WWOX deficiency impairs neurogenesis and neuronal function in human organoids

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   Review Commons

   Apr 15, 2025

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   Reply to the reviewers

   Reviewer 1:

   Evidence, reproducibility and clarity

   The manuscript "WWOX deficiency impairs neurogenesis and neuronal function in human organoids" by Aqeilan and co-workers provides impressive set of studies, mostly utilizing cerebral organoids (CO), gaining insights into the roles of the gene WWOX in neuronal development and molecular etiology of WOREE and SCAR12, two devastating rare diseases originating from mutations in WWOX. Further, therapeutic modalities through the neuron-specific gene therapy are investigated using the WWOX k/o and WOREE and SCAR12 patient-derived COs. Among the major findings of this work one can highlight the identification of …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Reviewer 1:

   Evidence, reproducibility and clarity

   The manuscript "WWOX deficiency impairs neurogenesis and neuronal function in human organoids" by Aqeilan and co-workers provides impressive set of studies, mostly utilizing cerebral organoids (CO), gaining insights into the roles of the gene WWOX in neuronal development and molecular etiology of WOREE and SCAR12, two devastating rare diseases originating from mutations in WWOX. Further, therapeutic modalities through the neuron-specific gene therapy are investigated using the WWOX k/o and WOREE and SCAR12 patient-derived COs. Among the major findings of this work one can highlight the identification of the main source of WWOX-expressing cells as radial glia (RG) cells; the discovery of the massive upregulation of Myc upon loss/decrease in WWOX expression in RGs; and the strong neuronal under-differentiation induced upon WWOX k/o and mutations. Regarding the latter finding, the authors report massive increase in RGs and concomitant drop in neuronal cells in WWOX k/o COs. In contrast, in WOREE and SCAR12 patient-derived COs, a more subtle under-differentiation is seen. Specifically, while WOREE but not SCAR12 patient-derived COs also show a certain increase in the RG proportion, both types of patient-derived COs demonstrate higher proportions of "young" neuronal cells as compared to wild-type COs. Thus, a picture can be drawn whereas complete loss of WWOX leads to strong under-differentiation mostly manifested as expansion of RGs and hence under-production of neuronal cells, while hypomorphic loss-of-function of WWOX in WOREE and in missense mutations in SCAR12 lead to the later defect in neuronal cell maturation. Overall, I find the work highly interesting, but I would like the authors to address one major issue and several minor ones.

   Major Comments

   Comment____:

   The major issue is related to the overall model the authors seem to build based on their data - or at least the overall model the reader may get from the paper. This model suggests that the loss / decrease in WWOX levels in RGs leads to Myc overexpression, that in turn affects the cell cycle and prevents neuronal differentiation. This model is highly attractive, but is probably incomplete, in the sense that it does not fully recapitulate the complicated picture. Indeed, all three types of mutated WWOX COs (WWOX k/o, WOREE patient-derived organoids, and SCAR12 patient-derived organoids) demonstrate strong - but equal levels of Myc upregulation. Yet the under-differentiation in each of these three types is different, as described above, and the disease manifestations among WOREE vs. SCAR12 patients are also different. Thus, another player (in addition to Myc) must be at place, that is differentially affected by the partial null mutations in WOREE and missense mutations in SCAR12. This point - ideally to be addressed experimentally - should be at least faced directly by the authors in the Discussion. Perhaps they can already point to such additional players based on their transcriptomics analysis.

   Response____:

   We thank the reviewer for this important point. We agree with the reviewer that the model of WWOX loss / decrease levels in RGs leading to MYC overexpression is incomplete, and that it is a limitation of our model. It seems plausible that other players have a high impact on the genotype and are potentially differently affected, resulting in this complexed phenotype. Following the reviewer advice, we plan to address this in the discussion as a limitation of the model, and we will compare how the expression levels of MYC change based on the genotype in comparison to the WT, using the single cell RNA-sequencing data. We would also like to clarify that MYC upregulation we observed in the patient lines in SOX2+/MYC+ populations, does not quantify expression levels of MYC, but rather positive/negative nuclear staining, in contrast to the high-resolution of scRNA-seq data.

   Minor Comments

   Comment____:

   1. It would be useful if a table (perhaps supplementary) describing the details of the WWOX__ mutations__ in all the COs models studied in this paper were presented.

   Response____*: *

   We thank the reviewer for this suggestion, and we plan to prepare a table summarizing all the mutations in the COs models presented in the paper.

   Comment____:

   1. For the new WOREE individual with complex genetics in WWOX: it is not clear why any WWOX protein is still present in this patient in Fig. S1D (please give an explanation or speculation); it is not clear which tissue was used for the Western blot in Fig. S1D; the data in Fig. S1D need to be quantified.

   Response____:

   We thank the reviewer for their observation and would like to clarify that the ‘upper’ band seen in WWOX bands in a nonspecific one that appears in the parent lines and the mutant offspring. We will* quantify the WB levels and* clearly state that they are the IPSCs in the figure legend.

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   Comment____:

   1. Western blot, quantified, should be performed on all COs under study, to compare the WWOX expression levels. Please also change the immunofluorescence shown in Fig. 1B (e.g. show WWOX in a different color), as the figure provided shows WWOX poorly in wild-type CO, and it is not clear how much it is removed in the mutant organoids. Why should there be no signal in the SCAR12 COs?

   Response____:

   We thank the reviewer for their observation, we will provide protein levels of WWOX in patient and KO cerebral organoids* which will better clarify the decreased WWOX levels, specifically in SCAR12 (see WB figure below). We will also perform any necessary changes to the figure to enhance visualization of WWOX.*

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   Significance

   The manuscript "WWOX deficiency impairs neurogenesis and neuronal function in human organoids" by Aqeilan and co-workers provides impressive set of studies, mostly utilizing cerebral organoids (CO), gaining insights into the roles of the gene WWOX in neuronal development and molecular etiology of WOREE and SCAR12, two devastating rare diseases originating from mutations in WWOX. Further, therapeutic modalities through the neuron-specific gene therapy are investigated using the WWOX k/o and WOREE and SCAR12 patient-derived COs. Among the major findings of this work one can highlight the identification of the main source of WWOX-expressing cells as radial glia (RG) cells; the discovery of the massive upregulation of Myc upon loss/decrease in WWOX expression in RGs; and the strong neuronal under-differentiation induced upon WWOX k/o and mutations. Regarding the latter finding, the authors report massive increase in RGs and concomitant drop in neuronal cells in WWOX k/o COs. In contrast, in WOREE and SCAR12 patient-derived COs, a more subtle under-differentiation is seen. Specifically, while WOREE but not SCAR12 patient-derived COs also show a certain increase in the RG proportion, both types of patient-derived COs demonstrate higher proportions of "young" neuronal cells as compared to wild-type COs. Thus, a picture can be drawn whereas complete loss of WWOX leads to strong under-differentiation mostly manifested as expansion of RGs and hence under-production of neuronal cells, while hypomorphic loss-of-function of WWOX in WOREE and in missense mutations in SCAR12 lead to the later defect in neuronal cell maturation.

   Overall, I find the work highly interesting, but I would like the authors to address one major issue and several minor ones.

   Response____:

   We sincerely thank the reviewer for their thoughtful and constructive comments, which have greatly helped us improve the clarity and rigor of our manuscript. We appreciate the recognition of our work’s significance and the careful evaluation of both our major findings and methodological details. We have addressed all the raised points to the best of our ability and believe the manuscript will be substantially strengthened as a result. We are grateful for the reviewer’s time and valuable insights.

   Reviewer 2:

   Evidence, reproducibility and clarity

   Summary

   In this study, Steinberg et al aim to elucidate the role of WWOX in human neurogenesis and model WOREE and SCAR12 syndromes which are rare neurodevelopmental disorders. They chose to investigate its function in human brain organoids after generating WWOX KO and patient-derived iPSC lines. Their major finding is that radial glial cells, the main neural progenitor population during corticogenesis, are affected. Via single-cell-RNA-sequencing, they try to decipher the perturbed molecular mechanisms identifying MYC, a proto-oncogene, as a major player. At the end of their study, they proceed to gene therapy restoration and suggest that this could become a potential therapeutic intervention for WOREE and SCAR12 syndromes. The study aims to elucidate major cellular and molecular mechanisms that modulate neurodevelopment and neurodevelopmental disorders. Although sc-RNA-seq could potentially be of great interest and unravel major mechanisms, the authors do not follow this part, but only discuss potential future avenues. Here are some suggestions that could be useful to the authros.

   Major comments

   Comment____:

   A big part of the paper focuses on generating the iPSCs and characterizing the generated brain organoids and gene restoration of the phenotype via restoration of the WWOX gene expression (Fig.1, Fig.6, Fig.S1, Fig.S8, Fig.S10 and potentially Fig.S9 - this figure is not included) however, this has already been done by the same authors (first and last authors) in a previous publication. What are the differences in the line that have been generated in previous publication (Steinberg et al 2021, EMBO Mol. Med.)? If there are differences, the authors should make a thought comparison and explain why they generated different lines. If there is no difference, the authors should reduce to minimum this part and place it to supplementary.

   Response____:

   We thank the reviewer for pointing this out. We would like to clarify that some iPSC lines used in this publication were not introduced in the previous one (Steinberg et al 2021, EMBO Mol. Med.), including the wildtype JH-iPS11, and the new compound heterozygous WOREE line LM-iPS. In this paper, we aimed to widen our understanding of the effects of WWOX mutations through advanced techniques not applied before, and by adding these lines we were able to better generalize our findings as we did not depend on a single patient for both WOREE and SCAR12. Additionally, WWOX rescue in this current paper relies on AAV9-hSynI targeting that is more clinically relevant to gene therapy, as opposed to lenti-WWOX and AAVS1 WWOX in the previous publication.* We will include the differences in a summary table.*

   Comment____:

   • Fig.1E: in the pictures shown, the majority of the Satb2+ cells are colocalized with SOX2. Although a small portion of neurons have been shown from many studies that in brain organoids are co-localized to SOX2, in the pictures depicted this percentage is big. Also in ctrl condition the VZ-CP like areas are not easily recognized. The authors should check if this co-localization is a more general phenotype and if not choose more representative pictures.

   Response____:

   We thank the reviewer* for their observation. We will check for the presence of a trend of colocalization between SATB2 and SOX2 and address the concern experimentally if needed. We will also choose pictures that better display VZ-CO areas in the control line.*

   Comment____: Information about the number of organoids per batch used in each figure is not included. This needs to be added for each experiment. Data (at least the majority of them) should be collected from brain organoids from at least two batches.

