Loss of HuD Sensitizes Neuroblastoma Cells to Palmitate-Driven Stress-Induced Premature Senescence via PPARα Downregulation and FAO Impairment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a substantial amount of experimental work, and the authors should be commended for the overall technical quality and clarity of the figures. The datasets are internally consistent and the experiments appear to have been performed with care and attention to detail. The study addresses an interesting question with potential relevance to metabolic stress responses and RNA-binding protein function, and the data are, at first sight, coherent within the chosen experimental framework.

 

However, while the experimental execution is solid, there are important conceptual issues regarding the neuronal relevance of the chosen pathways and interpretations. Specifically, the mechanistic rationale for focusing on certain targets in a neuronal context, as well as the interpretation of the observed stress responses as senescence, require further clarification and stronger support. These issues do not detract from the quality of the experimental work, but they do affect the biological conclusions that can be drawn.

 

Therefore, I have two major concerns that should be addressed to strengthen the manuscript and ensure that the conclusions are fully supported by the data. These points are detailed below.

 

Major Comment and concern 1:

I mis the rationale for focusing on PPARα-dependent FAO transcripts in neuronal cells.

 

In line 195 is stated that the previous work was performed in β-cells. In that context, the choice of PPARα-regulated FAO targets is fully understandable. However, it is less clear why the same target panel was transferred directly to a neuronal system.

 

While the RIP–qPCR experiments (Figures 2A and S1) demonstrate that HuD binds a set of canonical PPARα-regulated FAO transcripts (Pparα, Cpt1, Cpt2, Acsl1, Acadl, Acadm, Acads), the rationale for selecting this specific panel is not fully clear in the context of neuronal cells. In most neuronal and neuron-derived cell types, PPARδ (PPARD) is widely recognized as the predominant PPAR isoform, whereas PPARα expression is typically very low. Therefore, it is important to clarify why the authors chose to examine PPARα-dependent FAO transcripts without first establishing whether PPARα is sufficiently expressed or functionally relevant in N2a cells.

 

To strengthen the mechanistic interpretation and justify the choice of targets, I recommend that the authors:

• Provide baseline expression data (RNA and/or protein) for PPARα and PPARδ in their neuronal model;
• Explain the biological rationale for focusing specifically on PPARα-regulated transcripts in N2a cells; and
• Consider including PPARδ and its downstream FAO targets in the RIP–qPCR panel, or justify why these physiologically more relevant transcripts were not assessed.

 

This clarification is essential for interpreting the significance of HuD–mRNA interactions in a neuronal context and for determining whether the observed RIP enrichments reflect physiologically relevant regulatory relationships.

 

Major Comment and concern 2:

Although the authors quantify SA-β-gal–positive cells as evidence of senescence, it is important to note that SA-β-gal activity is not specific for senescence, particularly under metabolic or lipotoxic stress conditions such as palmitate exposure. SA-β-gal can be induced by mitochondrial dysfunction, lysosomal overload, ROS, and acute p53-dependent stress responses, all of which occur during palmitate treatment. Therefore, SA-β-gal positivity alone does not distinguish true, irreversible senescence from stress-induced, reversible checkpoint activation.

Combined with the relatively short incubation times and the induction of p21 and p16 response, the phenotype described here is more consistent with stress-induced cell-cycle arrest rather than bona fide senescence. To support a true senescence conclusion, the authors would need to demonstrate irreversibility, for example by removing palmitate and showing that the growth-arrest phenotype and SA-β-gal positivity persist despite relief from the stressor. (The cells look a bit “rounder” like the try to divide but cannot progress; this could easily been assessed using PI and the facs analysis; I expect a holdup in m-phase not a senescence)

 

Without such evidence, the data are more accurately interpreted as reflecting stress-induced p21 activation rather than canonical senescence.

 

Minor comments:

In the materials and methods, duration of the incubation time / harvest timing can be better explained / added this is typically different for protein and mRNA etc.

Line 88: Stable cells? Or N2a cells that stably expressing shHuD or control shRNA as previously described?

Line 88/90  also see comment supplementary data.

 

Line 191 good to mention the program used (R, SPSS or other)

Line 225 please ad “scale bar,…” at the correct spot; in (A).

 

Line 309 and supplementary info: In the p-TBK1 blot the original ECL shows a light spot exactly at the critical position. This suggests a partially failed ECL exposure. Please repeat the ECL; the background should be evenly grey to reliably conclude that the signal in the first lane (after the marker) is lower.

 

I also notice the β-actin control. However, this appears to be from a separate blot. While this confirms equal protein amounts in those samples, it does not validate loading equality for the other blots, even if they originate from the same samples.

 

Although the ECLs seem acceptable overall, I strongly recommend including a Ponceau S staining in future experiments. This provides a clear and direct demonstration of equal loading across the entire membrane, and also reveals any procedural artefacts during transfer or blotting.

 

Supplementary data: In the supplementary data, the sequences for siHuD and shHuD appear to differ markedly in their degree of complementarity to the HuD mRNA (based on the downregulation and the use of si/sh. The siHuD construct seems to resemble a miRNA-like design, with only the seed region (first ~7 nt) fully complementary, whereas the shHuD appears to be fully matched to the target sequence. If this interpretation is correct, the two constructs would not be mechanistically equivalent (miRNA-like modulation versus classical siRNA-like knockdown). Could you please clarify the exact design rationale for siHuD and shHuD, specify the degree of complementarity for each, and discuss how this might affect the comparability of the phenotypes observed (in the legenda of the figure)?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Ryu and co-authors describes the role of the RNA binding protein HuD (ELAVL4) in restraining metabolic stress and senescence. This study builds on their earlier work showing negative effects of HuD deletion on fatty acid oxidation (FAO) and senescence signaling.

