A2-Astrocyte Activation by Short-Term Hypoxia Rescues α-Synuclein Pre-Formed-Fibril-Induced Neuronal Cell Death

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Ha Nyeoung Choi et al., titled “A2-astrocyte activation by short term hypoxia rescue α-synuclein preformed fibril induced neuronal cell death,” is an exciting study. This manuscript is well-written and can be considered for publication with Major edits.

Major comments:

1. Figure 1 a, western blot images of the HIF-1α band at 0 hr are missing in astrocytes under long-term hypoxia; hence please provide a clear WB image.
2. In Figure 3. b, 3 e, 4b, 4e, neuronal morphology is not clear; hence, please provide the image with MAP-2 or Pan Neuronal Marker or any other clear marker showing proper cortical neuronal morphology. It will help readers appreciate the effect of A2-astrocytes on cortical neurons.
3. Authors can discuss the mechanism of A2-specific transcripts in neuroprotection in PD.

 

Minor Comments-

- Line 133, Can be renamed as "TUNEL staining/assay"

- Line 147, the method could be titled "Cell viability assay" or "AlamarBlue Assay"

- Lines 155, 160 and 176- Immunofluorescence, Western blot and Stastiscs may be numbered as 2.10, 2.11 and 2.12 respectively as they are part of subtitle 2. Materials and Methods

- Line 262- The result can be numbered as 3.5

Author Response

Point 1:  Figure 1 a, western blot images of the HIF-1α band at 0 hr are missing in astrocytes under long-term hypoxia; hence please provide a clear WB image.

Response 1: Thank you for your valuable comment. Hypoxia-inducible factor (HIF) is a nuclear transcription factor that acts as a key regulator in response to hypoxia. Hypoxia-inducible factor 1 (HIF-1) consists of HIF-1 alpha and HIF-1 beta, and is hydroxylated by dioxygenase prolyl hydroxylase (PHD) under normoxic conditions. Hydroxylated HIF is degraded in the proteasome by the von Hippel-Lindau tumor suppressor protein (VHL) through a polyubiquitination process [1-2]. Many studies, including in vivo and in vitro studies, have shown that Western blot bands do not appear under normoxic conditions [3-4]. We hope this explanation addresses your concern. Point 2: In Figure 3. b, 3 e, 4b, 4e, neuronal morphology is not clear; hence, please provide the image with MAP-2 or PanNeuronal Marker or any other clear marker showing proper cortical neuronal morphology. It will help readers appreciate the effect of A2-astrocytes on cortical neurons.   Response: We appreciate your comment and agree that the neuronal morphology in Figures 3b, 3e, 4b, and 4e could be improved for clarity. To address this, we have modified these images to better illustrate the cortical neuronal morphology. As for cortical neuronal markers, we used Tuj-1, which reacts with tubulin-beta 3, a widely utilized marker in both in vivo and in vitro studies to identify cortical neuronal morphology [1-3]. Tubulin-beta 3 is a microtubule protein that constitutes a major component of the cytoskeleton, is predominantly expressed in neurons, and plays a critical role in neuronal development and axonal growth [4-5]. Therefore, we believe that Tuj-1 is an appropriate and reliable marker to clearly demonstrate cortical neuronal morphology. We hope this explanation adequately addresses your concern.   Point 3: Authors can discuss the mechanism of A2-specific transcripts in neuroprotection in PD.   Response: Thank you for your valuable comment. We propose that suppressing neurotoxic A1 astrocytes while enhancing protective A2 astrocytes has a neuroprotective effect in PD. To address this, we have added a detailed explanation in the Discussion section as follows, linking A1/A2 astrocytes to PD pathogenesis.   FROM “Conversely, Tgm1, Ptx3, Sphk1, and Emp1 are A2 astrocyte markers that encode proteins regulating immune response, signal transduction, and cell cycle. These proteins demonstrated increased expression after ischemic insult in the in vivo model, which overlaps our results [7]. Recent studies have shown that astrocyte dysfunction contributes to PD pathogenesis, and controlling activated astrocyte phenotype could help ameliorate PD [17],[18]. It is also possible that there is a spectrum of activated astrocytes with A1 and A2 types being its extreme, and cellular milieu determines the predominant phenotype [8]. Our results suggest that conditioned hypoxia exhibits therapeutic potential for PD by promoting A2 astrocytes while suppressing A1 astrocytes.”   TO “Conversely, Tgm1, Ptx3, Sphk1, and Emp1 are A2 astrocyte markers that encode proteins involved in immune response regulation, signal transduction, and cell cycle control. These proteins show increased expression following ischemic insult in vivo, consistent with our findings [7]. Recent studies suggest that astrocyte dysfunction contributes to PD pathogenesis, and modulating astrocyte phenotypes may help mitigate disease progression [17],[18]. Specifically, microglia activate A1 astrocytes through IL-1α, TNF, and C1q, leading to complement cascade upregulation and secretion of neurotoxins that induce rapid neuronal death [19]. In contrast, A2 astrocytes promote neuronal recovery by upregulating neurotrophic factors and cytokines such as CLCF1, LIF, IL6, and thrombospondins, which support neuronal function [7,19]. Moreover, A2 astrocytes induce the neuroprotective nuclear factor erythroid 2-related factor (Nrf2) and enhance glutamate uptake, thereby preventing excitotoxicity [20]. Our results suggest that conditioned hypoxia holds therapeutic potential for PD by promoting A2 astrocytes and suppressing A1 astrocytes.”

