Extra-Neurological Characterization of Seckel Syndrome-Model Mice Harboring CEP152 Variants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper investigates extra-neurological manifestations associated with pathogenic CEP152 variants using two previously established Seckel syndrome mouse models. While previous studies focused mainly on neurodevelopmental abnormalities, this work extends the phenotypic characterization to reproductive, hematopoietic, and glial-associated alterations.

Overall, the manuscript is clearly written, the experiments are well performed, and the study provides useful information regarding the systemic consequences of CEP152 dysfunction. In particular, the comparative analysis of two distinct patient-derived Cep152 mutant models represents a strength of the study. The testicular phenotype is convincingly documented and supports the conclusion that CEP152 dysfunction affects proliferative progenitor populations beyond the nervous system.

The manuscript is suitable for publication in Cells after moderate revision. Some aspects of the interpretation could however be clarified and several conclusions slightly moderated.

1) One of the most interesting aspects of the manuscript is the observation that different tissues appear to show distinct sensitivities to CEP152 dysfunction despite shared defects in centrosome duplication and mitotic progression. This concept is mentioned in the Discussion but could be developed more clearly. In particular, the authors may wish to emphasize more explicitly the possibility of tissue-specific compensatory mechanisms or differential proliferative vulnerability across organs.

2) The observation of macrocytic anemia is interesting and relevant to the systemic manifestations of Seckel syndrome. However, the mechanistic interpretation should remain somewhat cautious, as the present analyses mainly rely on peripheral blood parameters. The statement that “CEP152 is required for erythropoiesis” may therefore be slightly stronger than warranted by the current data and could be moderated.

3) The reduction in Olig2-positive cells and Opalin expression is potentially interesting, but this part of the study remains relatively exploratory compared to the stronger testicular analyses. Since only Opalin reached statistical significance by qPCR, the authors should moderate the conclusions regarding oligodendrocyte differentiation and maturation defects.

4) The statistical methods are generally appropriate for this type of study. However, it would be useful for the authors to clarify whether the data distribution was inspected before applying parametric tests. This does not necessarily require additional analyses but would improve methodological transparency. 

Minor comments

1. Some sentences in the Discussion could be shortened to reduce repetition, particularly regarding “systemic” or “multi-organ” manifestations.
2. Figure legends are generally clear, although a few abbreviations could be defined directly in the legends for readability.
3. In Figure 5, the exploratory nature of the oligodendrocyte-related qPCR analysis could be acknowledged more explicitly.

In summary, this is a valuable and well-constructed study that expands the understanding of CEP152-associated Seckel syndrome beyond the nervous system and supports the concept that CEP152 dysfunction affects multiple proliferative cell populations in a tissue-dependent manner. After some modifications I consider that it might be considered for publication in "Cells".

Author Response

Reviewer #1

1) One of the most interesting aspects of the manuscript is the observation that different tissues appear to show distinct sensitivities to CEP152 dysfunction despite shared defects in centrosome duplication and mitotic progression. This concept is mentioned in the Discussion but could be developed more clearly. In particular, the authors may wish to emphasize more explicitly the possibility of tissue-specific compensatory mechanisms or differential proliferative vulnerability across organs.

We agree with the reviewer’s comment. We revised the second paragraph of the Discussion section to more explicitly discuss the possibility of tissue-specific compensatory mechanisms or differential proliferative vulnerability across organs (p. 11, lines 349 - 367).

2) The observation of macrocytic anemia is interesting and relevant to the systemic manifestations of Seckel syndrome. However, the mechanistic interpretation should remain somewhat cautious, as the present analyses mainly rely on peripheral blood parameters. The statement that “CEP152 is required for erythropoiesis” may therefore be slightly stronger than warranted by the current data and could be moderated.

We agree with the reviewer’s suggestion and revised the subheading “CEP152 is required for erythropoiesis” in the Results section to “Hematological abnormalities in Cep152 mutant mice” in the revised manuscript (p. 8, line 283).

3) The reduction in Olig2-positive cells and Opalin expression is potentially interesting, but this part of the study remains relatively exploratory compared to the stronger testicular analyses. Since only Opalin reached statistical significance by qPCR, the authors should moderate the conclusions regarding oligodendrocyte differentiation and maturation defects.