The data presented in the current manuscript make a compelling case for the likely role of HuD in protecting neuronal cells from oxidative stress and limiting senescence. The manuscript is well-written. However, the experimental approach is rather limited. The authors demonstrated the effects of HuD suppression, but experiments demonstrating effects of overexpressing or stimulating HuD on palmitate driven  senescence were not performed. This would strengthen the authors’ hypothesis that HuD plays an important role in neuronal metabolism and aging.

A few points that should be addressed in Discussion:

1. HuD does not exclusively bind to PPARα mRNA, but can also post-transcriptionally regulate mRNAs via other neuronal proteins. Are the effects of HuD on FAO and neuronal senescence exclusively mediated by PPARα, or can other mRNAs also be involved?
2. How is HuD regulated and in which disease conditions might HuD be involved? Are there any data on HuB upregulation in human conditions?

Minor concerns in the figures:

Fig.1 Legend: “N2a cells transfected with control siRNA (si Control and si HuD). Please remove control.

Fig.4F Protein band quantification is missing

Fig.5B should be replaced. The blue color is difficult to see.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In the manuscript entitled “Loss of HuD Sensitizes Neuroblastoma Cells to Palmitate- 2 Driven Stress-Induced Premature Senescence Via PPARα 3 Downregulation and FAO Impairment”, the experimental results of this paper are outstanding in both the depth and breadth of their mechanistic exploration, particularly in elucidating the regulatory relationship between HuD, miRNAs, and PPARα. The work in this area is very solid. However, the core causal logic chain has a significant gap due to the absence of a PPARα rescue experiment. Totally, this is an interesting work, and is also informative. However, there are some major problems that the authors should well address.

 

Limitations and Suggestions for Improvement

1.The study's conclusions are drawn exclusively from experiments using the Neuro2a neuroblastoma cell line. As a cancer cell line, its metabolic and proliferative properties are substantially different from those of normal, post-mitotic neurons. Therefore, generalizing findings on senescence from this model to physiological neuronal aging or neurodegenerative diseases is a significant leap that requires caution.

Suggestion: The authors should add a section in the Discussion to explicitly address the limitations of using the Neuro2a cell line. To significantly strengthen the study's conclusions and physiological relevance, validating key findings in primary neurons would be highly beneficial.

 

2.The "Missing Link" in the Causal Chain—The Rescue Experiment: This is the main weakness in the study's experimental design. The paper establishes a clear pathway: "HuD loss → PPARα downregulation → FAO impairment → mitochondrial dysfunction → cellular senescence". While each step in this logical chain is supported by data, the links remain correlational rather than definitively causal.

Suggested Improvement: It is essential to perform a key rescue experiment to establish causality. Specifically, in HuD-knockdown cells, the authors should exogenously overexpress a version of PPARα that is not regulated by HuD or the identified miRNAs. They should then observe if this overexpression can:

Restore FAO activity (e.g., the OCR value in a Seahorse assay)?

Alleviate mitochondrial damage (e.g., reduce ROS levels)?

Inhibit cellular senescence (e.g., reduce the percentage of SA-β-gal positive cells)?

If overexpressing PPARα reverses these phenotypes caused by HuD knockdown, the conclusion that "PPARα downregulation is the core reason for cellular senescence following HuD loss" would be definitively proven. Without this experiment, a reviewer could reasonably question whether these downstream phenotypes are caused by other, unknown targets regulated by HuD.

 

3.Insufficient Experimental Support for the "Feed-forward Loop" Concept: The discussion proposes an insightful "metabolic–senescence feed-forward loop" hypothesis, suggesting that senescence itself can, in turn, exacerbate metabolic dysfunction. However, all current experimental results only demonstrate a one-way path from "metabolic stress" to "senescence."

Suggested Improvement: To support the claim of a "vicious cycle," additional experiments are needed to prove the reverse pathway. For example, the authors could induce senescence using an alternative method (e.g., low-dose doxorubicin treatment) and then examine whether the expression and function of the HuD-PPARα axis are subsequently inhibited. If senescent cells themselves downregulate HuD or PPARα, then the "feed-forward loop" hypothesis would have experimental backing.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor and Authors,

Thank you for the thorough and careful revision. The manuscript has improved substantially, and my main concerns have been appropriately addressed.

In particular, the focus on the HuD PPARα axis is now well justified and clearly framed as model- and target- specific, and the senescence terminology has been properly revised to reflect a stress induced, senescence-like phenotype rather than irreversible arrest.

I only have two minor remaining suggestions:

The 6-hour recovery window used in the washout experiment is relatively short to assess stability of growth arrest. A longer-term recovery or proliferation assay (e.g., EdU/Ki67 after stress removal) would strengthen this point. However, since irreversibility is no longer claimed, this is acceptable in its current form.

Providing the exact shRNA targeting sequence(s) in the Supplementary Materials would further improve transparency and reproducibility.

These are minor points and do not affect the overall conclusions.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

This manuscript was returned to the authors for major revision; however, the major concerns raised by this reviewer do not appear to have been  addressed. The important  experiments are still missing.”

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your thorough and thoughtful revision. I am satisfied with your response.

Author Response

Please see the attachment.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was significantly improved and I don't have any further concerns.