   Response____:

   We thank the reviewer for their point, and we plan to better clarify the technical parts of the experiments, and if needed will include data from more batches.

   Comment____: The expression of WWOX in cortical development has been shown in the previous publication. Although sc-RNA data are validating the previous data and are adding more information, these data should be put as supplementary. Besides, in Fig.3G where authors aim to compare WWOX expression to MYC that fits nicely with their results depicting MYC as the most affected gene in KO and mutant line, when one looks at the WWOX expression only it seems that its expression is higher in CP that VZ. This is contrary to the conclusion that WWOX is mainly characterizes RGs. Why is that? Authors should at least discuss this.

   Response____:* We agree with the reviewer that Fig.3G can be misleading, and we acknowledge that it can lead to the opposite conclusion of WWOX being mainly characterized in RGs. In Fig.3G the x axis displays positive values on the left, and negative scores on the right. Following the reviewer’s suggestion, we modified the graph to show positive values on the right, demonstrating how WWOX expression is higher in the VZ compared to the CP.*

   Comment____: In this study, authors show that progenitors are reduced in WWOX-KO organoids, however in the previous publication SOX2 population is not majorly affected. Why are there such differences? Given that RGs are the main population affected as authors propose in this study, these differences must be at least discussed. Similar comments regarding neurons: in previous publication there is a minimal reduction of neurons in WWOX-KO brain organoids, while here authors describe major differences.

   Response____:

   We thank the reviewer for their remark and agree that it should be mentioned in our discussion. We believe that this is unfortunately due to the inherent issue of heterogeneity between organoids and could partly be attributed to the difference in the age of organoids at that timepoint (week 10 organoids in previous paper, week 7 organoids in this one), and the difference in control lines (WiBR3 hESCs vs JH-iPSCs). Additionally, while percentages of SOX2+ populations in WT organoids vary between the previous publication and this one, WWOX-KO organoids display similar levels of SOX2 upregulation in precious and current papers: 69% and 74%, respectively. We would also like to point out that calculations in scRNA-seq data convey the phenotype at a much higher resolution, as it identifies radial glia populations that are not necessarily SOX2+, further strengthening the validation of the SOX2+ RG quantifications that are present in this study.

   Comment____: Data from sc-RNA-seq analysis highlighting MYC as major differentially regulated gene are very interesting and seem to be key to the molecular pathway affected as authors suggest. Authors also validate this with immunostainings in brain orgnaoids. However, in Fig.3J MYC expression in ctrl is not depicted, even though in the respective graph it seems that 20% of SOX2+ cells co-express MYC. Please choose a more representative picture.

   Response____:

   We agree with the reviewer’s comments that the current image size and resolution limits the ability to appreciate the MYC staining in the control, and plan to use a more representative figure of the phenotype.

   Comment____: One of the main findings in this study is the cell cycle changes observed in WWOX-KO and mutant organoids. Given that the major novelty of the publication is the cellular and molecular mechanism implicated in WOREE and SCAR12 syndromes, authors should perform additional experiments towards this direction. One suggestion would be to perform stainings in brain organoids using markers of the different cell cycle phases (eg. KI67, cyclin a, BrdU/EdU, ph3). Also, treatment of organoids with different BrdU/EdU chase experiments would be important so as to measure exactly the length of each cell cycle phase.

   Response____:

   We appreciate the reviewer’s suggestions and plan to validate the findings through staining and quantifying percentages of proliferative RGs in WT vs mutant WWOX lines.

   Comment____: Regarding the molecular cascade, is WWOX directly affecting MYC of Wnt genes? Do they have information on upstream and downstream factors in the affected molecular pathway?

   Response____:

   We thank the reviewer for highlighting this important point. To address this question, encouraged by our results, we will compile genes belonging to the regulon of MYC and study the upstream and downstream factors in our transcriptional data. Additionally, we will look at protein expression levels of WNT genes in our organoid samples.

   Comment____: Restoration of phenotype via reinsertion of WWOX gene has already been done in the previous publications by the same authors. But what about MYC? Is MYC manipulation able to rescue the phenotype?

   Response____:

   *We thank the reviewer for this insightful suggestion. We fully agree that understanding the role of MYC in the observed phenotype is of great interest. However, due to the essential and widespread role of MYC in both radial glia and neurons, we refrained from direct perturbation of MYC levels—either through knockdown or overexpression—as such manipulations may have broad, uncontrolled effects that could confound the interpretation of our findings. The potential deleterious consequences of MYC modulation in radial glia have been **originally *discussed in the Discussion section of the manuscript. In our revisions, we will further explore the role of MYC regulons in our scRNA-seq dataset to better understand their contribution to the WWOX-related phenotype.

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   Comment____: Finally, MYC association to ribosome biogenesis as mentioned by the authors in discussion is very interesting. The authors should consider investigating this direction, as it will be a great addition to the mechanisms that regulate WOREE and SCAR12 syndromes which is the main focus of this study.

   Response____:

   We thank the reviewer for highlighting this point, and we agree that MYC's association with ribosome biogenesis is a fascinating topic to discuss. This could be connected to the alterations of the proliferative potential and to the anabolic state of the cell, and we plan to expand the discussion of this observation and its implication in the context of RGs and neurons*. *

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   Minor comments

   Comment:

   • Line 115: authors say that the data they discuss are found in Fig.S2A, maybe they mean Fig.S1A?

   Response:

   We thank the reviewer for their observation, we will correct Fig.S2A to Fig.S1A and B.

   Comment:

   • Fig.S9 is missing, in the current version this Fig is the same with Fig.S10. Please change it.

   Response____:

   We thank the reviewer for pointing this out and apologize for this oversight. We acknowledge the error and will correct the duplication by replacing Fig. S9 with the intended figure in the revised version of the manuscript.

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   Significance

   This study is the continuation of a previous publication the authors have published. The topic is very interesting and novel especially in modelling neurodevelopmental disorders in a human context, however, given that the main phenotype has already been published, the authors should include more effort in the molecular cascade. Clinical interventions if the molecular cascade is described would be of great importance to the field.

   Response____:

   We sincerely thank the reviewer for their thoughtful, constructive, and detailed review. We appreciate the time and effort taken to carefully read our manuscript and provide insightful suggestions, taking into consideration also our previous published work. The suggestion raised, especially regarding MYC-WNT axis and its potential link to ribosome biogenesis, will help us clarify, strengthen, and expand the scope of our study. We have carefully addressed each of the points raised and have incorporated the necessary experimental validations, clarifications, and revisions accordingly. We believe these changes have substantially improved the manuscript.

   Reviewer 3:

   Summary:

   The manuscript by Steinberg and colleagues describes cellular and molecular changes linked to mutations in WWOX, a gene implicated in rare neurodevelopmental disorders, WOREE and SCAR12 syndromes. By comparing immunofluorescene and single cell trascriptomics of unguided brain organoids from control and WWOX-knockout iPSCs, as well as 2D NSCs and in vivo fetal brain expression datasets, the authors identified radial glia as relevant cell types in which WWOX is expressed and affected by WWOX deficiency. Using immunofluorescence, single cell trascriptomics analysis and western blotting on week 16 organoids, the authors show that WWOX deficiency results in increased abundance of radial glia cells at the expenses of neuronal production. These changes are accompanied by accumulation of cells in G2/M and S phases, overexpression of c-MYC and Wnt activation. In addition to this, the authors characterize unguided brain organoids generated from iPSCs reprogrammed from patients affected by WOREE or SCARE12 syndromes. Using immunofluoresce and single cell trascriptomics, they find that, while RG abundance changes were very modest, patients's iPSC-derived neurons are enriched for signatures related to early development, suggesting delayed differentiation. Finally, the authors use patch clumping, calcium imaging and gene therapy in 16 weeks old organoids derived from control and patients-derived iPSCs, to demonstrate that WWOX restoration normalized hyperexcitability phenotypes in both WOREE and SCAR12 organoids. These results thus provide a proof-of-concept evidence that WWOX restoration in human cells is a valid strategy to correct for hyperexcitability pehnotypes in WWOX related syndromes.

   Major ____C____omments

   The study's main conclusions regarding neurodevelopmental phenotypes linked to WWOX deficiency and genotype-phenotype relationships are based on iPSC-derived brain organoid models analyzed using immunofluorescence, single-cell transcriptomics, and excitability recordings (cell-attached patch clamping, calcium imaging). While the analyses involve a diverse collection of iPSCs and two time points (7 and 16 weeks), the study falls short in providing sufficient experimental details and validation to fully support its conclusions. Additional quantification, replication, and functional validation would be necessary to solidify the study's conclusions. Some of these validations are achievable within a reasonable timeframe, while others would require a more substantial investment of time and resources as detailed below.

   Comment____:

   A key concern is the lack of experimental details and replicability. Number of individual organoids, number of images per organoid for IF, and whether multiple batches were used are only partially provided. While the authors report generating multiple WWOX knockout clones, the legends and methods do not specify whether multiple clones were used across different organoid experiments. The study states that four organoids were used for scRNA-seq, but it is unclear whether this means four organoids per genotype or one organoid per genotype was analyzed. These ambiguities make the claims appear rather preliminary.

   Response____:

   We thank the reviewer for pointing this out, and we acknowledge that the clarity of our description of the batch used in each experiment can be improved. Therefore, we will provide all these details, adding information on additional batches adopted for the different validations that were not included in the manuscript.

   Comment____:

   Another issue is the limited validation of scRNA-seq observations. Since scRNA-seq is often performed on a limited number of organoids, orthogonal validation is crucial to strengthen the findings. For example, changes in radial glia abundance and neuronal production observed in scRNA analyis (Figure 2-5) could be validated using immunofluorescence across genotypes and batches. Currently, IF stainings for Sox2 and TUBB3 are shown only at 7 weeks in Figure 1B, but no quantificative assessment is provided. Also, it is not clear if quantifications provided in Figure 1F refer to multiple organoids or batches.

   Response:

   We thank the reviewer for this important point. We would like to clarify that in Figure 1B, TUBB3 staining is primarily used for visualization purposes to provide anatomical context and delineate the overall architecture of the organoids, rather than for quantitative assessment of neuronal output. As such, the focus of our quantification in Figure 1F was on SOX2+ radial glial cells. That said, we agree that clearly stating the number of organoids and batches used in the quantification is important, and we will include this information in the figure legend for clarity.

