27? This significance of this could be explained better.

Point 4- Figure 2 needs to be bigger and better arranged for clarity

Point 5- Minor point- might be better to use the same font throughout e.g. same font used in the tables and main text.

Author Response

Dear Reviewer 1,

We greatly appreciate your thoughtful review of our manuscript and your constructive feedback. Please find below our responses to your comments and concerns. 

Comment_1: The introduction is very short and I feel non-specialists would benefit from the inclusion of a short paragraph about the plasma and molecular imaging biomarkers. This should include what they are, how they are linked to disease and why you have chosen these particular ones.

Response_1: Thank you for this recommendation. We have added an additional description of neurodegeration and AD biomarkers to our introduction.

Comment_2: Well written and explained methods. The inclusion and exclusion criteria are clear however it should be stated under what conditions and for how long these samples were stored. 

Response_2: We are pleased to hear that you found our methods to be clear and well-described. Per your recommendation, we have added information about sample storage (see Section 2.4), as well as discussing the storage time of our samples as a limitation to our study.

Comment_3: What are the significances (if any) in differences in GFAP levels between adolescents and young adults as shown in figure 1 and figures 2A and 2B, lines 25-27? This significance of this could be explained better.

Response_3: Thank you for this thoughtful question and identifying this omission. As you noted, we did not adequately interpret or describe our GFAP and NfL results in the discussion. We have now added our interpretations to the discussion.

Comment_4: Figure 2 needs to be bigger and better arranged for clarity

Response_4: Thank you for this suggestion. We have revised Figure 2 by splitting it in to Figure 2a and 2b, which allows us to make the components bigger. We hope that these changes are satisfactory. 

Comment_5: Minor point- might be better to use the same font throughout e.g. same font used in the tables and main text.

Response_5: We apologize for not catching the different fonts in the figures and tables. This has been corrected to be times new roman throughout. However, in our use of the journal-provided manuscript template, the formatting is a bit off. 

Reviewer 2 Report

The authors present an interesting, preliminary  longitudinal study in a novel area of research, such as biomarkers of neurodegeneration, in youth-onset diabetic patients.  My comments are the following:

The authors should discuss the potential effects of preanalytical issues related to the determination of neurodegeneration biomarkers as a limit of their study.  How long were serum samples stored? Even  storage at - 80C does not indefinitely prevent protein degradation. Were polypropylene tubes used? (This is important, in particular, for amyloid  beta).  Also, differences about methodologies, and little knowledge about intra- and inter-individual biological variability of the considered analytes can be  important issues to  cite in the discussion, as they  may represent a future challenge for scientists in the field, while leading to a cautious interpretation of the results.  Here some suggested reference papers:

• Perret-Liaudet A, Pelpel M, Tholance Y, Dumont B, Vanderstichele H, Zorzi W, ElMoualij B, Schraen S, Moreaud O, Gabelle A, Thouvenot E, Thomas-Anterion C, Touchon J, Krolak-Salmon P, Kovacs GG, Coudreuse A, Quadrio I, Lehmann S. Cerebrospinal fluid collection tubes: a critical issue for Alzheimer disease diagnosis. Clin Chem. 2012 Apr;58(4):787-9. doi: 10.1373/clinchem.2011.178368. Epub 2012 Feb 8. PMID: 22322825.

• Vecchio D, Puricelli C, Malucchi S, et al. Serum and cerebrospinal fluid neurofilament light chains measured by SIMOA™, Ella™, and Lumipulse™ in multiple sclerosis naïve patients. Multiple Sclerosis and Related Disorders. 2024 Feb;82:105412. DOI: 10.1016/j.msard.2023.105412. PMID: 38198989.

• Brum WS, Ashton NJ, Simrén J, di Molfetta G, Karikari TK, Benedet AL, Zimmer ER, Lantero-Rodriguez J, Montoliu-Gaya L, Jeromin A, Aarsand AK, Bartlett WA, Calle PF, Coşkun A, Díaz-Garzón J, Jonker N, Zetterberg H, Sandberg S, Carobene A, Blennow K. Biological variation estimates of Alzheimer's disease plasma biomarkers in healthy individuals. Alzheimers Dement. 2024 Feb;20(2):1284-1297. doi: 10.1002/alz.13518. Epub 2023 Nov 20. PMID: 37985230; PMCID: PMC10916965.

Can you provide data or information about amyloid beta 40/42 ratio? 

Minor issue: line 216/217: please add date of approval

Author Response

Dear Reviewer 2,

Thank you for your insight and helpful suggestions. Please find our responses to your comments below.

Comment_1: The authors should discuss the potential effects of pre-analytical issues related to the determination of neurodegeneration biomarkers as a limit of their study. How long were serum samples stored?

Response_1: Thank you for this suggestion. We have added a description of the sample storage duration for each of the included groups (Section 2.4). We have also added a discussion of how pre-analytical factors are a limitation to our study and results to the limitations section in the discussion.

Comment_2: Also, differences about methodologies, and little knowledge about intra- and inter-individual biological variability of the considered analytes can be  important issues to  cite in the discussion, as they  may represent a future challenge for scientists in the field, while leading to a cautious interpretation of the results.

Response_2: We greatly appreciate this suggestion and the provided references. We have included several references in our limitations section.

Comment_3: Can you provide data or information about amyloid beta 40/42 ratio? 

Response_3: Yes, we are happy to provide these results for the Reviewer. Please find PDF attached. Here, you can see that the ratio in Y-DM does not change between adolescence and young adulthood, suggesting that, while the averages at each time point are lower, compared to the age-similar control groups (Figures 1a and 1b), both AB-42 and AB-40 are changing similarly, relative to one-another, within the Y-DM group over time.

Comment_4: Minor issue: line 216/217: please add date of approval.

Response_4: Thank you for identifying this omission. We have added the date of approval.

Author Response File: Author Response.pdf