Differential Effects of Hypoglycemia and Excitotoxic Signals on SN56 Septal Cholinergic Neuronal Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please find the detailed comments via the attached documents 

Comments for author File: Comments.pdf

Author Response

Introduction remains in accord with general description of the work.

Acetyl-CoA is exclusive branching point between energy producing and energy consuming anabolic pathways in all cellular subcompartments.  Although final biochemical outputs and functional pathophysiological effects  have been extensively studied, the appearance of this cross road point remains poorly explored. As to our knowledge it is the first attempt to explore this point against hypoglycemia-like conditions.

1. Conceptual context and novelty

Ad 1. We have modified introduction to underline novelty of our work by addition of three appropriate sentences at end of the chapter. We also explained rationale for used glucose concentrations. (lines 95-199 and 103-107).

Ad 2 No effects of hypoglycemia on acetyl-CoA content were ever tested in SN56 and other neuronal and non-neuronal brain cells in our and other labs.

Ad 3. This glucose range is a compromise between in vivo experiments where about 5-7 mM glucose  is considered as physiological, and cell culture models in which 20-25 mM glucose is optimal for growth.  Thus this in vitro study compares in fact optimal (but hyperglycemic) and evidently hypoglycemic conditions. By the chance both 25 and 1 mM glucose may take place in humans under extremely pathological conditions.  In addition, glucose concentrations in extracellular space of the brain are 2/3 of those found in plasma. It is due to its avid uptake by neurons. Thus in heavy systemic hypoglycemia of 2 mM (36mg/dl) its level in CSF is 1.3 mM (24 mg/dl). In neonates this level may be lower. This issue is now described in first paragraph of discussion. We added problem of common neonate hypoglycemia for justification this study.

The conceptual advance seems to be explained in first paragraph of discussion

2. Physiological relevance

Ad 1. Fragments of first part of discussion were changed and supplemented with additional explanations concerning specificity of [glucose]in the brain.

Ad 2 Great majority of cell cultures is conducted in 25 mM glucose which is optimal for cell growth (Fig. 1A) but  in fact mimicking diabetic conditions with compensatory hyponatremia and apparent hyper-glycation in long term cultures. We did not studied potential hyperglycation artifacts like rest of investigators in preceding reports, as this report presents  effects of  hypoglycemia. However, discussion indicates that we were aware of such conditions. The alterations introduced in front of discussion hopefully meet this query.

3. Statistical analysis

Ad 1 Numbers of experiments are now displayed in parentheses in parentheses in parentheses individually for each plot

Ad 2. Fitting procedure performed by Prism5 program, and compatibility with straight line assumption is presented by statistically significant correlation coefficient r and p. It is obvious that it visualizes the strength and direction of relationships between numerical variables, but causality may be easily assessed if parameters are obviously interdependent (for instance acetyl-CoA against PDHC). Hence it rather confirms than excludes interdependency.

Ad 3 Data were always from independent experiments. it is now stated in methods and legends as well. Separate days of incubation and assays. It is now included in methods and legends

4. Acetyl-CoA dynamics

Ad 1. Acetyl-CoA level is resultant of rate its primary synthesis mainly                                                       by mitochondrial PDHC and utilization by citrate synthase (fast turnover) and its transport out of mitochondria indirectly as citrate for secondary re-synthesis by ATP-citrate lyase in cytoplasm and directly through PTP. In cytoplasm acetyl-CoA turnover rate is much slower than in mitochondria.

Ad 2. Experimental conditions do not limit acetyl-CoA quantification.  This assay is in our hands since 1987. We check effects compounds used on acetyl-CoA standard recovery.

Ad. 3. Rates synthesis and utilization in multiple reactions may contribute to acetyl-CoA level. We hope that indicating number of possible explanations in discussion we softened our primary claims.

 

5. Interpretation of Aβ25-35 toxicity

We described weak or none effects of A-beta. We now added some remark on ineffectiveness this compound in the experiment. Probably it is due to usage of 1 micromolar concentration which was detected with our experiments in vivo on demented transgenic mice (Bielarczyk et al. 2015 JCN). We added respective moderating frasses.