   Comment____:

   Furthermore, the observations on cell cycle arrest, DNA damage, senescence, metabolic alterations, Wnt activation obtained via scRNA-seq could be further validated on organoid tissues using specific antibodies that the lab used before (e.g. yH2AX antibody in PMID: 34268881) or assays that have been developed elsewhere (some examples are reviewed in PMID: 38759644). As for feasibility, immunofluorescence validation of existing tissues is realistic, requiring validated antibodies and procedures, some additional imaging time and analysis (estimated 1-2 months, with some budget to purchase antibodies and cover imaging time costs). Feasibility of efforts related to validation across organoids and batches depends on the number of organoids used so far and available tissues. Generating new organoids would be indeed more time-consuming (≈ 6 months) and expensive (but extact costs would depend on number of clones, organoids and batches used), but feasible.

   Response____:

   We appreciate the reviewer’s thoughtful feedback and for drawing our attention to the review by Sandoval, Soraya O., Anderson, Stewart, et al. We also thank the reviewer for their suggestions and intend to explore the proposed modifications through immunostaining, particularly to address questions related to cell cycle changes, and Wnt pathway. However, regarding DNA damage, senescence, and cell cycle arrest, we do not believe additional validation is necessary, as our current manuscript does not present findings related to these aspects.

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   Comment____:

   Another limitation is the lack of functional relevance of MYC alterations. The study confirms increased MYC expression via both scRNA-seq and immunofluorescence in organoid tissues. However, these results remain correlative and demonstrating the functional requirement of MYC overexpression in mediating WWOX-deficiency-related changes would significantly strengthen the study's conclusions. This would require additional differentiation experiments, including MYC overexpression or knockout models, to assess its direct impact. These efforts would represent a major conceptual advance by linking RG effects to MYC function and highlighting MYC-related therapeutic directions. These additonal experiments would require a substantial investment to generate the necessary regents (e.g. WWOX-KO and WT iPSCs with altered MYC levels) and additional time and costs for organoid analysis, mostly by immunofluorescence (estimated 6-8 months).

   Response____:

   We thank the reviewer for this insightful comment and fully agree that elucidating the functional contribution of MYC alterations in the context of WWOX deficiency would represent a major conceptual advance. We acknowledge that our current findings are correlative, based on scRNA-seq and immunostaining, and that direct manipulation of MYC could help establish causality.

   However, due to MYC’s essential and pleiotropic role in both progenitor and neuronal populations—including its regulation of cell cycle, metabolism, and apoptosis—we refrained from genetic overexpression or silencing approaches in this study. Such perturbations often lead to widespread, non-specific effects that can obscure the interpretation of lineage-specific phenotypes, particularly in a complex model like brain organoids.

   That said, we agree that further insight into the functional role of MYC is crucial. To this end, we plan to leverage our scRNA-seq dataset to analyze the activation state of MYC regulons across genotypes and cell types, and to assess how these regulons intersect with cell cycle dysregulation observed in WWOX-deficient radial glia. We also aim to integrate available transcriptomic data from primary cortical tissue to support the relevance of MYC pathway alterations in human development. While these analyses cannot replace experimental perturbation, we believe they can provide strong, hypothesis-generating evidence for MYC’s mechanistic involvement and help prioritize targeted experiments in future studies.

   Comment____:

   Another issue is the lack of patterning analysis in unguided organoids, which are known to exhibit high variability in regional identity (PMID: 28283582). While the authors acknowledge this limitation to some extent-abstaining from fine-resolution analysis (Lines 173-174)-this variability, combined with the limited number of organoids used, could be a major confounding factor in the phenotypic analyses, even at a broad resolution. Indeed, some of the reported differences across genotypes may stem from variability in organoid patterning rather than true genotype-driven effects. For example, the reduced SATB2 expression in KO and patient-derived organoids from Figure 1E-F could result from impaired cortical patterning rather than a direct effect of WWOX deficiency. Additionally, in Figure 6D and 6E, the fact that WOREE iPSC-derived organoids - but not SCAR12 organodis- show lower levels of both CTIP2 and SATB2, might reflect a shift toward a non-cortical identity rather than a direct WWOX-dependent phenotype. To rule out patterning variability as a contributing factor, the authors should analyze organoid regional identity across genotypes using immunostaining for dorsal and ventral forebrain markers. This would allow a more solid inference of genotype-specific effects on neurodevelopmental phenotypes. Patterning validation can be performed on existing organoid tissues (week 7) using validated antibodies (PMID: 28283582). As such, this analysis is expected to be relatively straightforward and feasible in a few weeks time. If the generation of new organoids is needed, such patterning validation should still be relatively feasible, as week 7 organoids are ideal for assessing regional identity. Analysis of patterning effects should also extend to 2D NSC cultures. In the 2D NSC models derived from WWOX-KO lines (Figure 3L, Figure S4A), the differentiation protocol includes patterning factors that promote ventral fates (SAG and IWP2). Interestingly, the quantification of MYC expression from unguided organoids and 2D NSCs (Figure 3K-L) reveals a major difference in the fraction of MYC-positive cells in WT conditions across the two culture models. A possible changes in the dorsal and ventral patterning of 3D and 2D cultures might explain these differences and implementing immunostaining for patterning markers in 2D would help clarify patterning contributions.

   Response____:

   We thank the reviewer for this thoughtful and constructive comment. We fully agree that regional identity variability in unguided cerebral organoids is a well-recognized challenge, and that systematic assessment of dorso-ventral patterning is important to confidently interpret genotype-driven phenotypes.

   We would like to clarify that the cerebral organoid protocol used here has consistently been shown to favor a dorsal forebrain identity (PMID: 23995685, 28562594, 32483384, 33328611), and in our previous work (PMID: 34268881), we demonstrated that WWOX mutations did not substantially alter dorsal identity in this model. Nevertheless, to directly address the reviewer’s concern, we plan to perform additional immunostaining for regional patterning markers on our existing week 7 organoid tissues and explore our scRNA-seq data to evaluate potential shifts in regional identity and rule out patterning-related confounders.

   Regarding the 2D NSC cultures, while the differentiation protocol included the ventralizing factor SAG, it did not include IWP2. We acknowledge the importance of validating patterning outcomes in this model as well and will do so using immunostaining.

   Comment____:

   There are also some concerns regarding WOREE and SCAR12 phenotypes. First, the genotypes of the patient-derived iPSCs are not clearly defined, making it difficult to establish clear genotype-phenotype relationships. The study uses iPSCs from four different patients (2 WOREE, 2 SCAR12), some of which were validated in a previous study (PMID: 34268881). However, it remains unclear how they were validated, and detailed genomic alterations of the four patients are not explicitly reported. Additionally, it is uncertain whether all variants result in a full loss of WWOX function, as protein loss evidence is only provided for one WOREE patient (Figure S1D). Also, the authors state that SCAR12 should have a milder phenotype (line 168), but it is unclear whether this claim is based on clinical evidence or genomic data from these specific patients. To improve genotype-phenotype comparisons, the authors should consider including a clear schematic summarizing the genomic alterations in all patient-derived lines and their expected disease severity.

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   Response____:

   We thank the reviewer for this suggestion, and we agree that including a schematic summarizing the genomic alterations in all patient-derived lines and their severity will improve the genotype-phenotype comparison. We will include this clarification and provide additional information on how the mutations affect the protein level, and the genotype-phenotype correlations in WWOX mutants based on clinical and genetic evidence.

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   Comment____:

   Second, the experimental design lacks appropriate controls for patient-derived iPSCs. All patient-derived iPSC comparisons are performed against a single reference male iPSC line, which is neither isogenic to WOREE nor SCAR12 iPSCs. This complicates the interpretation of differences between healthy and patient-derived organoids, as well as comparisons between WOREE and SCAR12 phenotypes. Given this design, it is impossible to draw solid conclusions about genotype-phenotype relationships. A more robust approach would involve including multiple healthy controls to account for genetic background variability or using isogenic parental or genetically corrected lines, which would provide a cleaner genetic comparison. A recent study (PMID: 36385170) discusses different study designs that could strengthen this aspect and might be useful for the authors to consult.

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   Response____:

   We thank the reviewer for highlighting this and pointing us to the work of Brunner, Lammertse, van Berkel et al. While we agree that isogenic controls for each mutant line would be the ideal wild-type reference, generating these through genomic editing is particularly challenging, specifically for the compound heterozygous mutants. Instead and as suggested, we plan to include additional wild-type lines derived from healthy individuals, collected from different batches. We will use these to validate our key findings, including analyses of RG and SATB2+ cell populations, as well as MYC expression through immunofluorescence.

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   Comment____:

   Third, the study presents seemingly conflicting results regarding WOREE and SCAR12 phenotypes. The authors present immunofluorescence (IF) and scRNA-seq data indicating that changes in radial glia (RG) abudance are not observed in these patient-derived organoids. However, using same methodologies, they indicate that neuronal production is affected, leading to the accumulation of early neuronal signatures in both WOREE and SCAR12 neurons. The study does not explore whether RG signatures might be altered in a way that could contribute to neuronal phenotypes. Also, Figure 1F suggests that while Sox2+ cell counts are not increased in SCAR12 organoids, SATB2 levels are still altered, indicating that Sox2 and SATB2 trends are not tightly coupled across genotypes.

   Furthermore, Figure 1 and 6 show that while both syndromes exhibit similar hyperexcitability, data in Figure 6 report that only WOREE organoids display reductions in SATB2 and CTIP2 counts and that this can be rescued by WWOX restoration. Some of these discrepancies could stem from patterning variability as discussed above. Also, neuronal firing rate across WOREE and SCAR12 iPSC-derived organoids (Figure 6B) was different at later stages, but was rather comparable at an earlier stages (Figure S1G). The reasons for these differences are not thoroughly discussed.

   To strengthen the discussion, the authors should address how RG alterations (if any) might contribute to neuronal phenotypes, provide a more detailed comparison between WOREE and SCAR12 organoids and the WWOX-KO model and elaborate on the distinct phenotypes of the two syndromes, including possible explanations for observed functional and molecular discrepancies.

   ---

   Response____:

   We thank the reviewer and agree that further investigation into the proposed link between WWOX deficiency and MYC-related alterations in radial glia would provide deeper insight into the downstream effects on neuronal populations. To this end, we will first illustrate how our model of radial glia alterations accounts for changes in neuronal production without affecting overall RG abundance. Second, we will expand our comparisons of RGs and MYC expression using patient-derived and control single-cell RNA-sequencing datasets. Third, we will address the discrepancies between neuronal hyperexcitability and SATB2/CTIP2 counts more comprehensively. Notably, while SATB2 is an early marker for several cortical neuron subtypes, it is not expressed in all neurons. In contrast, SOX2 is considered a pan-radial glia marker, which may help explain the differing expression trends observed.

   Comment:

   Lastly, the conclusions drawn about WWOX restoration via gene therapy are weakened by the lack of replication and validation (see points above).