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Choie et al. investigates the effects of short-term hypoxia on astrocytes and its potential neuroprotective role in Parkinson’s disease (PD). While the premise is intriguing, several significant issues render the study unsuitable for publication in its current form.

·       First, the claim that smoking may have a protective effect in PD due to intermittent hypoxia is scientifically flawed. Smoking is a well-established risk factor for various diseases, including PD, and suggesting a potential protective role due to hypoxia is misleading. This statement should be revised or removed to avoid misinterpretation.

·       Additionally, the study uses whole-brain astrocyte cultures but does not purify astrocytes from other cell types, which introduces confounding variables. Purifying astrocytes is essential, and the authors should validate the purity of their cultures, ideally excluding other neuronal cell types through methods such as flow cytometry.

·       There is also insufficient characterization of A1 and A2 astrocyte subtypes in response to hypoxia. To strengthen their findings, the authors should employ  purified astrocytes to measure both mRNA and protein expression of  the markers associated with  A1 and A2 astrocytes.  Again use the  flow cytometry to differentiate and quantify these subtypes.

·       Furthermore, the mechanism underlying the observed reduction in α-synuclein aggregation remains unclear.

·       The study does not involve dopaminergic neurons, which are particularly relevant in PD research. The authors should consider incorporating dopaminergic models to make the findings more directly applicable to PD. Moreover, the manuscript does not address the implications for GBA-associated PD, a major PD subtype linked to genetic mutations, which represents a significant portion of the PD population.

·       It is strongly recommended that the authors add a table summarizing the impact of hypoxia on cytokine production and the damage to various neuronal cell subsets, including microglial cells, primary neurons, and dopaminergic neurons, across mouse models, cell cultures, and PD patients. This table should also explain how these effects vary between genetic and idiopathic forms of PD. Additionally, Table 1 should be removed from the main text and moved to the supplementary material.

·       The manuscript contains typographical errors and unclear phrasing that require attention. For instance, the Animal section does not adequately explain the reference to ICR mice (Lines 82 and 93). The authors should clarify terms and ensure consistency throughout the manuscript.

·       Overall, the manuscript contains significant methodological flaws, and the experimental design lacks the necessary rigor. The authors must address these issues and provide more robust data before the manuscript can be reconsidered for publication.

Comments on the Quality of English Language

The grammar and clarity of the manuscript should be carefully reviewed and improved to ensure it meets the standards for publication.

Author Response

Point 1: First, the claim that smoking may have a protective effect in PD due to intermittent hypoxia is scientifically flawed. Smoking is a well-established risk factor for various diseases, including PD, and suggesting a potential protective role due to hypoxia is misleading. This statement should be revised or removed to avoid misinterpretation.

 

Response: We apologize for any misinterpretation caused by the original wording. As per your suggestion, we have removed “smoking” from the statement.