 

Minor comments

1. Language: We are going to correct language in Your translation office.
2. Figures legends have been supplemented with additional information.
3. Abbreviations for enzyme listed are first defined in summary.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The present study investigated if long-term changes in extracellular concentration of glucose may affect viability and transmitter functions of septum-originated SN56 cholinergic neuronal cells through alterations in their acetyl-CoA availability. This is an interesting  study focused on a very important effect of hypoglycemia on cholinergic neurons. Although I liked the study, I found several weaknesses. 1) In the initial pages (up to line 280) and later pages (from lie 340 onwards) of the manuscript, instead of references text “Error! Reference source not found.” is appearing. Without reference citations, it is difficult to review this manuscript. 2) The discussion section is extremely long and needs to be shortened. 3) I highly recommend authors to consult native language editor for this manuscript.

Comments on the Quality of English Language

I highly recommend authors to consult native language editor for this manuscript.

Author Response

Some repetitive parts of discussion  have been removed, but simultaneously additional text was inserted in response to reviewer’s queries

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript addresses an interesting question, namely whether metabolic differentiation alters the susceptibility of cholinergic neuronal cells to hypoglycemia and excitotoxic stress. The comparison between non-differentiated and differentiated SN56 cells is potentially informative, and the dataset includes several metabolic and functional readouts, including cell counts, trypan blue exclusion, LDH, PDHC, ACLY, ChAT, acetyl-CoA, and ATP. However, in its current form, the manuscript has substantial conceptual and methodological limitations. In particular, the mechanistic interpretation is considerably stronger than what the data directly support, the statistical strategy does not adequately match the experimental design, and the biological relevance of the model and stress paradigms needs to be more carefully justified. For these reasons, I recommend major revision.

Major comments

1. The main mechanistic conclusion is overstated.
   The manuscript concludes that a higher rate of glycolysis in differentiated cholinergic neurons may provide protection against hypoglycemia. However, glycolytic flux was not directly measured. The interpretation is inferred mainly from higher intracellular LDH activity in differentiated cells under low-glucose conditions and from correlation plots. The study does not include direct measurements of glucose uptake, lactate production, glycolytic flux, ECAR, or expression of key glucose transporters/glycolytic enzymes under the experimental conditions. Therefore, the data support an association, but not a direct demonstration, that enhanced glycolysis is the protective mechanism. The conclusion should either be substantially toned down or supported by additional mechanistic experiments.

2. The statistical analysis is not appropriate for the complexity of the design.
   According to the Methods, statistical analysis was performed using one-way ANOVA with Bonferroni correction or unpaired Student’s t-test. However, the experiments involve multiple factors, including differentiation state, glucose concentration, and in some experiments toxic treatment (Aβ, SNP, or both). A factorial design of this kind is better analyzed with two-way or three-way ANOVA, including interaction terms. As currently presented, the statistical framework does not adequately test whether the effect of glucose differs by differentiation status or toxic condition. In addition, the manuscript reports variable sample sizes such as “5–10 experiments” for several figures and “3–5 experiments” for Table 1, but it is not clear whether these represent independent biological replicates or technical repeats. This should be clarified for every experiment.

3. The biological model is limited, whereas the discussion extrapolates too far.
   The study uses SN56 neuroblastoma-derived cells differentiated for 48 h with dbcAMP and retinoic acid, followed by 24 h in media without the differentiating factors. This is a useful reductionist model, but it remains a transformed cell line rather than primary cholinergic neurons. The Discussion repeatedly extends the findings to ageing, diabetic encephalopathy, Alzheimer’s disease, and human cholinergic dysfunction. Such extrapolation is premature without validation in more physiological systems. At minimum, the authors should more clearly frame the study as an in vitro SN56-based model and temper the translational claims throughout the manuscript.

4. The glucose paradigm and toxin model require stronger justification.
   The authors contrast 1 mM and 25 mM glucose and describe the 1–25 mM range as pathophysiologically relevant. However, 25 mM glucose is itself a supraphysiological condition for most neuronal culture interpretations, and 1 mM represents profound hypoglycemia. While such extremes may be useful experimentally, the physiological and clinical relevance should be discussed more carefully. A similar concern applies to the toxicity model: Aβ25-35 alone appears to have limited effects in several readouts, whereas SNP is the dominant toxic factor in Table 1. This makes the model closer to NO donor toxicity under different glucose conditions than to a robust combined hypoglycemia-plus-amyloid paradigm. The rationale for using Aβ25-35 and the selected SNP concentration should be strengthened.