   First, the authors claim successful WWOX restoration in neurons, but provide limited evidence that the infected population consists of neurons. NeuN staining (Figure 6A and S10) suggests some neuronal expression, but quantification of WWOX+ NeuN+ / WWOX+ total cells is missing. Given that IF data are already available, this additional quantification could be completed within a few weeks and would significantly strengthen the claim.

   ---

   Response____:

   We thank the reviewer for the suggestion, and based on this, we will quantify the WWOX+ NeuN+ / WWOX+ total cells, *incorporating *data from additional batches to strengthen the analysis.

   ---

   Comment____:

   Second, the rationale for restoring WWOX in neurons is unclear, given that WT neurons do not normally express WWOX. Is WWOX being considered a functional neuronal maturation factor? If so, this should be explicitly discussed in the manuscript.

   Third, the authors propose that WWOX deficiency might lead to a delay in neuronal maturation. However, to demonstrate delayed maturation, the study should show that, given additional time, affected organoids can eventually produce late-stage neuronal signatures. Since this additional experiment may be technically challenging and time-consuming, the claim should instead be rephrased as speculative and discussed accordingly in the text.

   ---

   Response____:

   *We thank the reviewer for highlighting this point. We will discuss and re-phrase the rationale for restoring WWOX in the neurons and the WWOX deficiency-associated delayed maturation. *

   Comment____:

   Lastly, the study lacks key details necessary for reproducibility in multiple aspects. In addition to details about organoid numbers and batches discussed above, all IF images are shown as insets, making it difficult to assess broader reproducibility within the whole organoid tissue section. Also, whether distinct iPSC clones/sections/organoids were used across IF experiments - which is critical for ensuring reproducibility - is not specified.

   ---

   Response____:

   We thank the reviewer for mentioning this problem. We will include the details needed for reproducibility, including the number of batches and organoids.

   Comment____:

   As for details about experimental and bioinformatics methods, the bioinformatics pipeline is not fully described, making it impossible to verify or reproduce the computational analysis. No information is provided regarding batch correction procedures for scRNA-seq data (Lines 695-697) and on how clusters were mapped (lines 695-697) for cell type identification. Legends in Figure 1F, 2K-L, 6B, S10 do not specify what the error bars represent (e.g., standard deviation or standard error). Many catalog numbers for critical cell culture reagents are not provided, which is essential for experimental replication. The Western blot methods lack crucial details.

   ---

   Response____:

   *We thank the reviewer for highlighting this point. We acknowledge that our clarity in our methods could be improved, therefore, we will expand the bioinformatics pipeline description, the reagents used, and the details for the Western Blot. *

   Minor comments:

   Comment:

   • One relevant study (PMID: 32581702) that examines WWOX function in rat models and human fetal brains from patients has not been referenced or discussed. Notably, this study characterizes molecular changes associated with WWOX knockdown in human ESC-derived NPCs. Given its direct relevance to the current study, these findings should be acknowledged and integrated into the discussion to provide a more comprehensive understanding of WWOX-related neurodevelopmental alterations. Response____:

   We thank the reviewer for suggesting the work of Iacomino et al. which we are very aware of and shall cite appropriately in our revised version.

   Comment____:

   • For WKO-1C and 2C the exact mutations in exon1 identified by Sanger sequencing are not reported. Also, validation for WWOX protein loss in all the lines used is also missing. Information about cell line genome integrity check are also missing. Response____:

   *We thank the reviewer for bringing up these important points. We will provide the exact mutations identified in exon 1 of WKO-1C and WKO-2C as determined by Sanger sequencing and include this information in the revised manuscript. *Additionally, we will present data additional data regarding WWOX protein loss in all the cell lines used in the study.

   Comment:

   • Line 116 and 394, reference Steinberg et al is not formatted. Response:

   We apologize for this oversight and the formatting will be fixed.

   Comment:

   • Figure S1A: Localization of WWOX seems to be cytoplasmic and/or membrane-bound in organoids, while staining in IPSCs shows cytoplasmic and nuclear signals. Perphaps an orthogonal valiation with another anti-WWOX antibody would be appropriate to confirm subcellular localization. Response____:

   We thank the reviewer for their comment. WWOX localization was previously confirmed using anti-WWOX HPA050992 (Sigma), as reported in our prior publication (PMID: 34268881). While the images were not included due to a lack of novelty, we acknowledge the importance of confirming the observed patterns. The difference in localization between organoids (cytoplasmic/membrane-bound) and iPSCs (cytoplasmic/nuclear) may be attributed to differences in cell morphology, with RGs in 3D organoid sections exhibiting distinct characteristics compared to iPSCs cultured in 2D (Supplementary Figure S1A). In fact, in 2D cultures of NSCs (Supplementary Figure S4A), WWOX also shows a nuclear localization, similar to iPSCs. We will clarify this point in the manuscript.

   ---

   Comment____:

   In Figure 1, authors use week 7 organoids and claim that they are enriched for early born preplate neurons (line 141). However the authors decide to look at SATB2, which is not an eary-born preplate neuron. So while the rationale for using Satb2 is not clear, the reported staining in Figure 1E shows an unusal overlap beetween Sox2 and Satb2 nuclear signals in wt organoids. The authors needs to recheck that the correct antibodies were used in this analysis.

   Response____:

   We thank the reviewer highlighting this. We will better define the rationale for the usage of SATB2 as a marker expressed in many types of young neurons (not specifically preplate neurons), and add DCX as a marker for neurogenesis.

   Comment____:

   Figure 1 Panel F: legend states that n indiactes 3 neurons. Please specifify what n referes to.

   Response____:

   We thank the reviewer for the keen eyes and apologize for this mistake. We will correct the legend and specify that n is indeed referring to organoids and not single neurons.

   ---

   Comment____:

   Figure 3J: MYC staining appears to be nuclear in WWOX-KO organoids but more cytoplasmic in SCAR12 organoids. Also in WOREE organoids, both Sox2 and MYC staining appears different from what seen in other panels/ genotypes from the same figure panel.

   Response____:

   • *We thank the reviewer for their comment. Upon repeated staining, we consistently observe this MYC localization across organoids and more. Similarly, the differences in Sox2 and MYC staining in WOREE organoids are reproducible. While these results may seem divergent, they accurately represent the findings. We will, however, review the staining protocols and ensure that representative images are carefully selected to best reflect the data.

   ---

   Comment____:

   Figures 3 and related legend: Authors use the term w for weeks but they need to specify whether this refers to gestational weeks or post-conception weeks.

   Response____:

   *We thank the reviewer for pointing this out. We will add in the legend that “w” refers to post-conceptional weeks. *

   Comment____:

   Figure 4: The UMAP in B, E and G seems to be blurred in the bottom parts. Is this an intentional choice? If so, what would be intent? Also, title and legend for E mention metabolic alterations but data presented are not related to metabolic patwhays.

   Response____:

   We thank the reviewer for addressing this. The blurred parts of the UMAP are intentional, we will add a description of why and what it represents.

   Comment____:

   Figure 6. The same exact images from A and C are also reported in Figure S8 and S9 respectively.

   Response____:

   *We thank the reviewer for pointing this out, we will better clarify that figures S8 and S9 are an expansion of the panels shown in Figure 6, showing ROIs per cell line and rather than per genotype. *

   Comment____:

   Figure S1D: WWOX antibody seems to give an extra band at higehr molecular weight. This is also evident from S4B, where the upper band seems overrepresented in KO2. Also, are the healthy parents haploinsufficient for WWOX? what are the levels compared to wt (unrelated) controls?

   ---

   Response____:____* *

   • *We thank the reviewer for raising this point. We will quantify the bands in the WOREE patient samples and compare them to wild-type controls. We would like to clarify that the "upper" band is a nonspecific band, and its overrepresentation in KO2 samples is not indicative of WWOX expression. Additionally, we will address the question of WWOX haploinsufficiency in healthy parents and provide a comparison of WWOX levels to unrelated wild-type controls.

   Comment____:

   Figure S2: In B, what is the difference between top and bottom UMAPs? In C-D, what is NP? Correlation map suggests that the NP clusters 7 and 8 are different from cluster 11. What is the rational for labelling them all NP cluster?

   Response____:

   We would like to thank the reviewer, and we will add a clarification for the differences between top and bottom UMAPs and the rationale behind NP labeling.

   Comment____:

   Figure S6: In the legend, full description of cluster labels are missing. Also legends specifes A-D while the figure contains only A-C.

   Response____:

   We thank the reviewer and will alter the figure and its legend to clarify this.

   Comment:

   Figure S4A: The staining for TUBB3 is very different between KO1 and KO2.

   Response____:

   We thank the reviewer and will examine the pictures and if need be will replace them with more representative pictures.

   Comment____:

   Figure S8: The legend indicates n as 4 organoids but images are not quantified so there is no evidence that these patterns have been replicated in 4 organoids.

   Response____:

   We thank the reviewer for pointing this out. We will add the quantifications of NEUN+/WWOX+.

   Comment____:

   Figure S9: The title is duplicated and not corresponding to the data in the figure. The whole figure is duplicated in Figure S10 (which is wrongly labelled as Figure 10 in the legend).

   Response____:

   We thank the reviewer; we will fix the figures and corresponding titles and legends.

   Comment:

   Line 330: Figure S6 F-H should be corrected in Figure 6 F-H.

   Response____:

   We thank and agree with the observation; we will correct it.

   Comment____:

   Line 353: reference needs to be added for "our earlier findings”.

   Response____:

   We thank the reviewer, and we will re-phrase to clarify.

   Comment____:

   Lines 383 and 392: The authors describe several possible MYC roles but which ones could relevant in this contex is not discussed.

   Response____:

   We thank the reviewer and agree with their observation and would like to clarify that as we are not aware of any relevant literature examining the relationship between WWOX and MYC in non-tumor settings, we refrained from drawing any conclusions in any one direction without further experimental exploration. Nonetheless, we will re-phrase the sentences to draw clearer conclusions.

   Comment____:

   Lines 402 and 403: The authors state that the study "highlights the critical role of Wnt signalling" but they fail to provide evidence that Wnt is functionally involved, as Wnt perturbation experiments are not applied.

   Response____:

   • *We thank the reviewer for their comment. We agree that further clarification is needed regarding the functional involvement of Wnt signaling. While we have previously shown that Wnt is inappropriately activated in WWOX-KO, WOREE, and SCAR12 organoids (PMID: 34268881), and demonstrated Wnt activation in RGs via our scRNA-seq data (Figure 4I), we recognize that direct perturbation experiments would strengthen this aspect. In light of this, we will examine the levels of Wnt target genes in our transcriptomic data to provide more direct evidence of Wnt signaling involvement and its functional relevance in the context of WWOX deficiency.

