Author response:
Reviewer #1:
We thank Reviewer #1 for the thoughtful critique. We have revised the manuscript to clarify the physiological rationale for the experimental system, the potential relevance of mitochondrial STX–VDAC signaling to neurodegeneration, and the appropriate scope of our conclusions.
(1) Lack of Rationale
The study provides no justification for investigating sex-specific aspects of Alzheimer's disease by focusing on VDAC-mediated mitochondrial dysfunction in POMC neurons. These hypothalamic neurons are not recognized as early or primary sites of AD vulnerability, making the biological premise unclear.
We thank the reviewer for raising this important point. We agree that the original manuscript did not sufficiently explain the rationale for using POMC neurons.
Our rationale is primarily physiological rather than disease-specific. POMC neurons are a well-characterized, metabolically sensitive, and estrogen-responsive neuronal population in which membrane-initiated estrogen signaling and the actions of STX have been extensively characterized. They therefore provide a physiologically relevant neuronal system for identifying the molecular mechanisms through which STX regulates mitochondrial function.
The potential relevance to neurodegeneration is supported by evidence that hypothalamic and POMC neuronal function can be disrupted in neurodegenerative disease models. Do and colleagues (2018) reported hypothalamic neurodegeneration, increased inflammatory and apoptotic markers, and reduced POMC neuronal populations in 3xTg-AD mice and further showed that exercise attenuated hypothalamic apoptosis and restored POMC neuronal populations (Do, Laing et al. 2018). In addition, Shen and colleagues (2016) demonstrated disruption of POMC/MC4R signaling in APP/PS1 mice and showed that restoration of this pathway improved synaptic function (Shen, Tian et al. 2016).
These studies do not establish POMC neurons as a primary site of AD pathology, but they demonstrate that POMC-related neuronal systems can be vulnerable to neurodegenerative processes. This provides a broader biological context for investigating mitochondrial mechanisms in this neuronal population.
There is also a strong physiological rationale for examining estrogen-sensitive mechanisms in POMC neurons. These neurons are established targets of 17β-estradiol and are highly responsive to metabolic and mitochondrial state. STX is a non-steroidal estrogenic compound that activates membrane-initiated estrogen signaling, and our previous studies demonstrated neuroprotective and mitochondrial effects of STX in a neurodegenerative disease model.
Thus, POMC neurons were used because they provide a well-defined estrogen-responsive and metabolically sensitive neuronal population in which STX signaling can be mechanistically investigated—not because we consider them an initiating site of AD pathology.
We have revised the Introduction and Discussion accordingly. The revised manuscript now emphasizes the physiological significance of STX–VDAC signaling for neuronal mitochondrial function and presents its potential relevance to neurodegeneration as an important area for future investigation.
(2) Weak Link to AD Pathogenesis
Although mitochondrial dysfunction is well established in AD, the authors do not convincingly demonstrate a mechanistic or pathological connection between VDAC2 and AD. VDACs are not established contributors to AD etiology, and the manuscript does not strengthen this association.
We agree that the present experiments do not establish VDAC2 as an etiological or pathological driver of AD. This was not the objective of the present study.
The primary goal was to identify the molecular target(s) through which STX influences mitochondrial function. Using BF-STX chemoproteomic capture and competition experiments together with single-cell gene-expression analysis, planar lipid membrane electrophysiology, and mitochondrial metabolic analyses, we identify VDAC proteins as mitochondrial targets of STX and demonstrate functional effects of STX on VDAC channel properties and mitochondrial bioenergetics.
Our previous studies demonstrated neuroprotective and mitochondrial effects of STX in the 5xFAD model (Lee, Bostick et al. 2025), providing a neurodegenerative context that motivated the present mechanistic investigation. However, the current findings should not be interpreted as establishing VDAC2 as an AD pathogenic mechanism.
We have revised the manuscript accordingly. The STX–VDAC interaction is now presented principally as a mitochondrial mechanism with potential relevance to neuronal physiology and neurodegeneration. Whether this pathway contributes to the neuroprotective actions of STX in disease models will require direct experimental testing.
(3) Unclear Relevance to AD Contexts
While the data support an interaction between STX and VDAC2 affecting mitochondrial parameters (ATP production, membrane potential, glycolysis, respiration) in POMC neurons, the study does not show whether this mechanism is relevant to mitochondrial dysfunction in AD. No validation is provided in AD-related models or in contexts related to sex-specific AD phenotypes.
We agree that the present experiments do not directly establish the relevance of the STX–VDAC interaction to mitochondrial dysfunction in AD.
The current study was designed to identify and characterize the molecular mechanism underlying the mitochondrial actions of STX, rather than to test this pathway in a specific neurodegenerative disease model. Our findings demonstrate that STX interacts with VDAC proteins, modifies VDAC channel properties, and alters mitochondrial bioenergetics.