5. Several interpretations may be confounded by survival bias and normalization issues.
   Low glucose markedly reduces cell number, and the manuscript also documents strong effects on viability and LDH release. At the same time, enzyme activities and metabolites are expressed per mg protein. Under severe injury conditions, this can bias interpretation toward the surviving cell population rather than the original cell population. This issue is particularly relevant when the authors interpret relatively modest decreases in acetyl-CoA or PDHC alongside much larger changes in cell count and viability. The manuscript should discuss this limitation explicitly and avoid overinterpreting preserved metabolite levels as evidence of homeostatic compensation without additional data.

6. Correlation analyses are used as evidence of causality more often than is justified.
   The manuscript repeatedly argues for “putative causative links” based on reciprocal or direct correlations between LDH, acetyl-CoA, ATP, PDHC, ChAT, and viability parameters. These analyses are descriptive and useful, but they do not establish directionality or causality. This is especially important because several of the correlation plots are derived from condition means across a limited number of glucose concentrations or treatment groups, rather than from independent mechanistic perturbations. The language in the Results and Discussion should be revised accordingly.

7. The cholinergic phenotype is inferred mainly from ChAT activity and prior work rather than directly demonstrated here under stress conditions.
   The manuscript relies heavily on ChAT as a surrogate of cholinergic function and refers to prior studies for increased ACh content, quantal release, VAChT, and high-affinity choline transporter after differentiation. However, in the present study these endpoints were not measured under the experimental low-glucose and toxic conditions. Therefore, statements about preservation or loss of transmitter function should be softened unless directly supported by additional data.

Minor comments

1. The manuscript would benefit from substantial language editing. There are frequent grammatical problems, awkward phrasing, punctuation errors, and several sentences that are difficult to follow.

2. The rationale for using Lineweaver–Burk and Dixon-type plots for cellular dose-response data should be explained more clearly, as these plots are unconventional in this context and may not be the most appropriate way to estimate IC50-like values.

3. The figure legends should specify exact n values for each dataset rather than broad ranges.

4. Please clarify whether osmolality correction with NaCl was verified experimentally and whether this correction itself affected cell viability or metabolism.

5. The text should be checked carefully for typographical inconsistencies, including notation for glucose concentration, enzyme names, and spacing around statistical symbols.

Author Response

The mechanistic conclusion overstated: We discuss LDH issue as indirect marker of glycolysis more carefully with support of respective references. There are multiple evidences that LDH  activity tightly adjusts to glycolysis rate. We added few appropriate references [        .

                                            

Statistical analysis In figures we either comparison between two [glucose]  corresponding points in DC and NC, or One way Anova for multiple [glucose] in one plot, what is appropriate. Now we changed to nonparametric Mann-Whitney and Kruskal-Wallis, respectively. There is general assumption that below 10 tests nonparametric analysis is more appropriate. Also in Table we compared either two values in one row or 4 values in one column. No diagonal analyses were performed. Data are means from independent experiments performed in different days. It is now stressed in every figure legend and number of experiments marked by numbers in parentheses on the plots.

Biological model limited: We modified discussion toward in vitro model and weakened extrapolation to AD.

The glucose paradigm. We addressed [glucose] issue in response to Reviewer 1. On the other hand active fragment of amyloid-beta was used  in 0.001mM concentration because it corresponds to level A-beta 1-42 in number of Tg mice including our data from Tg2576 (Bielarczyk et al. 2015, JAD 48, 1083).

Several interpretations: Discussion of acetyl-CoA changes vs viability has been modified to correct partially questioned interpretation. Our interpretation results from our earlier studies on PDHC/acetyl-CoA appearance in different experimental conditions.

Correlation analyses: We used wide range of [glucose] to obtain easily distinguishable data. Concordant directions of different plots seems to indicate the existence causal direct or indirect interactions, basing upon known data.

Results and discussion language will be corrected professionally after acceptance of the manuscript.

Cholinergic phenotype. Statement on loss/preservation transmitter function has been softened, but significance of ChAT as a marker cholinergic injury should remain untouched.

 

Minor points

Language will be corrected by Editors translation office..

Figures present  n values

Osmolality of  each [glucose] medium was checked with osmometer. Thanks to Na correction omolality in 1 mM and 25 mM glucose was the same9295-298 mOsm/kg water.. It is now marked in Methods. Reciprocal alterations of plasma [sodium] against [glucose] is physiological  mechanism of iso-osmolality maintenance of extracellular compartment. We mimicked it by increasing additions of NaCl  with decreasing [glucose].

