TrkB Agonist Treatment Decreases Hippocampal Testosterone Contents in a Sex-Dependent Manner Following Neonatal Hypoxia and Ischemia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting and well-written manuscript addressing sex-specific neurosteroid regulation following neonatal hypoxia–ischemia (HI) and its modulation by the TrkB agonist 7,8-DHF. The topic is timely, the methodological approach (LC-MS/MS for hippocampal neurosteroid quantification) is solid, and the manuscript is generally clear and well-structured.

However, while the experimental design is sound and the data set is rich, the manuscript requires substantial revision to strengthen mechanistic interpretation, improve contextualization within the neurotrophin literature, and refine several aspects of the discussion.

First of all, I'd like to underline the strengths: 

• The study is technically rigorous, particularly the steroid quantification pipeline, which is a major improvement over classical immunoassays.

• The inclusion of both sexes, two time points, and multiple HI conditions (sham, HO, HI, HI+DHF) adds robustness.

• Figures are well designed and data presentation is clear.

• The question addressed is highly relevant.

• My point-by-point revision:

  1. The Introduction and/or Discussion still miss critical concepts on:

  • Neurotrophin–steroid crosstalk, including how neurotrophins interact with androgen/estrogen signalling;

  • Neurite-outgrowth mechanisms involving small GTPases inactivation/activation;

  • The interplay between Trk receptors and cytoskeletal remodeling proteins ?!?

  These topics directly relate to the authors’ core hypothesis on sex-dependent signaling and would help frame their findings.
  Several studies have demonstrated non-canonical interactions between neurotrophin receptors, androgens, and cytoskeletal mediators, and citing such work would considerably strengthen the foundation of the manuscript.

   

  2. The Discussion often does not adequately explain the results:

  • Why does DHF lower hippocampal testosterone specifically in females?

  • How might local aromatase activity be influenced by neurotrophin signaling?

  • How does shift in neurosteroid profiles relate to the well-documented sex-dependent downstream signalling of Trk receptors?

  A stronger mechanistic narrative is needed. For example, integrating known pathways wherein neurotrophins modulate small GTPases, steroid-dependent receptor complexes, or cytoskeletal scaffolds during neuronal differentiation and injury responses.

  3. The manuscript heavily focuses on TrkB but omits important parallels from TrkA research, especially regarding:

  • androgen-dependent modulation of neurotrophin signaling,

  • neurite outgrowth 

  • bidirectional communication between neurotrophins and steroid signaling systems.

  Bringing in these aspects would contextualise their findings in a wider neurotrophin-steroid signalling framework.

  4. Some conclusions, particularly those suggesting a direct causal link between DHF-induced testosterone reduction and neuroprotection, are presented too strongly.
  Given the correlative nature of the dataset, these statements should be toned down or more clearly qualified.

  • Please, Some repetition in the Introduction could be removed for conciseness.

  • Methods are extremely detailed; consider moving some descriptions (e.g., homogenization steps) to Supplementary Methods.

  • Several p-value descriptions in the Results could be streamlined for readability.

   

  To improve mechanistic depth, the authors should consider citing studies on:

  • androgen/estrogens- and NGF-regulated neurite outgrowth via RhoA/Rac inactivation/activation;

  • AR/cytoskeleton proteins–TrkA cross-talk controlling neurotrophin-dependent differentiation;

  • neurotrophin-steroid interactions in systems beyond development (including pathological contexts where NGF/TrkA signaling promotes cellular plasticity and migration).

  These works would allow the authors to more effectively position their findings within the established neurotrophin signaling landscape and highlight conceptual parallels between TrkB and other Trk family members.

  The manuscript has clear potential, but it requires major revision to deepen the mechanistic interpretation, integrate essential neurotrophin biology, and contextualize the sex-specific effects observed. Once these issues are addressed, the study will provide a more compelling contribution to the field of neonatal neuroprotection and neurosteroid biology. 

   

  - In addition to the conceptual points discussed above, I believe the manuscript would benefit greatly from a few targeted experiments that could clarify some of the mechanistic questions that currently remain open. I list them here as constructive suggestions that would substantially strengthen the study 

  First, since the central interpretation of the manuscript relies on TrkB activation by 7,8-DHF, it would be extremely helpful to include a specificity control using a TrkB antagonist such as ANA-12. What does it happen after the co-administration of a TrkB blocker if we observe the DHF-induced reduction in hippocampal testosterone (and related molecular readouts)? It would provide strong evidence that the observed effects are indeed TrkB-dependent rather than off-target consequences of DHF.