 

FROM

“Smoking and exercise, well-known protective lifestyle factors in PD, have in common that the brain is exposed to acute intermittent hypoxia, which supports hypoxia-mediated disease-modifying effects [4].”

 

TO

“Exercise, a well-known protective lifestyle factor in PD, involves exposing the brain to acute intermittent hypoxia, which supports hypoxia-mediated disease-modifying effects [4].”

Point 2: Additionally, the study uses whole-brain astrocyte cultures but does not purify astrocytes from other cell types, which introduces confounding variables. Purifying astrocytes is essential, and the authors should validate the purity of their cultures, ideally excluding other neuronal cell types through methods such as flow cytometry.

 

Response: Thank you for your comment. We appreciate your suggestion and agree that purified astrocytes could provide a clearer understanding of their contributions to PD. However, we have employed the same methodology in this study as in our previous research—separating an astrocyte-rich fraction from a microglia-rich fraction using the EasySep mouse CD11b positive selection kit (StemCell, Canada) and culturing the pour-off fraction of astrocytes [1-5]. This approach has proven effective in demonstrating astrocyte activation in the past. Additionally, obtaining purified astrocytes would require at least three months, which is not feasible given our current time constraints. We hope this explanation addresses your concern.

Point 3: There is also insufficient characterization of A1 and A2 astrocyte subtypes in response to hypoxia. To strengthen their findings, the authors should employ purified astrocytes to measure both mRNA and protein expression of the markers associated with A1 and A2 astrocytes. Again use the flowcytometry to differentiate and quantify these subtypes.

 

Response: Thank you for your comment. As we stated in the previous response above, we have employed the same methodology in this study as in our previous research which has proven effective in demonstrating astrocyte activation in the past [1-5]. Additionally, obtaining purified astrocytes would require at least three months, which is not feasible given our current time constraints. We hope this explanation addresses your concern.

Point4: Furthermore, the mechanism underlying the observed reduction in α-synuclein aggregation remains unclear.

 

Response: Thank you for your insightful comment. In our study, we demonstrated that short-term hypoxia reduces toxic p-α-syn expression by activating protective A2 astrocytes while suppressing neurotoxic A1 astrocytes. As you noted, we did not perform additional experiments to directly establish a link between A2 astrocytes and p-α-syn reduction. However, it is well established that both microglia and astrocytes play critical roles in PD pathogenesis, and we have added a detailed explanation in the Discussion section, linking A1/A2 astrocytes to PD pathogenesis as follows.

 

FROM

“Recent studies have shown that astrocyte dysfunction contributes to PD pathogenesis, and controlling activated astrocyte phenotype could help ameliorate PD [17],[18]. It is also possible that there is a spectrum of activated astrocytes with A1 and A2 types being its extreme, and cellular milieu determines the predominant phenotype [8]. Our results suggest that conditioned hypoxia exhibits therapeutic potential for PD by promoting A2 astrocytes while suppressing A1 astrocytes.”

 

TO

“Recent studies suggest that astrocyte dysfunction contributes to PD pathogenesis, and modulating astrocyte phenotypes may help mitigate disease progression [17,18]. Specifically, microglia activate A1 astrocytes through IL-1α, TNF, and C1q, leading to complement cascade upregulation and secretion of neurotoxins that induce rapid neuronal death [19]. In contrast, A2 astrocytes promote neuronal recovery by upregulating neurotrophic factors and cytokines such as CLCF1, LIF, IL6, and thrombospondins, which support neuronal function [7,19]. Moreover, A2 astrocytes induce the neuroprotective nuclear factor erythroid 2-related factor (Nrf2) and enhance glutamate uptake, thereby preventing excitotoxicity [20]. Our results suggest that conditioned hypoxia holds therapeutic potential for PD by promoting A2 astrocytes and suppressing A1 astrocytes.”

Point5: The study does not involve dopaminergic neurons, which are particularly relevant in PD research. The authors should consider incorporating dopaminergic models to make the findings more directly applicable to PD. Moreover, the manuscript does not address the implications for GBA-associated PD, a major PD subtype linked to genetic mutations, which represents a significant portion of the PD population.