   Comment:

   Line 473: "at X concentration" needs to be correct to specify the concentration used.

   Response:

   *We thank the reviewer for noticing this missing information and we will add the final puromycin concentration (1 **mg/ml) *.

   Comment____:

   Line 478: The authors state that "inform consent is under approval". Does this mean that the study was conducted before approval was obatined?

   ---

   Response____:

   We thank the reviewer for raising this concern. To clarify, approval was obtained prior to the commencement of experimentation. The sentence should read: "Skin biopsies and blood samples were obtained with informed consent, under the approval of the Kaplan Medical Center Helsinki Committee," indicating that the study was conducted in full compliance with ethical requirements, with prior approval from the committee.

   Comment____:

   Line 525: which orbital shaker and which speed was used?

   Response____:

   We thank the reviewer and will add orbital shaker details and speed.

   Comment____:

   Line 537: what is GC in GC/ul?

   Response____:

   We thank the reviewer and clarify that this is the accepted units for viral load. GC is Genome Copies, and this is often used in qPCR assays to estimate the amount of viral genetic material in a sample. It is often used interchangeably with vg/µL (Viral Genome per microliter).

   Comment____:

   Line 629: samples were centrifgues at which speed and for how long?

   Response____:

   We thank the reviewer and will fix to include details about centrifugation.

   Comment____:

   Line 639: "All primer sequence" should be plural.

   Response____:

   We thank the reviewer and will correct the typing mistake.

   Referees cross-commenting

   All four reviews appear fair and complementary to each other. Reviewers have consistently highlighted concerns regarding unclear genomic alterations in patients' iPSCs and experimental reproducibility in organoid cultures, emphasizing the need for further validation of the reported findings and the underlying molecular cascade. Additionally, they have noted some inconsistencies, with Reviewer #2 specifically identifying a major discrepancy in the WWOX-KO phenotypes compared to those previously described by the same team.

   General assessment:

   The strengths of this study lie in its focus on disease phenotypes in a human context and the use of patient-derived iPSC lines, which provide valuable translational relevance. Additionally, the study employs a complementary set of analyses, including functional assays, immunofluorescence (IF), and single-cell RNA sequencing (scRNA-seq), which enhance its depth. However, the study has several critical weaknesses, primarily related to suboptimal experimental design and limited reproducibility. These are discussed in section A and also indicated below:

   • Lack of isogenic controls or patient-derived lines and presence of conflicting data for patient-derived organoids, making genotype-based comparisons for patients' lines less robust; examples of studies using iPSC isogenic controls for dissecting neurodevelopmental disorders are found here (PMID: 35084981; PMID: 26186191).
   • Limited reproducibility, due to a small number of organoids used and the lack of orthogonal validation for key findings.
   • Absence of functional validation for MYC's contribution, making its proposed role unclear. Advance:

   This study builds upon and expands previous efforts by the same team to characterize brain organoid models obtained from patient-derived iPSCs, as well as to explore gene therapy restoration approaches (PMID: 34268881, PMID: 34747138). Some of the bioinformatics analyses appear to have been developed elsewhere, and technically, the study offers only a limited methodological advance.

   Instead, the key advancement of this work is more conceptual: it proposes potential underlying mechanisms of WWOX-related neurodevelopmental disorders. If the study's limitations were addressed, it could provide valuable insights into WWOX's role as a key regulator of radial glia proliferation and differentiation, as well as potential functions in neuronal maturation. These findings would be relatively novel in the context of WWOX-related neurodevelopmental disorders. WWOX has been extensively studied in rodent models, where WWOX -/- mice exhibit growth retardation and brain malformations (PMID: 32000863, PMID: 18487609, PMID: 15026124). Additionally, studies in rats and human fetal cortical tissue from patients (PMID: 32581702) have linked WWOX deficiency to migration defects and cortical cytoarchitectural alterations. Previous work in mice by the same team suggested that neurons are the key population affected, linking WWOX deficiency to hyperexcitability and intractable epilepsy (PMID: 33914858). However, the relevance of radial glia and cell-type specific molecular alterations linked to WWOX mutations have remained poorly defined. Through scRNA-seq, this study offers some insights into cell-type-specific molecular changes, especially in radial glia cells. These changes are linked to MYC fucntion, cell cycle arrest and altered differentiation trajectories. However, these insights remain preliminary due to the study's design limitations.

   Another potential advancement of this study is its exploration of syndrome-specific alterations in WOREE and SCAR12 patients and their rescue through WWOX gene therapy-an aspect that has been difficult to study in animal models and remains largely unexplored. While the brain organoid model offers a promising approach, the true conceptual advance of this study remains uncertain, as its current limitations hinder the ability to draw definitive conclusions.

   Audience: This study could be particularly relevant to a specialized audience, including basic research scientists working in developmental biology and the molecular basis of neurodevelopmental disorders, as well as those interested in translational approaches. Additionally, given WWOX's known roles beyond neurodevelopment and potential involvement of MYC, the findings may also be of interest to cancer biologists.

   Expertise: My expertise lies in iPSCs and brain organoid modeling of neurodevelopmental disorders, with a strong focus on organoid phenotypic analysis, particularly immunofluorescence and transcriptomics. However, I do not have a strong background in bioinformatics and therefore lack sufficient expertise to evaluate the bioinformatic methodologies utilized in the study.

   Response:

   We thank the reviewer for their valuable feedback and for acknowledging the strengths of our study. We agree with the reviewer that additional validation and replication are needed to strengthen our conclusions. We acknowledge the limitations in experimental design, and we are committed to enhancing the reproducibility of our findings. We also appreciate the reviewer's comments on the study's conceptual advancements, which we believe offer new insights into WWOX's role in neurodevelopmental disorders.

   We are confident that with the additional experiments outlined, our study will provide valuable contributions to understanding WWOX-related syndromes. Thank you again for your thoughtful suggestions.

   __ __

   ---

   Reviewer 4:

   Summary

   The article deals with WWOX gene deficiency related neural diseases such as WOREE and SCAR12 syndromes. While there is no available drugs for treatment, the authors used organoid approach to study the development of the potential of disease development. The authors utilized neural organoids and single-cell transcriptomics and identified radial glial cells (RGs) as preferentially affected. The RG cells have disrupted cell cycle arrests in the leading G2/M and S phases, along with MYC overexpression and concomitant reduction in neuronal generation. The study also included detecting neural hyperexcitability and restoring defective WWOX gene for functional assessment. The study is important in understanding the function of WWOX and its mutated states, especially in identifying RG in the potential disease progression.

   My concerns are:

   Comment____:

   1.Although organoids are good models for in vitro simulation of disease progression, I am not convinced that RG is the only cell type affected initially.

   Response____:

   We thank the reviewer for their thoughtful comment. We would like to clarify that we do not suggest that only radial glia cells are affected. As mentioned in both the current manuscript and our previous work (Steinberg et al., EMBO Mol. Med. 2021, and Repudi et al., Brain, 2021), other cell populations, including neurons and oligodendrocytes, are also impacted by WWOX deficiency. WWOX is widely expressed in the mature brain, and we are actively investigating whether these effects are cell-autonomous. In this study, we focus on WWOX in RGs due its high expression and possible importance in maintaining RG homeostasis. We will further clarify this point in the revised manuscript.

   Comment____:

   Functional characterization of RG needs further strengthening. I suggest utilizing a proteomic approach to compare the diseased-ongoing RG versus regular RG and identify which proteins are involved for functional characterization. Finally, the functional alterations in the mitochondria due to WWOX deficiency should be checked.

   Response____:

   We thank the reviewer for their suggestion and agree that performing proteomic analysis on RG populations will strengthen our understanding of the underlying mechanism, however, the experiment itself was attempted and proved to be technically challenging at this size, and for now is beyond the scope of this paper.

   Comment____:

   WWOX-deficient radial glia cells are expected not to guide neurons' migration normally during neural development. Please note that neuronal heterotopia occurs frequently in the WWOX deficiency. Neurons tend to exhibit groups of cells coming together in the neocortex. Purified RG cells are used to run versus typical neurons or RG cells. One can expect WWOX-deficient cells to run away from the normal cells, and they may kill each other, leading to compromise. The authors should run the real-time cell migration experiments using normal neurons versus WWOX-deficient radial glia cells and see the behavior of both cell types upon encountering each other. This will provide better insight regarding the deficiency of WWOX in radial glia cells.

   Response____:

   We thank the reviewer for their insightful suggestion regarding the validation of neuronal heterotopia in WWOX-deficient cells through real-time migration experiments. While we recognize the potential value of this approach for investigating the behavior of WWOX-deficient radial glia cells, we believe that such experiments would extend beyond the scope of the current study. However, we are considering them as part of our future research to further explore the impact of WWOX deficiency on cell migration and neuronal positioning. Thank you again for your valuable input.

   Significance

   The study is significant in our understanding the progression of syndromes associated with WWOX deficiency. My suggestions are shown in the above section.

   Response____:

   We thank the reviewer for their thoughtful and constructive feedback. We especially appreciate the suggestions regarding the broader involvement of additional cell types and the importance of exploring radial glia function through real-time migration assays. These insights will help us refine the focus and interpretation of our findings, and we will address the relevant clarifications and improvements in the revised manuscript.

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   Referee #4

   Evidence, reproducibility and clarity

   The article deals with WWOX gene deficiency related neural diseases such as WOREE and SCAR12 syndromes. While there is no available drugs for treatment, the authors used organoid approach to study the development of the potential of disease development. The authors utilized neural organoids and single-cell transcriptomics and identified radial glial cells (RGs) as preferentially affected. The RG cells have disrupted cell cycle arrests in the leading G2/M and S phases, along with MYC overexpression and concomitant reduction in neuronal generation. The study also included detecting neural hyperexcitability and restoring defective WWOX …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #4

   Evidence, reproducibility and clarity

   The article deals with WWOX gene deficiency related neural diseases such as WOREE and SCAR12 syndromes. While there is no available drugs for treatment, the authors used organoid approach to study the development of the potential of disease development. The authors utilized neural organoids and single-cell transcriptomics and identified radial glial cells (RGs) as preferentially affected. The RG cells have disrupted cell cycle arrests in the leading G2/M and S phases, along with MYC overexpression and concomitant reduction in neuronal generation. The study also included detecting neural hyperexcitability and restoring defective WWOX gene for functional assessment. The study is important in understanding the function of WWOX and its mutated states, especially in identifying RG in the potential disease progression. My concerns are:

   1. Although organoids are good models for in vitro simulation of disease progression, I am not convinced that RG is the only cell type affected initially.
   2. Functional characterization of RG needs further strengthening. I suggest utilizing a proteomic approach to compare the diseased-ongoing RG versus regular RG and identify which proteins are involved for functional characterization. Finally, the functional alterations in the mitochondria due to WWOX deficiency should be checked.
   3. WWOX-deficient radial glia cells are expected not to guide neurons' migration normally during neural development. Please note that neuronal heterotopia occurs frequently in the WWOX deficiency. Neurons tend to exhibit groups of cells coming together in the neocortex. Purified RG cells are used to run versus typical neurons or RG cells. One can expect WWOX-deficient cells to run away from the normal cells, and they may kill each other, leading to compromise. The authors should run the real-time cell migration experiments using normal neurons versus WWOX-deficient radial glia cells and see the behavior of both cell types upon encountering each other. This will provide better insight regarding the deficiency of WWOX in radial glia cells.