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 

Manuscript Title: Differential Effects of Hypoglycemia and Excitotoxic Signals on SN56 Septal Cholinergic Neuronal Cells

The authors have submitted a revised version of the manuscript. While the revision introduces some improvements—particularly in methodological clarification and minor linguistic corrections—the manuscript still falls short of the standards required for publication. Importantly, several critical issues from the previous version remain unresolved, and new editorial and formatting problems have been introduced.

Overall, the current version does not yet provide the level of clarity, rigor, and technical completeness expected for a journal. Therefore, I recommend major revision.

Major Concerns

1. Persistent Reference and Citation Errors

A significant number of in-text citations still appear as “Error! Reference source not found.” throughout the manuscript, including in the Introduction and Results sections.

This issue is unacceptable at this stage and undermines the scientific credibility of the work, as it prevents proper evaluation of the supporting literature.

All references must be fully restored, verified, and consistently formatted.

2. Severe Formatting and Editorial Problems in the Revised Version

The revised manuscript contains new structural and formatting issues, including:

1. Duplicated or partially repeated paragraphs
2. Residual tracked changes or editorial artifacts
3. Repeated figure legends
4. Disrupted text flow around figures (especially in early sections)

These problems suggest that the revision was not carefully finalized and significantly affect readability.

A thorough editorial cleanup is necessary before further evaluation.

3. Statistical Methodology Remains Problematic

The statistical section has been modified; however, the current description is methodologically incorrect or unclear. For instance:

1. The manuscript refers to a “Kruskal–Wallis normality test,” which is conceptually incorrect
2. The rationale for switching from parametric to non-parametric tests is not justified
3. There is no clear indication of how assumptions (normality, variance homogeneity) were assessed

The statistical analysis must be revised, clearly justified, and correctly described, including:

1. Proper identification of statistical tests
2. Justification for test selection
3. Clarification of sample size (n) and independent replicates

4. Lack of Substantial Scientific Revision

Despite the revision, the core scientific narrative and interpretation remain largely unchanged. The manuscript still lacks:

1. Deeper mechanistic insight into the differential vulnerability of NC vs DC phenotypes
2. A clearer integration of acetyl-CoA metabolism with broader neurodegenerative frameworks
3. A more critical discussion of limitations and translational relevance

The revision appears primarily editorial rather than conceptual, which is insufficient given the complexity of the topic.

The Discussion should be strengthened to provide:

1. Mechanistic interpretation beyond descriptive findings
2. Clear positioning within the context of neurodegeneration (e.g., Alzheimer’s disease)
3. Explicit limitations and future directions

5. Figure Presentation and Clarity

Although some figure descriptions were slightly improved, issues remain:

1. Redundant or inconsistent figure legends
2. Lack of clarity in explaining kinetic plots (e.g., Lineweaver–Burk, Dixon plots)
3. Insufficient explanation of biological interpretation of IC50/AC50 values

Figures and legends should be revised for clarity, consistency, and interpretability.

 

Minor Comments

1. Several grammatical and stylistic issues persist and require careful proofreading by a fluent English speaker.
2. Terminology should be standardized (e.g., “glycaemia” vs “glucose concentration”).
3. Some sentences remain overly long and difficult to follow.

While the authors have made an effort to revise the manuscript, the current version does not adequately address key concerns and introduces additional issues that affect both readability and scientific rigor.

Recommendation: Major Revision

A substantially improved version—with corrected references, clean formatting, rigorous statistical description, and strengthened scientific interpretation—will be necessary before the manuscript can be reconsidered for publication.

 

 

 

Author Response

Answer to reviewer 1 report v2.

1. and 2. points concerning formatting problems appeared in editorial office. And has been corrected at least in our copy of the manuscript. They still persist in current manuscript

 

3. Statistical methodology

We have these changes following suggestions of first round. But we agree that usage of ANOVA+Dunnets more appropriate for 3 and more experimental points. We enclosed description of statistical approaches which should be correct. See below:

Results are shown as means ± SEM from 3-10 independent culture experiments. Particular number is given in parentheses in figures. Calculations of glucose concentration-response relationships ([IC₅₀/AC₅₀])were performed with non-linear regression analysis using Graph Pad Prism Software 5.0 (Graph Pad Software Inc.La Jolla, CA USA) by fitting data to log{glucose] to data obtained for series of glucose concentrations. Differences between glycemia-derived data were estimated using one-way ANOVA followed by pos-hoc Dunnetts test. Differences between pairs of parallel NC and DC data were assessed with Student’s t test. Correlations between different variables were determined using linear regression with regression coefficients as an indicator statistically significant relationship.