 

Response: Thank you for your precious comment.

First, we acknowledge that incorporating dopaminergic neurons would make this research more directly relevant to PD. However, dopaminergic neurons account for less than 1% of the total neuronal population in the brain, requiring a substantial number of animal sacrifices. In future studies, we plan to use human iPS cell-derived dopaminergic neurons to address this limitation, which we have now included in the Discussion section as a study limitation as follows.

 

FROM

“The most critical limitation of this study is the lack of in vivo data. However, we used primary cells in which genomic and phenotypic stability was well validated.”

 

TO

“The most critical limitation of our study is the lack of in vivo data. Nevertheless, we used primary cells with well-validated genomic and phenotypic stability. In the future, we aim to extend our research to PD mouse models or human iPS cell-derived dopaminergic neurons to further validate our findings.”

Second, we fully agree that GBA-associated PD (GBA-PD) represents a significant subset of the overall PD population. GBA is a well-established genetic risk factor for PD, with a reported prevalence of 7%–10%, depending on ethnic variability. GBA mutations lead to reduced GCase activity in lysosomes, resulting in lysosomal dysfunction, impaired α-synuclein degradation, and enhanced α-synuclein aggregation.

In the Introduction, we specifically mentioned DJ-1, PINK1, PRKN, and LRRK2 because these genes are more directly linked to mitochondrial function, which may be particularly sensitive to hypoxia. While we acknowledge that GBA mutations indirectly affect mitochondrial function through dynamic mitochondria-lysosome interactions, we believe our findings can be broadly applied to both genetic and sporadic PD since astrocytes, as common glial cells, play a central role in the CNS. For this reason, we did not specifically focus on GBA-PD. Exploring whether short-term hypoxia has a unique beneficial role in GBA-PD is an intriguing direction for future research.

Point6: It is strongly recommended that the authors add a table summarizing the impact of hypoxia on cytokine production and the damage to various neuronal cell subsets, including microglial cells, primary neurons, and dopaminergic neurons, across mouse models, cell cultures, and PD patients. This table should also explain how these effects vary between genetic and idiopathic forms of PD. Additionally, Table 1 should be removed from the main text and moved to the supplementary material.

 

Response: Thank you for your insightful comment. We agree that summarizing the impact of hypoxia on cytokines, cells, mouse models, and PD patients would greatly enhance readers’ understanding of this field. However, the significant variation in hypoxia intensities and protocols (acute, intermittent, or chronic) across studies and study subjects makes it difficult to comprehensively present all findings within the scope of our study. We believe this would be an excellent topic for a dedicated review article.

Furthermore, to the best of our knowledge, no research has directly compared the effects of hypoxia between genetic and idiopathic PD patients. However, we agree that certain genetic PD subtypes may exhibit greater sensitivity to hypoxia. For instance, PD patients with DJ-1 mutations are known to have reduced HIF-1α levels and impaired cellular ROS defense, which could influence their response to hypoxic conditions.

Finally, we moved Table 1 to Supplementary material (Table S1).

Point7: The manuscript contains typographical errors and unclear phrasing that require attention. For instance, the Animal section does not adequately explain the reference to ICR mice (Lines 82and 93). The authors should clarify terms and ensure consistency throughout the manuscript.

 

Response: Thank you for your detailed comments. I have revised the references and terminology for ICR mouse to clarify based on your suggestions.

 

FROM

“2.1. Animal

All experimental procedures were under the guidelines of the Laboratory Animal Manual, which was approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (No. GNU-210125-M0005). Our experiments were performed to reduce the number of mice and minimize their pain or discomfort. Mice were acclimated to a 12 hr light/dark cycle and had access to food and water ad libitum. The ambient temperature was maintained at 21 ± 23 °C, and humidity was maintained at 60 ± 10%.”

 

TO

“All experimental procedures complied with the guidelines outlined in the Laboratory Animal Manual and were approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (Approval No. GNU-210125-M0005). Efforts were made to minimize the number of mice used and to reduce their pain or discomfort throughout the study.