   Significance

   The study is significant in our understanding the progression of syndromes associated with WWOX deficiency. My suggestions are shown in the above section.

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   ---

   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   The manuscript by Steinberg and colleagues describes cellular and molecular changes linked to mutations in WWOX, a gene implicated in rare neurodevelopmental disorders, WOREE and SCAR12 syndromes. By comparing immunofluorescene and single cell trascriptomics of unguided brain organoids from control and WWOX-knockout iPSCs, as well as 2D NSCs and in vivo fetal brain expression datasets, the authors identified radial glia as relevant cell types in which WWOX is expressed and affected by WWOX deficiency. Using immunofluorescence, single cell trascriptomics analysis and western blotting on week 16 organoids, the authors show …

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   The manuscript by Steinberg and colleagues describes cellular and molecular changes linked to mutations in WWOX, a gene implicated in rare neurodevelopmental disorders, WOREE and SCAR12 syndromes. By comparing immunofluorescene and single cell trascriptomics of unguided brain organoids from control and WWOX-knockout iPSCs, as well as 2D NSCs and in vivo fetal brain expression datasets, the authors identified radial glia as relevant cell types in which WWOX is expressed and affected by WWOX deficiency. Using immunofluorescence, single cell trascriptomics analysis and western blotting on week 16 organoids, the authors show that WWOX deficiency results in increased abundance of radial glia cells at the expenses of neuronal production. These changes are accompanied by accumulation of cells in G2/M and S phases, overexpression of c-MYC and Wnt activation. In addition to this, the authors characterize unguided brain organoids generated from iPSCs reprogrammed from patients affected by WOREE or SCARE12 syndromes. Using immunofluoresce and single cell trascriptomics, they find that, while RG abundance changes were very modest, patients's iPSC-derived neurons are enriched for signatures related to early development, suggesting delayed differentiation. Finally, the authors use patch clumping, calcium imaging and gene therapy in 16 weeks old organoids derived from control and patients-derived iPSCs, to demonstrate that WWOX restoration normalized hyperexcitability phenotypes in both WOREE and SCAR12 organoids. These results thus provide a proof-of-concept evidence that WWOX restoration in human cells is a valid strategy to correct for hyperexcitability pehnotypes in WWOX related syndromes.

   Major comments:

   The study's main conclusions regarding neurodevelopmental phenotypes linked to WWOX deficiency and genotype-phenotype relationships are based on iPSC-derived brain organoid models analyzed using immunofluorescence, single-cell transcriptomics, and excitability recordings (cell-attached patch clamping, calcium imaging). While the analyses involve a diverse collection of iPSCs and two time points (7 and 16 weeks), the study falls short in providing sufficient experimental details and validation to fully support its conclusions. Additional quantification, replication, and functional validation would be necessary to solidify the study's conclusions. Some of these validations are achievable within a reasonable timeframe, while others would require a more substantial investment of time and resources as detailed below. A key concern is the lack of experimental details and replicability. Number of individual organoids, number of images per organoid for IF, and whether multiple batches were used are only partially provided. While the authors report generating multiple WWOX knockout clones, the legends and methods do not specify whether multiple clones were used across different organoid experiments. The study states that four organoids were used for scRNA-seq, but it is unclear whether this means four organoids per genotype or one organoid per genotype was analyzed. These ambiguities make the claims appear rather preliminary. Another issue is the limited validation of scRNA-seq observations. Since scRNA-seq is often performed on a limited number of organoids, orthogonal validation is crucial to strengthen the findings. For example, changes in radial glia abundance and neuronal production observed in scRNA analyis (Figure 2-5) could be validated using immunofluorescence across genotypes and batches. Currently, IF stainings for Sox2 and TUBB3 are shown only at 7 weeks in Figure 1B, but no quantificative assessment is provided. Also, it is not clear if quantifications provided in Figure 1F refer to multiple organoids or batches. Furthermore, the observations on cell cycle arrest, DNA damage, senescence, metabolic alterations, Wnt activation obtained via scRNA-seq could be further validated on organoid tissues using specific antibodies that the lab used before (e.g. yH2AX antibody in PMID: 34268881) or assays that have been developed elsewhere (some examples are reviewed in PMID: 38759644). As for feasibility, immunofluorescence validation of existing tissues is realistic, requiring validated antibodies and procedures, some additional imaging time and analysis (estimated 1-2 months, with some budget to purchase antibodies and cover imaging time costs). Feasibility of efforts related to validation across organoids and batches depends on the number of organoids used so far and available tissues. Generating new organoids would be indeed more time-consuming (≈ 6 months) and expensive (but extact costs would depend on number of clones, organoids and batches used), but feasible. Another limitation is the lack of functional relevance of MYC alterations. The study confirms increased MYC expression via both scRNA-seq and immunofluorescence in organoid tissues. However, these results remain correlative and demonstrating the functional requirement of MYC overexpression in mediating WWOX-deficiency-related changes would significantly strengthen the study's conclusions. This would require additional differentiation experiments, including MYC overexpression or knockout models, to assess its direct impact. These efforts would represent a major conceptual advance by linking RG effects to MYC function and highlighting MYC-related therapeutic directions. These additonal experiments would require a substantial investment to generate the necessary regents (e.g. WWOX-KO and WT iPSCs with altered MYC levels) and additional time and costs for organoid analysis, mostly by immunofluorescence (estimated 6-8 months). Another issue is the lack of patterning analysis in unguided organoids, which are known to exhibit high variability in regional identity (PMID: 28283582). While the authors acknowledge this limitation to some extent-abstaining from fine-resolution analysis (Lines 173-174)-this variability, combined with the limited number of organoids used, could be a major confounding factor in the phenotypic analyses, even at a broad resolution. Indeed, some of the reported differences across genotypes may stem from variability in organoid patterning rather than true genotype-driven effects. For example, the reduced SATB2 expression in KO and patient-derived organoids from Figure 1E-F could result from impaired cortical patterning rather than a direct effect of WWOX deficiency. Additionally, in Figure 6D and 6E, the fact that WOREE iPSC-derived organoids - but not SCAR12 organodis- show lower levels of both CTIP2 and SATB2, might reflect a shift toward a non-cortical identity rather than a direct WWOX-dependent phenotype. To rule out patterning variability as a contributing factor, the authors should analyze organoid regional identity across genotypes using immunostaining for dorsal and ventral forebrain markers. This would allow a more solid inference of genotype-specific effects on neurodevelopmental phenotypes. Patterning validation can be performed on existing organoid tissues (week 7) using validated antibodies (PMID: 28283582). As such, this analysis is expected to be relatively straightforward and feasible in a few weeks time. If the generation of new organoids is needed, such patterning validation should still be relatively feasible, as week 7 organoids are ideal for assessing regional identity. Analysis of patterning effects should also extend to 2D NSC cultures. In the 2D NSC models derived from WWOX-KO lines (Figure 3L, Figure S4A), the differentiation protocol includes patterning factors that promote ventral fates (SAG and IWP2). Interestingly, the quantification of MYC expression from unguided organoids and 2D NSCs (Figure 3K-L) reveals a major difference in the fraction of MYC-positive cells in WT conditions across the two culture models. A possible changes in the dorsal and ventral patterning of 3D and 2D cultures might explain these differences and implementing immunostaining for patterning markers in 2D would help clarify patterning contributions. There are also some concerns regarding WOREE and SCAR12 phenotypes. First, the genotypes of the patient-derived iPSCs are not clearly defined, making it difficult to establish clear genotype-phenotype relationships. The study uses iPSCs from four different patients (2 WOREE, 2 SCAR12), some of which were validated in a previous study (PMID: 34268881). However, it remains unclear how they were validated, and detailed genomic alterations of the four patients are not explicitly reported. Additionally, it is uncertain whether all variants result in a full loss of WWOX function, as protein loss evidence is only provided for one WOREE patient (Figure S1D). Also, the authors state that SCAR12 should have a milder phenotype (line 168), but it is unclear whether this claim is based on clinical evidence or genomic data from these specific patients. To improve genotype-phenotype comparisons, the authors should consider including a clear schematic summarizing the genomic alterations in all patient-derived lines and their expected disease severity. Second, the experimental design lacks appropriate controls for patient-derived iPSCs. All patient-derived iPSC comparisons are performed against a single reference male iPSC line, which is neither isogenic to WOREE nor SCAR12 iPSCs. This complicates the interpretation of differences between healthy and patient-derived organoids, as well as comparisons between WOREE and SCAR12 phenotypes. Given this design, it is impossible to draw solid conclusions about genotype-phenotype relationships. A more robust approach would involve including multiple healthy controls to account for genetic background variability or using isogenic parental or genetically corrected lines, which would provide a cleaner genetic comparison. A recent study (PMID: 36385170) discusses different study designs that could strengthen this aspect and might be useful for the authors to consult. Third, the study presents seemingly conflicting results regarding WOREE and SCAR12 phenotypes. The authors present immunofluorescence (IF) and scRNA-seq data indicating that changes in radial glia (RG) abudance are not observed in these patient-derived organoids. However, using same methodologies, they indicate that neuronal production is affected, leading to the accumulation of early neuronal signatures in both WOREE and SCAR12 neurons. The study does not explore whether RG signatures might be altered in a way that could contribute to neuronal phenotypes. Also, Figure 1F suggests that while Sox2+ cell counts are not increased in SCAR12 organoids, SATB2 levels are still altered, indicating that Sox2 and SATB2 trends are not tightly coupled across genotypes. Furthermore, Figure 1 and 6 show that while both syndromes exhibit similar hyperexcitability, data in Figure 6 report that only WOREE organoids display reductions in SATB2 and CTIP2 counts and that this can be rescued by WWOX restoration. Some of these discrepancies could stem from patterning variability as discussed above. Also, neuronal firing rate across WOREE and SCAR12 iPSC-derived organoids (Figure 6B) was different at later stages, but was rather comparable at an earlier stages (Figure S1G). The reasons for these differences are not thoroughly discussed. To strengthen the discussion, the authors should address how RG alterations (if any) might contribute to neuronal phenotypes, provide a more detailed comparison between WOREE and SCAR12 organoids and the WWOX-KO model and elaborate on the distinct phenotypes of the two syndromes, including possible explanations for observed functional and molecular discrepancies. Lastly, the conclusions drawn about WWOX restoration via gene therapy are weakened by the lack of replication and validation (see points above). First, the authors claim successful WWOX restoration in neurons, but provide limited evidence that the infected population consists of neurons. NeuN staining (Figure 6A and S10) suggests some neuronal expression, but quantification of WWOX+ NeuN+ / WWOX+ total cells is missing. Given that IF data are already available, this additional quantification could be completed within a few weeks and would significantly strengthen the claim. Second, the rationale for restoring WWOX in neurons is unclear, given that WT neurons do not normally express WWOX. Is WWOX being considered a functional neuronal maturation factor? If so, this should be explicitly discussed in the manuscript. Third, the authors propose that WWOX deficiency might lead to a delay in neuronal maturation. However, to demonstrate delayed maturation, the study should show that, given additional time, affected organoids can eventually produce late-stage neuronal signatures. Since this additional experiment may be technically challenging and time-consuming, the claim should instead be rephrased as speculative and discussed accordingly in the text. Lastly, the study lacks key details necessary for reproducibility in multiple aspects. In addition to details about organoid numbers and batches discussed above, all IF images are shown as insets, making it difficult to assess broader reproducibility within the whole organoid tissue section. Also, whether distinct iPSC clones/sections/organoids were used across IF experiments - which is critical for ensuring reproducibility - is not specified. As for details about experimental and bioinformatics methods, the bioinformatics pipeline is not fully described, making it impossible to verify or reproduce the computational analysis. No information is provided regarding batch correction procedures for scRNA-seq data (Lines 695-697) and on how clusters were mapped (lines 695-697) for cell type identification. Legends in Figure 1F, 2K-L, 6B, S10 do not specify what the error bars represent (e.g., standard deviation or standard error). Many catalog numbers for critical cell culture reagents are not provided, which is essential for experimental replication. The Western blot methods lack crucial details.