The study builds on our previous findings in the 5xFAD model, in which STX reduced amyloid-β-associated pathology and affected mitochondrial function (Lee, Bostick et al. 2025). The identification of VDAC proteins as STX targets therefore provides a mechanistic foundation for future studies examining whether this pathway contributes to neuronal protection under neurodegenerative conditions.
We have revised the Discussion to acknowledge the absence of direct disease-model validation. Future studies using conditional or neuron-specific manipulation of VDAC isoforms will be important for determining the physiological and neuroprotective significance of STX–VDAC signaling in vivo.
Accordingly, our conclusions now emphasize what is directly supported by the present experiments: STX interacts with VDAC proteins and modulates VDAC channel function and mitochondrial bioenergetics. The relevance of this mechanism to neurodegenerative disease remains to be established.
(4) Interpretation of Competitive Binding Data
The competitive binding results in Figure S4B are not adequately interpreted. The dose-dependent competition observed for VDAC3 suggests it may be a stronger candidate than VDAC2, yet this possibility is not addressed.
We thank the reviewer for highlighting this important point. We agree that the original manuscript placed too much emphasis on VDAC2 based on the chemoproteomic data.
Our chemoproteomic experiments identified VDAC1, VDAC2, and VDAC3 as STX-interacting proteins. Competition with unlabeled STX produced a particularly clear reduction in VDAC3 labeling, including complete loss of the VDAC3 signal at three molar equivalents of unlabeled STX. We therefore agree that VDAC3 represents an important candidate STX target.
We have revised the Results and Discussion so that the competition experiment is no longer interpreted as demonstrating preferential or exclusive binding to VDAC2.
However, the persistence of VDAC1 and VDAC2 labeling may also be influenced by properties of the BF-STX photoprobe as alkyl diazirine probes can preferentially photolabel membrane proteins (Kleiner, Heydenreuter et al. 2017). We now present this only as a possible technical consideration rather than an explanation established by our data.
Our subsequent emphasis on VDAC2 was based on the integration of several observations. Single-cell qPCR demonstrated that Vdac2 is the predominant transcript in the native POMC neurons examined (revised Figure 3B), with an approximate expression hierarchy of Vdac2 > Vdac3 >> Vdac1. In addition on a technical note, recombinant VDAC3 is more difficult to reconstitute reliably into artificial membranes because of its lower stability in detergent.
The revised manuscript therefore recognizes all three VDAC isoforms as candidate STX targets but does not claim that VDAC2 is the exclusive or preferential target. Direct comparisons of STX binding and functional modulation among the three isoforms will be required to establish isoform selectivity.
Reviewer #2:
We thank Reviewer #2 for the positive assessment of our chemoproteomic strategy and multidisciplinary characterization of the STX–VDAC interaction. We also appreciate the reviewer’s identification of important limitations and directions for future investigation.
(1) Physiological Relevance of Cell-Line Experiments
Most experiments were performed in immortalized cell lines rather than primary neurons or in vivo models, limiting their physiological relevance.
We agree that the use of immortalized neuronal cell models represents an important limitation.
However, these models provided the cellular material, reproducibility, and experimental control required for chemoproteomic target capture and Seahorse metabolic measurements. Importantly, we complemented these studies with single-cell analysis of native POMC neurons and electrophysiological characterization of recombinant VDAC channels.
Nevertheless, these approaches do not substitute for direct demonstration of STX–VDAC signaling in primary POMC neurons or in vivo. We have therefore revised the Discussion to emphasize that establishing the physiological significance of this mitochondrial pathway will require validation in primary neuronal preparations and whole-animal models.
(2) Structural Binding Site of STX on VDAC
The exact structural binding site of STX on VDAC remains unresolved.
We agree. BF-STX chemoproteomics identifies proteins interacting with STX in a cellular environment but does not resolve the amino acid residues or structural pocket responsible for binding.
We have clarified this limitation in the Discussion. Determining the STX-binding site will require complementary approaches such as targeted mutagenesis, direct binding measurements with purified VDAC proteins, and structural studies in membrane-like environments, including lipid nanodiscs. Such studies should also determine whether STX recognizes a conserved feature among VDAC isoforms or exhibits isoform selectivity.
(3) Lack of VDAC2 Loss-of-Function Experiments
No loss-of-function experiments (e.g., VDAC2 knockdown) were performed to establish a direct causal link between VDAC2 and STX's bioenergetic and neuroprotective effects.
We agree that VDAC loss-of-function experiments would provide an important additional test of causality.
Our present evidence is convergent: chemoproteomics identifies VDAC proteins as STX-interacting targets; single-cell analyses demonstrate VDAC expression in POMC neurons; electrophysiological studies show that STX modifies VDAC channel properties; and metabolic analyses demonstrate STX-dependent changes in mitochondrial bioenergetics. Together, these findings support a STX–VDAC mechanism but do not establish that VDAC2 alone is necessary for the mitochondrial or neuroprotective actions of STX.
We have revised the manuscript accordingly and explicitly identify the absence of loss-of-function experiments as a limitation.