  - The authors repeatedly suggest that changes in aromatase might underlie the sex-specific steroid modulation, yet aromatase expression or activity is never directly measured. Assessing aromatase levels and enzymatic activity in hippocampal tissue could clarify whether increased T→E2 conversion contributes to the phenotype, or whether alternative metabolic pathways should be considered. Even a basic analysis (qPCR, Western blot, or an activity assay) would add valuable mechanistic depth.

  - A rescue experiment would considerably reinforce the link between reduced testosterone and neuroprotection. For instance, supplementing HI + DHF animals with exogenous testosterone (or conversely, inhibiting aromatase) could test whether restoring testosterone levels reverses the protective effects of DHF. If protection is lost under these conditions, the causal chain becomes much more compelling.

  In addition, the manuscript would benefit from examining downstream cytoskeletal signaling. The neurotrophin literature has repeatedly demonstrated that RhoA and related cytoskeletal pathways are modulated following Trk activation and influence neurite growth, plasticity, and injury responses. Assessing RhoA-GTP levels, or markers such as phospho-cofilin or phospho-FAK, could help explain how DHF differentially affects male and female hippocampi at a cellular level.

  A complementary approach that the authors might consider is exploring the interaction between Trk receptors and steroid signaling components. There is growing evidence that neurotrophin receptors engage in cross-talk with androgen receptors and cytoskeletal scaffolding proteins such as Filamin A. Co-immunoprecipitation or proximity ligation assays from hippocampal tissue could reveal whether such complexes are altered after HI or DHF treatment, providing a bridge between neurotrophin signaling and sex hormone regulation.

  Finally, the authors could gain additional insight by identifying the specific cell types responsible for neurosteroid modulation. Whether the observed changes are primarily neuronal or glial in origin has important implications for interpretation. 

  I emphasize that these recommendations are offered to help the authors sharpen the mechanistic narrative and increase the impact of this very interesting study. Even addressing one or two of these points (particularly the TrkB specificity experiment or the aromatase analyses) would substantially strengthen the manuscript.

Author Response

Comments :

This is an interesting and well-written manuscript addressing sex-specific neurosteroid regulation following neonatal hypoxia–ischemia (HI) and its modulation by the TrkB agonist 7,8-DHF. The topic is timely, the methodological approach (LC-MS/MS for hippocampal neurosteroid quantification) is solid, and the manuscript is generally clear and well-structured.

However, while the experimental design is sound and the data set is rich, the manuscript requires substantial revision to strengthen mechanistic interpretation, improve contextualization within the neurotrophin literature, and refine several aspects of the discussion.

First of all, I'd like to underline the strengths: 

• The study is technically rigorous, particularly the steroid quantification pipeline, which is a major improvement over classical immunoassays.
• The inclusion of both sexes, two time points, and multiple HI conditions (sham, HO, HI, HI+DHF) adds robustness.
• Figures are well designed and data presentation is clear.
• The question addressed is highly relevant.
• My point-by-point revision:

1. The Introduction and/or Discussion still miss critical concepts on:
   • Neurotrophin–steroid crosstalk, including how neurotrophins interact with androgen/estrogen signalling;
   • Neurite-outgrowth mechanisms involving small GTPases inactivation/activation;
   • The interplay between Trk receptors and cytoskeletal remodeling proteins ?!?

These topics directly relate to the authors’ core hypothesis on sex-dependent signaling and would help frame their findings.
Several studies have demonstrated non-canonical interactions between neurotrophin receptors, androgens, and cytoskeletal mediators, and citing such work would considerably strengthen the foundation of the manuscript.

Response:

We agree with the reviewer that the Introduction and Discussion would be improved by expanding their scope to include the roles of neurotrophin-steroid crosstalk and the interplay of Trk receptors and cytoskeletal proteins. We have included the following paragraphs starting at line 95 in the introduction (in red) as written below.

“It is noteworthy, that a body of experimental research indicates that sex steroids play an essential role in neuronal circuit formation and maintenance, via cross talk with the neurotrophin BDNF (Murawska-Cialowicz, Inter. J. Mol. Sci, 2025). In the hippocampus, BDNF has a role in neuronal proliferation, differentiation, and homeostasis throughout the brain’s developmental stages. It has been established that estrogen regulates BDNF expression via both classical nuclear receptors and by the activation of membrane-associated ERs and subsequent up regulation of second messenger signaling systems (Lannigan, Steroids, 2003). It has been proposed that the estrogen/ER system is able to regulate the BDNF/TrkB system, while at the same time the inhibitory effect of the TrkB/PI3K/AKT pathway on ERE transcription implies that the BDNF signal is able to limit and control BDNF expression (C. S. Weickert, Mol Cell.Neurosci, 2011). While the progesterone receptor has been less studied, it has been shown that activation of the classical nuclear progesterone receptor in neuronal cells increases BDNF synthesis (Singh, Endocrinology, 2009). On the other hand, while testosterone does have a role in regulating the BDNF gene it is indirect, via the activation of classical androgen receptors which upregulate androgen-responsive genes upstream of BDNF (Z. Wang, Am. J. Clin. Exp Urol, 2021). In addition, it is interesting that the BDNF/TrkB system positively regulates the expression of androgen receptors in neurons, enhancing testosterone’s regulation of the BDNF gene (D. R. Sengelaub, Endocrinology, 2004).