   Minor comments:

   • One relevant study (PMID: 32581702) that examines WWOX function in rat models and human fetal brains from patients has not been referenced or discussed. Notably, this study characterizes molecular changes associated with WWOX knockdown in human ESC-derived NPCs. Given its direct relevance to the current study, these findings should be acknowledged and integrated into the discussion to provide a more comprehensive understanding of WWOX-related neurodevelopmental alterations.
   • For WKO-1C and 2C the exact mutations in exon1 identified by Sanger sequencing are not reported. Also, validation for WWOX protein loss in all the lines used is also missing. Information about cell line genome integrity check are also missing.
   • Line 116 and 394, reference Steinberg et al is not formatted.
   • Figure S1A: Localization of WWOX seems to be cytoplasmic and/or membrane-bound in organoids, while staining in IPSCs shows cytoplasmic and nuclear signals. Perphaps an orthogonal valiation with another anti-WWOX antibody would be appropriate to confirm subcellular localization.
   • In Figure 1, authors use week 7 organoids and claim that they are enriched for early born preplate neurons (line 141). However the authors decide to look at SATB2, which is not an eary-born preplate neuron. So while the rationale for using Satb2 is not clear, the reported staining in Figure 1E shows an unusal overlap beetween Sox2 and Satb2 nuclear signals in wt organoids. The authors needs to recheck that the correct antibodies were used in this analysis.
   • Figure 1 Panel F: legend states that n indiactes 3 neurons. Please specifify what n referes to.
   • Figure 3J: MYC staining appears to be nuclear in WWOX-KO organoids but more cytoplasmic in SCAR12 organoids. Also in WOREE organoids, both Sox2 and MYC staining appears different from what seen in other panels/ genotypes from the same figure panel.
   • Figures 3 and related legend: Authors use the term w for weeks but they need to specify whether this refers to gestational weeks or post-conception weeks.
   • Figure 4: The UMAP in B, E and G seems to be blurred in the bottom parts. Is this an intentional choice? If so, what would be intent? Also, title and legend for E mention metabolic alterations but data presented are not related to metabolic patwhays.
   • Figure 6. The same exact images from A and C are also reported in Figure S8 and S9 respectively.
   • Figure S1D: WWOX antibody seems to give an extra band at higehr molecular weight. This is also evident from S4B, where the upper band seems overrepresented in KO2. Also, are the healthy parents haploinsufficient for WWOX? what are the levels compared to wt (unrelated) controls?
   • Figure S2: In B, what is the difference between top and bottom UMAPs? In C-D, what is NP? Correlation map suggests that the NP clusters 7 and 8 are different from cluster 11. What is the rational for labelling them all NP cluster?
   • Figure S6: In the legend, full description of cluster labels are missing. Also legends specifes A-D while the figure contains only A-C.
   • Figure S4A: The staining for TUBB3 is very different between KO1 and KO2.
   • Figure S8: The legend indicates n as 4 organoids but images are not quantified so there is no evidence that these patterns have been replicated in 4 organoids.
   • Figure S9: The title is duplicated and not corresponding to the data in the figure. The whole figure is duplicated in Figure S10 (which is wrongly labelled as Figure 10 in the legend).
   • Line 330: Figure S6 F-H should be corrected in Figure 6 F-H.
   • Line 353: reference needs to be added for "our earlier findings"
   • Lines 383 and 392: The authors describe several possible MYC roles but which ones could relevant in this contex is not discussed.
   • Lines 402 and 403: The authors state that the study "highlights the critical role of Wnt signalling" but they fail to provide evidence that Wnt is functionally involved, as Wnt perturbation experiments are not applied.
   • Line 473: "at X concentration" needs to be correct to specify the concentration used.
   • Line 478: The authors state that "inform consent is under approval". Does this mean that the study was conducted before approval was obatined?
   • Line 525: which orbital shaker and which speed was used?
   • Line 537: what is GC in GC/ul?
   • Line 629: samples were centrifgues at which speed and for how long?
   • Line 639: "All primer sequence" should be plural

   Referees cross-commenting

   All four reviews appear fair and complementary to each other. Reviewers have consistently highlighted concerns regarding unclear genomic alterations in patients' iPSCs and experimental reproducibility in organoid cultures, emphasizing the need for further validation of the reported findings and the underlying molecular cascade. Additionally, they have noted some inconsistencies, with Reviewer #2 specifically identifying a major discrepancy in the WWOX-KO phenotypes compared to those previously described by the same team.

   Significance

   General assessment:

   The strengths of this study lie in its focus on disease phenotypes in a human context and the use of patient-derived iPSC lines, which provide valuable translational relevance. Additionally, the study employs a complementary set of analyses, including functional assays, immunofluorescence (IF), and single-cell RNA sequencing (scRNA-seq), which enhance its depth. However, the study has several critical weaknesses, primarily related to suboptimal experimental design and limited reproducibility. These are discussed in section A and also indicated below:

   • Lack of isogenic controls or patient-derived lines and presence of conflicting data for patient-derived organoids, making genotype-based comparisons for patients' lines less robust; examples of studies using iPSC isogenic controls for dissecting neurodevelopmental disorders are found here (PMID: 35084981; PMID: 26186191).
   • Limited reproducibility, due to a small number of organoids used and the lack of orthogonal validation for key findings.
   • Absence of functional validation for MYC's contribution, making its proposed role unclear.

   Advance:

   This study builds upon and expands previous efforts by the same team to characterize brain organoid models obtained from patient-derived iPSCs, as well as to explore gene therapy restoration approaches (PMID: 34268881, PMID: 34747138). Some of the bioinformatics analyses appear to have been developed elsewhere, and technically, the study offers only a limited methodological advance. Instead, the key advancement of this work is more conceptual: it proposes potential underlying mechanisms of WWOX-related neurodevelopmental disorders. If the study's limitations were addressed, it could provide valuable insights into WWOX's role as a key regulator of radial glia proliferation and differentiation, as well as potential functions in neuronal maturation. These findings would be relatively novel in the context of WWOX-related neurodevelopmental disorders. WWOX has been extensively studied in rodent models, where WWOX -/- mice exhibit growth retardation and brain malformations (PMID: 32000863, PMID: 18487609, PMID: 15026124). Additionally, studies in rats and human fetal cortical tissue from patients (PMID: 32581702) have linked WWOX deficiency to migration defects and cortical cytoarchitectural alterations. Previous work in mice by the same team suggested that neurons are the key population affected, linking WWOX deficiency to hyperexcitability and intractable epilepsy (PMID: 33914858). However, the relevance of radial glia and cell-type specific molecular alterations linked to WWOX mutations have remained poorly defined. Through scRNA-seq, this study offers some insights into cell-type-specific molecular changes, especially in radial glia cells. These changes are linked to MYC fucntion, cell cycle arrest and altered differentiation trajectories. However, these insights remain preliminary due to the study's design limitations. Another potential advancement of this study is its exploration of syndrome-specific alterations in WOREE and SCAR12 patients and their rescue through WWOX gene therapy-an aspect that has been difficult to study in animal models and remains largely unexplored. While the brain organoid model offers a promising approach, the true conceptual advance of this study remains uncertain, as its current limitations hinder the ability to draw definitive conclusions.

   Audience: This study could be particularly relevant to a specialized audience, including basic research scientists working in developmental biology and the molecular basis of neurodevelopmental disorders, as well as those interested in translational approaches. Additionally, given WWOX's known roles beyond neurodevelopment and potential involvement of MYC, the findings may also be of interest to cancer biologists.

   Expertise: My expertise lies in iPSCs and brain organoid modeling of neurodevelopmental disorders, with a strong focus on organoid phenotypic analysis, particularly immunofluorescence and transcriptomics. However, I do not have a strong background in bioinformatics and therefore lack sufficient expertise to evaluate the bioinformatic methodologies utilized in the study.