Because whole-body VDAC2 loss-of-function is associated with severe developmental consequences, future studies will require conditional or neuron-specific approaches. Parallel evaluation of VDAC1 and VDAC3 will also be important because all three isoforms were identified by chemoproteomics and potential functional redundancy may complicate single-isoform manipulations.
(4) Non-linear Dose-Response at Higher STX Concentrations
The non-linear dose-response at higher STX concentrations also requires further investigation.
We agree that the non-linear concentration-response warrants further investigation and have revised the Discussion to avoid interpreting the STX response as a simple monotonic concentration-response relationship.
One possibility is that STX engages multiple mitochondrial targets with different apparent affinities and opposing effects on respiration. At lower concentrations, STX may preferentially engage a higher-affinity target, potentially VDAC, whereas higher concentrations may recruit lower-affinity targets that constrain this response. Consistent with this possibility, our BF-STX dataset (Supplemental Tables) identified several mitochondrial proteins involved in oxidative phosphorylation and metabolite transport, including ATP5F1C, NNT, SLC25A4, SLC25A5 and SLC25A3. ATP5F1C is required for efficient mitochondrial ATP production (Fiorillo, Scatena et al. 2021), and estrogenic regulation of ATP synthase has been reported (Massart, Paolini et al. 2002, Moreno, Moreira et al. 2013). NNT, SLC25A4, SLC25A5 and SLC25A3 also regulate mitochondrial respiration, redox balance, and ATP production (Mayr, Merkel et al. 2007, Lopert and Patel 2014). However, BF-STX enrichment does not establish direct STX binding, relative affinity, or functional modulation of these proteins. We therefore present the multi-target explanation only as a hypothesis. Direct binding and concentration-dependent target-engagement studies will be required to determine whether the non-linear response reflects recruitment of a lower-affinity mitochondrial target, concentration-dependent effects on VDAC itself, or downstream mitochondrial feedback.
We thank the editors and reviewers again for their constructive comments. The revised manuscript more clearly defines the physiological rationale for the experimental system, establishes the scope of the STX–VDAC mitochondrial mechanism supported by our data, and places its potential relevance to neurodegeneration in an appropriately forward-looking context.
References cited in the response
Do, K., B. T. Laing, T. Landry, W. Bunner, N. Mersaud, T. Matsubara, P. Li, Y. Yuan, Q. Lu and H. Huang (2018). "The effects of exercise on hypothalamic neurodegeneration of Alzheimer's disease mouse model." PLoS One 13(1): e0190205.
Fiorillo, M., C. Scatena, A. G. Naccarato, F. Sotgia and M. P. Lisanti (2021). "Bedaquiline, an FDA-approved drug, inhibits mitochondrial ATP production and metastasis in vivo, by targeting the gamma subunit (ATP5F1C) of the ATP synthase." Cell Death Differ 28(9): 2797-2817.
Kleiner, P., W. Heydenreuter, M. Stahl, V. S. Korotkov and S. A. Sieber (2017). "A Whole Proteome Inventory of Background Photocrosslinker Binding." Angew Chem Int Ed Engl 56(5): 1396-1401.
Lee, H.-J., Z. Bostick, J. Doherty, T. L. Swanson, M. J. Kelly, J. F. Quinn, N. E. Gray and P. F. Copenhaver (2025). "Neuroprotection against beta-amyloid toxicity by the novel estrogen receptor modulator STX requires convergent signaling pathways." Frontiers in Molecular Neuroscience Volume 18 - 2025.
Lopert, P. and M. Patel (2014). "Nicotinamide nucleotide transhydrogenase (Nnt) links the substrate requirement in brain mitochondria for hydrogen peroxide removal to the thioredoxin/peroxiredoxin (Trx/Prx) system." J Biol Chem 289(22): 15611-15620.
Massart, F., S. Paolini, E. Piscitelli, M. L. Brandi and G. Solaini (2002). "Dose-dependent inhibition of mitochondrial ATP synthase by 17 beta-estradiol." Gynecol Endocrinol 16(5): 373-377.
Mayr, J. A., O. Merkel, S. D. Kohlwein, B. R. Gebhardt, H. Böhles, U. Fötschl, J. Koch, M. Jaksch, H. Lochmüller, R. Horváth, P. Freisinger and W. Sperl (2007). "Mitochondrial phosphate-carrier deficiency: a novel disorder of oxidative phosphorylation." Am J Hum Genet 80(3): 478-484.
Moreno, A. J., P. I. Moreira, J. B. Custódio and M. S. Santos (2013). "Mechanism of inhibition of mitochondrial ATP synthase by 17β-estradiol." J Bioenerg Biomembr 45(3): 261-270.
Shen, Y., M. Tian, Y. Zheng, F. Gong, A. K. Y. Fu and N. Y. Ip (2016). "Stimulation of the Hippocampal POMC/MC4R Circuit Alleviates Synaptic Plasticity Impairment in an Alzheimer's Disease Model." Cell Rep 17(7): 1819-1831.