Modified paragraph is underlined. It contains usual details included in such sections elsewhere

We changed  method of [IC50]/[AC50] estimation from “classic kinetic” to non-linear regression, which revealed same key differences between NC and DC (qualitatively). We have used L-B and Dixons method before,  because they are simpler  for interpretation as giving straight line plots with intercepts of x axis. But non linear regression fits better to complicated models and indirect multiple effects. So now we used this method in Fig. 2DDFH and Fig. 3D, with respective alterations in the text.

The Discussion is supplemented with 3 additional inserts (underlined)

Figures: legends has been changed where applicable, kinetic analysis is made by non-linear regression.

Some biological interpretations are included inside inserts.

Minor points. We are going to follow advises by giving manuscript to professional translation MDPI office. We standardized  nomenclature to “glycemia

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

I have no further concerns.

Author Response

No commments from Reviewer 3

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

 

The authors have submitted multiple revised versions of the manuscript entitled “Differential Effects of Hypoglycemia and Excitotoxic Signals on SN56 Septal Cholinergic Neuronal Cells.” The current version (v3) shows clear improvements compared to the initial submission and the intermediate revision.

General Assessment

Overall, the manuscript has improved in terms of clarity, organization, and methodological transparency. In particular, the Materials and Methods section has been strengthened. The authors now provide more explicit details regarding medium composition, osmolarity adjustment, and experimental conditions (e.g., glucose range, NaCl compensation, and treatment parameters), which enhances reproducibility and scientific rigor.

In addition, some editorial inconsistencies present in the previous version (v2)—such as duplicated text blocks and formatting artifacts—have been partially resolved in the current version. The experimental design is now more clearly presented, and the description of treatments (Aβ25–35 and SNP) is more precise.

Remaining Concerns

Despite these improvements, several important issues remain:

1. Unresolved Reference Errors
   A major concern is the persistent presence of multiple “Error! Reference source not found.” statements throughout the manuscript, particularly in the Introduction. These errors indicate broken cross-references and significantly affect readability, credibility, and overall presentation. This issue must be fully corrected before publication.
2. Language and Formatting Issues
   The manuscript still contains several grammatical inconsistencies, punctuation errors, and formatting problems (e.g., missing spaces between sentences, abrupt sentence transitions, and minor typographical artifacts). While these do not fundamentally compromise the scientific content, they detract from the professional quality expected for publication.
3. Clarity of Scientific Narrative
   Although improved, some sections—particularly in the Introduction and Results—would benefit from further refinement to enhance logical flow and readability. Certain sentences remain overly complex or imprecise, and minor polishing would improve clarity.

The study addresses an important topic related to neuronal energy metabolism and cholinergic vulnerability under hypoglycemic and excitotoxic conditions. The experimental approach using differentiated vs. non-differentiated SN56 cells is appropriate, and the findings regarding acetyl-CoA metabolism and phenotype-dependent susceptibility are potentially valuable. The scientific content appears sound, and the revisions have improved the methodological robustness.

Final Recommendation

The manuscript has clearly progressed through revision; however, it is not yet ready for acceptance in its current form.

I recommend: Minor Revision (Mandatory Editorial Correction)

The remaining issues are primarily technical and editorial, but they are essential to resolve. In particular:

1. All reference/cross-reference errors must be corrected.
2. The manuscript should undergo careful language editing and proofreading.

Once these points are addressed, the manuscript would be suitable for publication.

 

 

Author Response

Dear Editors and Reviewers:

Thank You very much for prompt processing our article:

Following remarks of Scientific Editor and Reviewer we have done corrections:

1. Reference errors have been removed and numbers placed back
2. We did not try to correct language/formatting issues relaying on the professional services of Editorial Office. Our University is on the list of institutions eligible for 20% institutional discount. If You think that our submission requires extensive language correction except of standard one, we will agree.
3. We have shortened some sentences dividing them for clarity: -lines: 414-415, 493-495, 510-511, 530-532, 538-539, 583-584. During previous correction we also made few such improvements (no track).
4. We accept Your proforma.

 

If our explanations and corrections are satisfactory please send us appropriate proforma to start administrative attempts for payments.

 

Sincerely Yours,

Andrzej Szutowicz

Author Response File: Author Response.pdf