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   Apr 15, 2025

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   In this study, Steinberg et al aim to elucidate the role of WWOX in human neurogenesis and model WOREE and SCAR12 syndromes which are rare neurodevelopmental disorders. They chose to investigate its function in human brain organoids after generating WWOX KO and patient-derived iPSC lines. Their major finding is that radial glial cells, the main neural progenitor population during corticogenesis, are affected. Via single-cell-RNA-sequencing, they try to decipher the perturbed molecular mechanisms identifying MYC, a proto-oncogene, as a major player. At the end of their study, they proceed to gene therapy restoration and …

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   In this study, Steinberg et al aim to elucidate the role of WWOX in human neurogenesis and model WOREE and SCAR12 syndromes which are rare neurodevelopmental disorders. They chose to investigate its function in human brain organoids after generating WWOX KO and patient-derived iPSC lines. Their major finding is that radial glial cells, the main neural progenitor population during corticogenesis, are affected. Via single-cell-RNA-sequencing, they try to decipher the perturbed molecular mechanisms identifying MYC, a proto-oncogene, as a major player. At the end of their study, they proceed to gene therapy restoration and suggest that this could become a potential therapeutic intervention for WOREE and SCAR12 syndromes. The study aims to elucidate major cellular and molecular mechanisms that modulate neurodevelopment and neurodevelopmental disorders. Although sc-RNA-seq could potentially be of great interest and unravel major mechanisms, the authors do not follow this part, but only discuss potential future avenues. Here are some suggestions that could be useful to the authros.

   Major comments:

   • A big part of the paper focuses on generating the iPSCs and characterizing the generated brain organoids and gene restoration of the phenotype via restoration of the WWOX gene expression (Fig.1, Fig.6, Fig.S1, Fig.S8, Fig.S10 and potentially Fig.S9 - this figure is not included) however, this has already been done by the same authors (first and last authors) in a previous publication. What are the differences in the line that have been generated in previous publication (Steinberg et al 2021, EMBO Mol. Med.)? If there are differences, the authors should make a thought comparison and explain why they generated different lines. If there is no difference, the authors should reduce to minimum this part and place it to supplementary.
   • Fig.1E: in the pictures shown, the majority of the Satb2+ cells are colocalized with SOX2. Although a small portion of neurons have been shown from many studies that in brain organoids are co-localized to SOX2, in the pictures depicted this percentage is big. Also in ctrl condition the VZ-CP like areas are not easily recognized. The authors should check if this co-localization is a more general phenotype and if not choose more representative pictures.
   • Information about the number of organoids per batch used in each figure is not included. This needs to be added for each experiment. Data (at least the majority of them) should be collected from brain organoids from at least two batches.
   • The expression of WWOX in cortical development has been shown in the previous publication. Although sc-RNA data are validating the previous data and are adding more information, these data should be put as supplementary. Besides, in Fig.3G where authors aim to compare WWOX expression to MYC that fits nicely with their results depicting MYC as the most affected gene in KO and mutant line, when one looks at the WWOX expression only it seems that its expression is higher in CP that VZ. This is contrary to the conclusion that WWOX is mainly characterizes RGs. Why is that? Authors should at least discuss this.
   • In this study, authors show that progenitors are reduced in WWOX-KO organoids, however in the previous publication SOX2 population is not majorly affected. Why are there such differences? Given that RGs are the main population affected as authors propose in this study, these differences must be at least discussed. Similar comments regarding neurons: in previous publication there is a minimal reduction of neurons in WWOX-KO brain organoids, while here authors describe major differences.
   • Data from sc-RNA-seq analysis highlighting MYC as major differentially regulated gene are very interesting and seem to be key to the molecular pathway affected as authors suggest. Authors also validate this with immunostainings in brain orgnaoids. However, in Fig.3J MYC expression in ctrl is not depicted, even though in the respective graph it seems that 20% of SOX2+ cells co-express MYC. Please choose a more representative picture.
   • One of the main findings in this study is the cell cycle changes observed in WWOX-KO and mutant organoids. Given that the major novelty of the publication is the cellular and molecular mechanism implicated in WOREE and SCAR12 syndromes, authors should perform additional experiments towards this direction. One suggestion would be to perform stainings in brain organoids using markers of the different cell cycle phases (eg. KI67, cyclin a, BrdU/EdU, ph3). Also treatment of organoids with different BrdU/EdU chase experiments would be important so as to measure exactly the length of each cell cycle phase.
   • Regarding the molecular cascade, is WWOX directly affecting MYC of Wnt genes? Do they have information on upstream and downstream factors in the affected molecular pathway?
   • Restoration of phenotype via reinsertion of WWOX gene has already been done in the previous publications by the same authors. But what about MYC? Is MYC manipulation able to rescue the phenotype?
   • Finally, MYC association to ribosome biogenesis as mentioned by the authors in discussion is very interesting. The authors should consider investigating this direction, as it will be a great addition to the mechanisms that regulate WOREE and SCAR12 syndromes which is the main focus of this study.

   Minor comments:

   • Line 115: authors say that the data they discuss are found in Fig.S2A, maybe they mean Fig.S1A?
   • Fig.S9 is missing, in the current version this Fig is the same with Fig.S10. Please change it.

   Significance

   This study is the continuation of a previous publication the authors have published. The topic is very interesting and novel especially in modelling neurodevelopmental disorders in a human context, however, given that the main phenotype has already been published, the authors should include more effort in the molecular cascade. Clinical interventions if the molecular cascade is described would be of great importance to the field

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   Referee #1

   Evidence, reproducibility and clarity

   The manuscript "WWOX deficiency impairs neurogenesis and neuronal function in human organoids" by Aqeilan and co-workers provides impressive set of studies, mostly utilizing cerebral organoids (CO), gaining insights into the roles of the gene WWOX in neuronal development and molecular etiology of WOREE and SCAR12, two devastating rare diseases originating from mutations in WWOX. Further, therapeutic modalities through the neuron-specific gene therapy are investigated using the WWOX k/o and WOREE and SCAR12 patient-derived COs. Among the major findings of this work one can highlight the identification of the main source of …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   ---

   Referee #1

   Evidence, reproducibility and clarity

   The manuscript "WWOX deficiency impairs neurogenesis and neuronal function in human organoids" by Aqeilan and co-workers provides impressive set of studies, mostly utilizing cerebral organoids (CO), gaining insights into the roles of the gene WWOX in neuronal development and molecular etiology of WOREE and SCAR12, two devastating rare diseases originating from mutations in WWOX. Further, therapeutic modalities through the neuron-specific gene therapy are investigated using the WWOX k/o and WOREE and SCAR12 patient-derived COs. Among the major findings of this work one can highlight the identification of the main source of WWOX-expressing cells as radial glia (RG) cells; the discovery of the massive upregulation of Myc upon loss/decrease in WWOX expression in RGs; and the strong neuronal under-differentiation induced upon WWOX k/o and mutations. Regarding the latter finding, the authors report massive increase in RGs and concomitant drop in neuronal cells in WWOX k/o COs. In contrast, in WOREE and SCAR12 patient-derived COs, a more subtle under-differentiation is seen. Specifically, while WOREE but not SCAR12 patient-derived COs also show a certain increase in the RG proportion, both types of patient-derived COs demonstrate higher proportions of "young" neuronal cells as compared to wild-type COs. Thus, a picture can be drawn whereas complete loss of WWOX leads to strong under-differentiation mostly manifested as expansion of RGs and hence under-production of neuronal cells, while hypomorphic loss-of-function of WWOX in WOREE and in missense mutations in SCAR12 lead to the later defect in neuronal cell maturation. Overall, I find the work highly interesting, but I would like the authors to address one major issue and several minor ones. The major issue is related to the overall model the authors seem to build based on their data - or at least the overall model the reader may get from the paper. This model suggests that the loss / decrease in WWOX levels in RGs leads to Myc overexpression, that in turn affects the cell cycle and prevents neuronal differentiation. This model is highly attractive, but is probably incomplete, in the sense that it does not fully recapitulate the complicated picture. Indeed, all three types of mutated WWOX COs (WWOX k/o, WOREE patient-derived organoids, and SCAR12 patient-derived organoids) demonstrate strong - but equal levels of Myc upregulation. Yet the under-differentiation in each of these three types is different, as described above, and the disease manifestations among WOREE vs. SCAR12 patients are also different. Thus, another player (in addition to Myc) must be at place, that is differentially affected by the partial null mutations in WOREE and missense mutations in SCAR12. This point - ideally to be addressed experimentally - should be at least faced directly by the authors in the Discussion. Perhaps they can already point to such additional players based on their transcriptomics analysis.

   The minor issues are as follows.

   1. It would be useful if a table (perhaps supplementary) describing the details of the WWOX mutations in all the COs models studied in this paper were presented.
   2. For the new WOREE individual with complex genetics in WWOX: it is not clear why any WWOX protein is still present in this patient in Fig. S1D (please give an explanation or speculation); it is not clear which tissue was used for the Western blot in Fig. S1D; the data in Fig. S1D need to be quantified.
   3. Western blot, quantified, should be performed on all COs under study, to compare the WWOX expression levels. Please also change the immunofluorescence shown in Fig. 1B (e.g. show WWOX in a different color), as the figure provided shows WWOX poorly in wild-type CO, and it is not clear how much it is removed in the mutant organoids. Why should there be no signal in the SCAR12 COs?

   Significance

   The manuscript "WWOX deficiency impairs neurogenesis and neuronal function in human organoids" by Aqeilan and co-workers provides impressive set of studies, mostly utilizing cerebral organoids (CO), gaining insights into the roles of the gene WWOX in neuronal development and molecular etiology of WOREE and SCAR12, two devastating rare diseases originating from mutations in WWOX. Further, therapeutic modalities through the neuron-specific gene therapy are investigated using the WWOX k/o and WOREE and SCAR12 patient-derived COs. Among the major findings of this work one can highlight the identification of the main source of WWOX-expressing cells as radial glia (RG) cells; the discovery of the massive upregulation of Myc upon loss/decrease in WWOX expression in RGs; and the strong neuronal under-differentiation induced upon WWOX k/o and mutations. Regarding the latter finding, the authors report massive increase in RGs and concomitant drop in neuronal cells in WWOX k/o COs. In contrast, in WOREE and SCAR12 patient-derived COs, a more subtle under-differentiation is seen. Specifically, while WOREE but not SCAR12 patient-derived COs also show a certain increase in the RG proportion, both types of patient-derived COs demonstrate higher proportions of "young" neuronal cells as compared to wild-type COs. Thus, a picture can be drawn whereas complete loss of WWOX leads to strong under-differentiation mostly manifested as expansion of RGs and hence under-production of neuronal cells, while hypomorphic loss-of-function of WWOX in WOREE and in missense mutations in SCAR12 lead to the later defect in neuronal cell maturation. Overall, I find the work highly interesting, but I would like the authors to address one major issue and several minor ones.

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6. 

   Version published to 10.1101/2024.12.22.630016 on bioRxiv

   Dec 25, 2024