Pregnant ICR mice (13-14 days) were purchased from Koatech Inc.(Gyeonggi-do, South Korea). Mice were acclimated to a 12 hr light/dark cycle and had access to food and water ad libitum. The ambient temperature was maintained at 21 ± 23 °C, and humidity was maintained at 60 ± 10%.”

Point8: Overall, the manuscript contains significant methodological flaws, and the experimental design lacks the necessary rigor. The authors must address these issues and provide more robust data before the manuscript can be reconsidered for publication.

The grammar and clarity of the manuscript should be carefully reviewed and improved to ensure it meets the standards for publication.

 

Response: Thank you for your thorough and valuable comment. Your feedback has allowed us to improve our manuscript to the best of our ability. We hope that the revised version will be positively received by you and the journal.

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

With pleasure I am writing to you regarding your recently submitted manuscript. The paper focuses on astrocytes’ adaptation to hypoxia and its impact in Parkinson disease-like conditions in-vitro and is therefore much relevant to the field. Despite the manuscript being a well-written report of interesting new data, I think improvements can be made.

 Briefly, the “discussion” explores the therapeutic potential of inducing a hypoxia-like state as treatment or preventing treatment for PD (line 329-336), I think this paragraph focuses on hypoxic conditions which are not easily appliable to animal models or patients. Moreover, this paragraph does not acknowledge the molecular mechanisms of hypoxia response which could be a more viable target for therapeutic intervention in astrocytes and the central nervous system at large.

Furthermore, strong proof has been given of astrocytes phenotype change through qPCR for different markers, however, no consideration has been given to astrocytes morphology as astrocytes may vary significantly in shape following a variety of stimuli and treatments. It may be interesting to briefly explore astrocytes morphology change (or lack thereof) in-vitro or to quickly acknowledge this aspect in the “discussion” section.

Hoping my comments will be useful in improving the second draft of this already remarkable manuscript.

Author Response

Point1: Briefly, the “discussion” explores the therapeutic potential of inducing a hypoxia-like state as treatment or preventing treatment for PD (line 329-336), I think this paragraph focuses on hypoxic conditions which are not easily appliable to animal models or patients. Moreover, this paragraph does not acknowledge the molecular mechanisms of hypoxia response which could be a more viable target for therapeutic intervention in astrocytes and the central nervous system at large.

 

Response: Thank you for your insightful comment. As suggested, we revised the paragraph accordingly as follows.

 

FROM

“To date, few studies have applied hypoxia to cellular or mouse models of PD, and even more limited human studies have used hypoxia as a therapeutic intervention in PD [14],[19]. Moreover, optimal hypoxic conditions have not yet been established. Thus, it is important to verify an ideal hypoxic condition that maximises protective effects and minimises adverse effects in the following research. We would have obtained different results if we had adopted different hypoxia regimens, for example, increasing the frequency of hACM treatment per day or extending the duration of hACM treatment. Furthermore, the interval between the administration of PFF and hACM may have affected the overall results. In this study, hACM was added 10 days after PFF administration. Despite this delay, hACM demonstrated a protective effect against p-α-syn aggregation and neuronal death. It is hard to know whether hACM ceased p-α-syn accumulation, reversed the toxic aggregates, or both. However, it seems that hACM or short-term hypoxia potentially exhibits a disease-modifying effect for PD as 10 days post-PFF treatment is when p-α-syn induced neuronal dysfunction starts. Considering that the first PD motor symptoms appear when more than 50% of the dopaminergic neurons are lost [20], it is crucial to investigate whether hACM can halt PD progression in future studies.”

 

TO

“Mitochondrial dysfunction and oxidative stress are pivotal contributors to PD pathogenesis [21]. Dopaminergic neurons in the substantia nigra are particularly vulnerable to oxidative stress, and the antioxidant deficit observed in PD may exacerbate reactive oxygen species accumulation, ultimately leading to neuronal cell death [22,23]. Adaptive responses to hypoxia, including activation of HIF and Nrf2, mitigate oxidative stress, enhance α-synuclein clearance, and reduce nigrostriatal neurodegeneration [24,25]. These mechanisms have been targeted in several therapeutic interventions for PD [14]. Additionally, although it is unclear whether hACM ceased p-α-syn accumulation, reversed the toxic aggregates, or both, based on our study, hACM or short-term hypoxia potentially exhibits a disease-modifying effect for PD as 10 days post-PFF treatment marks the initiation of p-α-syn induced neuronal dysfunction. Considering that the first PD motor symptoms appear when more than 50% of the dopaminergic neurons are lost [26], it is crucial to investigate whether hACM can halt PD progression in future studies.”