In addition, it has also been reported besides TrkB, the TrkA receptor has a role in the crosstalk between sex receptors and neurotrophins that can promote neuritogenesis. In unchallanged PC12 cells, both the synthetic androgen, R1881, and nerve growth factor can promote neurite outgrowth via inducing the formation of the TrkA/androgen receptor/fliamin A complex and subsequent inactivation of RhoA. When the RhoA effector, ROCK, is pharmacologically inhibited, it promotes both androgen and nerve growth factor-induced neurite outgrowth thus confirming the importance of TrkA in sex receptor/neurotrophin interaction (Donato, Cells, 2023).”

2. The Discussion often does not adequately explain the results:

• Why does DHF lower hippocampal testosterone specifically in females?
• How might local aromatase activity be influenced by neurotrophin signaling?
• How does shift in neurosteroid profiles relate to the well-documented sex-dependent downstream signalling of Trk receptors?

A stronger mechanistic narrative is needed. For example, integrating known pathways wherein neurotrophins modulate small GTPases, steroid-dependent receptor complexes, or cytoskeletal scaffolds during neuronal differentiation and injury responses.

3. The manuscript heavily focuses on TrkB but omits important parallels from TrkA research, especially regarding:

• androgen-dependent modulation of neurotrophin signaling,
• neurite outgrowth 
• bidirectional communication between neurotrophins and steroid signaling systems.

Bringing in these aspects would contextualise their findings in a wider neurotrophin-steroid signalling framework.

Response:

We thank this reviewer for bringing this issue up. We have addressed this by adding a paragraph in the Introduction  highlighting the role of TrkA in androgen/neurotrophin crosstalk (line 113) in red.

4. Some conclusions, particularly those suggesting a direct causal link between DHF-induced testosterone reduction and neuroprotection, are presented too strongly.
   Given the correlative nature of the dataset, these statements should be toned down or more clearly qualified.

• Please, Some repetition in the Introduction could be removed for conciseness.

Response:

We thank the reviewer for this comment and have removed several sentences in the Introduction to make it more concise.

 

• Methods are extremely detailed; consider moving some descriptions (e.g., homogenization steps) to Supplementary Methods

 

Response:

We agree with the reviewer that the LCMS-MS method is extremely detailed. We have moved the entire method to Supplementary Methods and revised the LCMS-MS method descripted in the text (line 195).

 

Several p-value descriptions in the Results could be streamlined for readability.

We have addressed this by editing the paragraphs describing the multivaritate analysis to help with readability.

 To improve mechanistic depth, the authors should consider citing studies on:

• androgen/estrogens- and NGF-regulated neurite outgrowth via RhoA/Rac inactivation/activation;
• AR/cytoskeleton proteins–TrkA cross-talk controlling neurotrophin-dependent differentiation;
• neurotrophin-steroid interactions in systems beyond development (including pathological contexts where NGF/TrkA signaling promotes cellular plasticity and migration).

These works would allow the authors to more effectively position their findings within the established neurotrophin signaling landscape and highlight conceptual parallels between TrkB and other Trk family members.

The manuscript has clear potential, but it requires major revision to deepen the mechanistic interpretation, integrate essential neurotrophin biology, and contextualize the sex-specific effects observed. Once these issues are addressed, the study will provide a more compelling contribution to the field of neonatal neuroprotection and neurosteroid biology. 

- In addition to the conceptual points discussed above, I believe the manuscript would benefit greatly from a few targeted experiments that could clarify some of the mechanistic questions that currently remain open. I list them here as constructive suggestions that would substantially strengthen the study 

First, since the central interpretation of the manuscript relies on TrkB activation by 7,8-DHF, it would be extremely helpful to include a specificity control using a TrkB antagonist such as ANA-12. What does it happen after the co-administration of a TrkB blocker if we observe the DHF-induced reduction in hippocampal testosterone (and related molecular readouts)? It would provide strong evidence that the observed effects are indeed TrkB-dependent rather than off-target consequences of DHF.

- The authors repeatedly suggest that changes in aromatase might underlie the sex-specific steroid modulation, yet aromatase expression or activity is never directly measured. Assessing aromatase levels and enzymatic activity in hippocampal tissue could clarify whether increased T→E2 conversion contributes to the phenotype, or whether alternative metabolic pathways should be considered. Even a basic analysis (qPCR, Western blot, or an activity assay) would add valuable mechanistic depth.

- A rescue experiment would considerably reinforce the link between reduced testosterone and neuroprotection. For instance, supplementing HI + DHF animals with exogenous testosterone (or conversely, inhibiting aromatase) could test whether restoring testosterone levels reverses the protective effects of DHF. If protection is lost under these conditions, the causal chain becomes much more compelling.

In addition, the manuscript would benefit from examining downstream cytoskeletal signaling. The neurotrophin literature has repeatedly demonstrated that RhoA and related cytoskeletal pathways are modulated following Trk activation and influence neurite growth, plasticity, and injury responses. Assessing RhoA-GTP levels, or markers such as phospho-cofilin or phospho-FAK, could help explain how DHF differentially affects male and female hippocampi at a cellular level.

A complementary approach that the authors might consider is exploring the interaction between Trk receptors and steroid signaling components. There is growing evidence that neurotrophin receptors engage in cross-talk with androgen receptors and cytoskeletal scaffolding proteins such as Filamin A. Co-immunoprecipitation or proximity ligation assays from hippocampal tissue could reveal whether such complexes are altered after HI or DHF treatment, providing a bridge between neurotrophin signaling and sex hormone regulation.

Finally, the authors could gain additional insight by identifying the specific cell types responsible for neurosteroid modulation. Whether the observed changes are primarily neuronal or glial in origin has important implications for interpretation. 

I emphasize that these recommendations are offered to help the authors sharpen the mechanistic narrative and increase the impact of this very interesting study. Even addressing one or two of these points (particularly the TrkB specificity experiment or the aromatase analyses) would substantially strengthen the manuscript.

Response:

We thank the reviewer for the many insight comments on future directions for our research. While we are not currently able to perform the suggested experiments, we agree that it would be helpful to mention these limitations and the questions we hope to address in subsequent investigations. Thus, the following sentences have been added to the discussion, begining at line 455 as written below.

“There are several limitations to this study that need to be addressed. First, we cannot rule out that the observed effects of DHF are not only TrkB-dependent but could be explained by off-target consequences of the drug. In future experiments, we plan to use the TrkB antagonist ANA-12 post- HI to answer this question.  Second, in this study we do not directly measure aromatase expression. Determination of aromatase activity would confirm the primary importance of T to E2 conversion to explaining our observations as opposed to alternative metabolic pathways. Future studies will include examination of hippocampal aromatase levels or activity to improve the mechanistic strength of our conclusions. Third, the link between reduced T and neuroprotection would be confirmed by applying exogenous T in the presence of DHF post-HI to see if that protection is lost. Future directions in our research will also include experiments to examine the effect of TrkB activation on cytoskeletal pathways such as RhOA which could influence injury responses and explain the sexually differential effect of DHF (Migliaccio, Cells, 12, page 373, 2023). In addition, investigating the effect of DHF on possible cross-talk between androgen receptors and cytoskeletal scaffolding proteins post-HI could illuminate the interaction between Trk receptors (TrkB and TrkA) and sex steroids (Migliaccio, Cells, 12, page 373, 2023)(Murawska-Cialowicz, In J. Mol Sci, 26, 2532, 2025). It is interesting that it has been reported that in PC12 cells that TrkA regulates neurite outgrowth via interaction with a complex involving the androgen receptor, the signaling effector, filamin A, andb1 integrin. The complex has a role in regulating GTPase signalling and inducing neuritogeneis in these cells. (Donato, Mol Bio of the Cell, 2015).   Lastly, this study cannot differentiate between cell types in the hippocampus. Further experiments, using RNAseq techniques would be invaluable in determining the relative contributions of neurons and glial cells to our observations.”

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for this work, which provides a better understanding of the pathophysiological mechanisms of hypoxia-ischemia.
I have no specific comments, but simply regret that the link with potential clinical applications or future areas of research is not mentioned in the discussion.

Author Response

Comments and Suggestions for Authors

Thank you for this work, which provides a better understanding of the pathophysiological mechanisms of hypoxia-ischemia.
I have no specific comments, but simply regret that the link with potential clinical applications or future areas of research is not mentioned in the discussion.

Response:

To address the reviewers concerns we have added a paragraph in the Discussion outlining our future areas of research (line 456).