 

Point2: Furthermore, strong proof has been given of astrocytes phenotype change through qPCR for different markers, however, no consideration has been given to astrocytes morphology as astrocytes may vary significantly in shape following a variety of stimuli and treatments. It may be interesting to briefly explore astrocytes morphology change (or lack thereof) in-vitro or to quickly acknowledge this aspect in the “discussion” section.

 

Response: We sincerely appreciate the reviewer’s thorough evaluation of our manuscript. We agree that astrocyte morphology can be influenced by various stimuli, and numerous studies, including both in vivo and in vitro research, have reported morphological changes in astrocytes in response to hypoxia-related stimuli [1-7]. However, our results did not reveal significant changes in astrocyte morphology under hypoxic conditions. This discrepancy may be due to inconsistencies between the hypoxia conditions used in the reference studies and those employed in our study. As such, we cannot rule out the possibility that the lack of morphological changes observed in our study reflects differences in experimental conditions compared to the references. Furthermore, while not specific to astrocytes, variations in cell density across different cell types may influence intracellular responses or produce different outcomes under external stimuli [8-13]. We also consider that astrocyte density in our experimental setup may have contributed to the absence of observable morphological changes.

We recognize this as a clear limitation of our study and have addressed it in the Discussion section, as follows.

 

FROM

“The most critical limitation of this study is the lack of in vivo data. However, we used primary cells in which genomic and phenotypic stability was well validated. Although we only searched for astrocyte transcripts, other mechanisms, such as inhibiting pro-inflammatory cytokines or oxidative stress, are also possible [21],[22]. As our qPCR data indicate we believe the immune response is deeply involved in the adaptive response to hypoxia. Accordingly, microglia, an upstream marker of astrocyte activation, or inflammatory markers could also be investigated for further mechanistic analyses [23].”

 

TO

“The most critical limitation of our study is the lack of in vivo data. Nevertheless, we used primary cells with well-validated genomic and phenotypic stability. In the future, we aim to extend our research to PD mouse models or human iPS cell-derived dopaminergic neurons to further validate our findings. In addition, while astrocytes are known to present morphological changes in response to external stimuli such as hypoxia, our study did not observe the expected changes. This discrepancy may be due to differences in experimental protocol, such as the hypoxic exposure setup and cell culture conditions. To address this limitation, future studies should investigate the morphological changes of astrocytes in response to both long- and short-term hypoxia exposure and correlate these findings with changes in astrocytic phenotype. Finally, although we only searched for astrocyte transcripts, other mechanisms, such as inhibiting pro-inflammatory cytokines or oxidative stress, are also possible [27,28]. As our qPCR data indicate, we believe the immune response is deeply involved in the adaptive response to hypoxia. Accordingly, microglia, an upstream marker of astrocyte activation, or inflammatory markers could also be investigated for further mechanistic analyses [29].”

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1.       If we agreed to the argument provided by the authors then how can authors seen a band ( Hif expression)  in western blot image at  0 (zero) time point in figure 2 A. Please provide an explanation for contradictory results between figure 1.a and figure 2.a HIF expression at 0 time point

2.       Primary cortical neuronal images are not clear, please see the earlier published literatures for the Tuj-1 staining in cortical neurons and provide images showing clear neuronal morphology 

 

Ref:

https://doi.org/10.3390/ijms24087106

                DOI: 10.1371/journal.pone.0089310

  PMCID: PMC6492790  PMID: 27333812

Author Response

Thank you for your insightful comments on our manuscript. We greatly appreciate your feedback and have made revision to the text based on your comments which we believe have significantly improved the quality of our paper. The author’s responses are written below each comment.

The manuscript by Ha Nyeoung Choi et al., titled “A2-astrocyteactivation by short term hypoxia rescue α-synuclein preformedfibril induced neuronal cell death,” is an exciting study. This manuscript is well-written and can be considered for publicationwith Major edits.

Point 1:  If we agreed to the argument provided by the authors then how can authors seen a band ( Hif expression)  in western blot image at  0 (zero) time point in figure 2 A. Please provide an explanation for contradictory results between figure 1.a and figure 2.a HIF expression at 0 time point.

Response 1:

We sincerely appreciate Reviewer 1's invaluable suggestion. To address Reviewer 1’s concerns thoroughly, we conducted additional experiments. As shown in Figure 1a, HIF-1α was faintly expressed at 0 time point (normoxia). Many studies, including in vivo and in vitro studies, have shown that HIF-1α protein is present in the nucleus even under normoxic conditions [1-3]. We hope this explanation satisfactorily addresses your concern.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Dr. Yun and Kim,

I understand the time limitations as generating most of the cellular data I suggested, such as protein level characterization of A1 and A2 astrocytes, including dopaminergic neurons, and testing the impact of intermittent hypoxia on different Parkinson's disease subtypes will require several months. I believe you have addressed most of my points effectively. However, I would like to emphasize that most Parkinson's disease studies focus primarily on dopaminergic neurons and alpha-synuclein aggregation. Your study is unique in that it explores the different populations of astrocytes and their connection to hypoxia. This approach is especially significant given the current global environmental concerns highlighted at COP29. This makes your study not only timely but also highly relevant to the field. I therefore recommend acceptance of your paper.

Author Response

Thank you for your insightful comments on our manuscript. We greatly appreciate your feedback and have made revision to the text based on your comments which we believe have significantly improved the quality of our paper. The author’s responses are written below each comment.

The manuscript by Ha Nyeoung Choi et al., titled “A2-astrocyteactivation by short term hypoxia rescue α-synuclein preformedfibril induced neuronal cell death,” is an exciting study. This manuscript is well-written and can be considered for publicationwith Major edits.

Point 1: I understand the time limitations as generating most of the cellular data I suggested, such as protein level characterization of A1 and A2 astrocytes, including dopaminergic neurons, and testing the impact of intermittent hypoxia on different Parkinson's disease subtypes will require several months. I believe you have addressed most of my points effectively. However, I would like to emphasize that most Parkinson's disease studies focus primarily on dopaminergic neurons and alpha-synuclein aggregation. Your study is unique in that it explores the different populations of astrocytes and their connection to hypoxia. This approach is especially significant given the current global environmental concerns highlighted at COP29. This makes your study not only timely but also highly relevant to the field. I therefore recommend acceptance of your paper.

Response: Thank you for your understanding of the limitations inherent in most cellular data. Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. While extensive research has focused on dopaminergic neurons and alpha-synuclein aggregation, the relationship between PD and astrocytes—the most abundant glial cells in the brain—remains poorly understood. To address this gap, we investigated the relationship between hypoxia and different populations of astrocytes. Additionally, we fully agree with reviewer 2's comment on global environmental issues, as recent studies suggest a potential link between the increasing global burden of PD and environmental contamination.

We are deeply grateful for reviewer 2’s invaluable insights into our study and sincerely appreciate the thoughtful review and acceptance of our paper.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

In order to accept this work for publication, the authors still need to address the following  concerns.

 

1.       The authors should provide for all the figures overlayed Western Blot with molecular weight marker in the supplement

2.       The bar graph and the representative figures in Figure 3.b for tunnel staining do not match. Additionally, a correct image showing the morphology of neurons should be provided. 

3.       A clear representative image with p-α-syn (green) staining should be included in figure 3.e.

4.       The bar graph and the representative figures in Figure 4.b for tunnel staining do not match. Additionally, a correct image showing the morphology of neurons should be provided. 

5.       A clear representative image with p-α-syn (green) staining should be included in figure 4.e.

6.       Additionally, a clear neuronal morphology (representative) image (PBS hACM, PFF nACm, PFF hACm,) should be provided in Figure 4e.

Author Response

We would like to express our sincere gratitude to the reviewer for their valuable feedback, which has significantly enhanced the value and significance of our manuscript. We have carefully considered the reviewer's comments and believe that our manuscript is now much stronger as a result. The following is our response to the reviewer's comments.