Reviewer 3 Report

Comments and Suggestions for Authors

In the study, Aycan and colleagues described the use of a high-specific technique (LC-MS/MS) to detect alterations in plasma and hippomcapi of steroids in mice exposed to HI model in the seearch for sexually-dimporhic differences differences. Authors used also an agonist of Trk-B receptor to check its protective potential in plasma and HC. The study is well designed, the results are sound and can contribute to the better understanding of the neurosteroids role during development in both normal and injured animals. The literature presented covers relevant publcations on the theme and sustain the conclusions in part of the results. A few major commments need to be addressed before publication.

Major:

1 - The sentence "Both hippocampi were then identified and harvested [31,32]. The 164 hippocampi (IL and CL) from two mice (n=1) are separately pooled and stored at -80C to 165 optimize the LC-MS/MS measurements (Figure 1)." Did authors optimize LC-MS using an HI animal? Isn't it counterintuitive, since you oberved effects of the hypoxia-only as weel for most of measurements, and using only one animal? Naïve animals (of both sexes) should have been be considered for this purpose since even animals with only hypoxia showed alterations in most of the analysis.

2 - The weakness of statistical power of a 4-way anova with so few (and different) number of animals / group needs to be assumed and stated in the description of the statistical analysis. Authors must describe in more detail the distribution of animals in each measurement (Table 1, for instance, could include the n on the side of each age - P10 and P12). 

4 - Authors could highlight the need for a long-term behavioral and histological follow-up in order to describe if DHF has efective neurprotective effects in the model.

Minor

1 - Please standardize the use of the (*) in the figures, or delete the non-significant differences. 

Author Response

Comments and Suggestions for Authors

In the study, Aycan and colleagues described the use of a high-specific technique (LC-MS/MS) to detect alterations in plasma and hippomcapi of steroids in mice exposed to HI model in the seearch for sexually-dimporhic differences differences. Authors used also an agonist of Trk-B receptor to check its protective potential in plasma and HC. The study is well designed, the results are sound and can contribute to the better understanding of the neurosteroids role during development in both normal and injured animals. The literature presented covers relevant publcations on the theme and sustain the conclusions in part of the results. A few major commments need to be addressed before publication.

Major:

1 - The sentence "Both hippocampi were then identified and harvested [31,32]. The 164 hippocampi (IL and CL) from two mice (n=1) are separately pooled and stored at -80C to 165 optimize the LC-MS/MS measurements (Figure 1)." Did authors optimize LC-MS using an HI animal? Isn't it counterintuitive, since you oberved effects of the hypoxia-only as weel for most of measurements, and using only one animal? Naïve animals (of both sexes) should have been be considered for this purpose since even animals with only hypoxia showed alterations in most of the analysis.

Response:

We agree with the reviewer that this sentence needs clarification. Starting at line 182,  the text now reads: “LC-MS/MS method optimization was performed using tissue from naive mice of both sexes to establish baseline parameters before analyzing experimental samples.The hippocampi (IL and CL) from two mice (n=1) are separately pooled and stored at -80C to 165 until the analysis using the LC-MS/MS (Figure 1).”

2 - The weakness of statistical power of a 4-way anova with so few (and different) number of animals / group needs to be assumed and stated in the description of the statistical analysis.

Response:

 

We thank the reviewer for bring this to our addtion. For clarifcation we have added the following sentences to the Statistical Anaylsis.. “Although the sample sizes may be too small to detect very subtle differences, we believe that the four-way ANOVA, along with the corresponding sliced interaction contrasts, provides the most sensitive approach for the comparisons of interest. This modeling strategy offers a comprehensive framework and results in smaller standard errors.” Line 234)

Authors must describe in more detail the distribution of animals in each measurement (Table 1, for instance, could include the n on the side of each age - P10 and P12). 

Response:

We thank the reviewer for bringing this to our antention. We agree that the sample sizes for the various measurements and experimental conditions are important pieces of information. The sample sizes for each measurement and condition are shown in Table 1 in the parenthesis , following the means± standard errors. We added to totals in the top row of the revised Table 1.

4 - Authors could highlight the need for a long-term behavioral and histological follow-up in order to describe if DHF has efective neurprotective effects in the model.

Response:

We draw the reviewers attention to the second paragraph on the Introduction where we cite studies that show DHF treatment post-neonatal HI improves hippocampal-dependent memory and learning in young adult females.

Minor

1 - Please standardize the use of the (*) in the figures, or delete the non-significant differences.

Response:

For clarity, we indicated significant differences between sexes in the same group by the use (*). Specific p values are used in the text. We do agree that p values greater than 0.05 should be eliminated from figures and have done so.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Ok