Point 1: The authors should provide for all the figures overlayed Western Blot with molecular weight marker in the supplement.

Response 1: Thank you for your valuable comment. We sincerely apologize for providing an incomplete Western blot image in the supplement. As suggested by the reviewer's suggestion, we have overlaid the molecular weight marker and included it in the supplement for the Western blot image as follows. We appreciate your insightful comments that have contributed to improving our manuscript. Point 2: The bar graph and the representative figures in Figure 3.b for tunnel staining do not match. Additionally, a correct image showing the morphology of neurons should be provided.  Response 2: We express sincere respect and appreciation to our Reviewer 1 for clearly understanding our manuscript as a whole and giving a meticulous review. We apologize for the discrepancies between the bar graph and the representative figures provided. The representative figure has been revised according to the Reviewer 1 comments as follows. We revised the representative figure of TUNEL staining and also provided the fluorescent images of Tuj-1, TUNEL, and DAPI respectively to better show the neuronal morphology. In addition, many studies including in vivo and in vitro research, have shown that treatment of preformed fibrils (PFFs) to primary neurons leads to the formation of intraneuronal aggregates composed of mouse α-syn in a time- and dose-dependent manner, which impairs synaptic activity and accelerates neuronal death [1-4].] Point 3: A clear representative image with p-α-syn (green) staining should be included in figure 3.e. Response 3: We sincerely thank Reviewer 1 for making a great point. We apologize for any confusion caused by the unclear p-α-syn image. According to the previous research, it shows clear p-α-syn using the p-α-syn (Biolegend, USA) antibody we used [1-5]. Point 4: The bar graph and the representative figures in Figure 4.b for tunnel staining do not match. Additionally, a correct image showing the morphology of neurons should be provided.  Response 4: We would like to thank you for bringing this to our attention and pointing out the shortcomings of our manuscript. Again, we sincerely apologize for the discrepancy between the graph and the representative figure. We have reviewed the issue and revised Figure 4b accordingly. In addition, we corrected the representative values ​​of TUNEL staining and also worked on image enhancement to better show neuronal morphology, and provided fluorescent images of Tuj-1, TUNEL, and DAPI. Point 5:  A clear representative image with p-α-syn (green) staining should be included in figure 4.e. Response 5: We appreciate Reviewer 1’s interest in our results and are grateful for your attention to our manuscript. We apologize for providing an unclear image of p-α-syn in Figure 4e such as Figure 3e. We have revised Figure 4e based on the reviewer's comments as follows. As we mentioned in response to Comments 3, it shows clear p-α-syn using the p-α-syn (Biolegend, USA) antibody we used [1-5]. We have taken into full account the reviewers' comments and made efforts to improve the images, and provided fluorescence images of Tuj-1, p-α-syn, and DAPI respectively to show clearer images. Point 6: Additionally, a clear neuronal morphology (representative) image (PBS hACM, PFF nACm, PFF hACm,) should be provided in Figure 4e.   Response 6: Thank you very much for taking the time to review our manuscript and for providing valuable feedback. We have strived to provide a more correct representation of the morphology of neurons. We have also revised Figure 4e based on the comments of Reviewer 1. As noted in comments 2 and 4, many studies, including in vivo and in vitro studies, have shown that addition of preformed fibers (PFFs) to primary neurons leads to the formation of intraneuronal aggregates composed of mouse α-syn, which impairs synaptic activity and accelerates neuronal death [1-4]. Also we provided fluorescent images of Tuj-1, TUNEL, and DAPI, respectively, to better show the neuronal morphology, and the modified figure shows more accurate neuronal morphology. As mentioned in points 2 and 4, our results show that neurite breakage in the PFF group is a consequence of nerve damage.   We are grateful for your time and consideration in evaluating our manuscript. Your insightful feedback certainly improved our manuscript’s clarity and accuracy, and we are genuinely grateful for your scientific contributions to our work.

Author Response File: Author Response.pdf

Round 4

Reviewer 1 Report

Comments and Suggestions for Authors

No more suggestions or comments