We agree with the reviewer’s suggestion. We revised the final paragraph of the Discussion section in the revised manuscript, in which we toned down the interpretation and moderated the conclusions regarding oligodendrocyte differentiation and maturation defects.

4) The statistical methods are generally appropriate for this type of study. However, it would be useful for the authors to clarify whether the data distribution was inspected before applying parametric tests. This does not necessarily require additional analyses but would improve methodological transparency. 

We agree with the reviewer’s important comment. In response, we clarified in the revised manuscript that data normality was not formally assessed prior to the application of parametric tests, no formal test for outliers was conducted, and no data were excluded from the analyses (p.4, lines 146-147).

Minor comments

1) Some sentences in the Discussion could be shortened to reduce repetition, particularly regarding “systemic” or “multi-organ” manifestations.

As suggested by the reviewer, we revised the Discussion section in the revised manuscript to reduce repetitive descriptions regarding “systemic” or “multi-organ” manifestations (p. 11, line 337– p. 11, 381).

2) Figure legends are generally clear, although a few abbreviations could be defined directly in the legends for readability.

In response to the reviewer’s suggestion, the abbreviations (pHH3 and DAPI in Figure 3, and RT-PCR in Figure 5) have been defined directly in the respective figure legends to improve readability in the revised manuscript. Also, we revised the subheading of RT-PCR (p.3, line 124).

3) In Figure 5, the exploratory nature of the oligodendrocyte-related qPCR analysis could be acknowledged more explicitly.

We agree with the reviewer’s comment. We have revised the corresponding statements to avoid overinterpretation and have more explicitly acknowledged the exploratory nature of the oligodendrocyte-related qPCR analysis in the revised manuscript (p. 11, lines 332–335).

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, authors reported extra-neuronal tissues consequences of CEP152 dysfunction in two mouse strains Cep152W105*/K897*carrying a compound heterozygous variants and Cep152Q32P/Q32P a homozygous variant. They identified hematological abnormalities indicative of macrocytic anemia as well as impaired spermatogenesis, characterized by defective mitosis and increased apoptosis in spermatogonia. They also found reduced expression of Opalin, a gene involved in oligodendrocyte differentiation, and decreased numbers of Olig2-positive oligodendrocytes, suggesting broader glial deficits among neurodevelopmental defects.

Hematological abnormalities are among the recognized manifestations of Seckel syndrome, although  only a subgroup. Abnormalities of spermatogenesis may be observed, but they are not constant. The most clearly reported sign is cryptorchidism, which can secondarily alter spermatogenesis. Glial involvement exists, but they are heterogeneous and poorly documented in humans.

The reported data are interesting and innovative. They extended the spectrum of neural and non-neural damage observed in Seckel syndrome.

Comments

Could you specify why you test the mice at P60?

Do you study seminiferous tubules, Leydig and Sertoli cells  at P10 because this age corresponds to the period of their establishment and functioning? It would be useful to mention it.

Why compare the results with mice at P60, which corresponds rather to a period of reduced functionality?

Figure 3 : For ease of reading, it would be more logical to place figure 3 after the text.

Discussion session ″ two knock-in mouse models″ : typo error ?

Author Response

Reviewer #2

1)  Could you specify why you test the mice at P60?

We thank the reviewer for this comment. P60 was selected because we aimed to evaluate the final adult phenotypes in the mutant mice. In addition, cortical tissues at this stage were already being collected for other analyses in our previous study (Hamada et al., EMBO Mol. Med. 2026, Ref 26), allowing us to examine extra-neurological phenotypes under the same experimental conditions.

2)  Do you study seminiferous tubules, Leydig and Sertoli cells at P10 because this age corresponds to the period of their establishment and functioning? It would be useful to mention it.

We thank the reviewer for this important comment. P10 was selected because this developmental stage corresponds to the early establishment and functional maturation of seminiferous tubules and their associated cell populations. Around this stage, spermatogonia begin to differentiate into the first generation of spermatocytes, marking the onset of active spermatogenesis. In addition, Sertoli cells begin to express CLDN11 at approximately P10, which contributes to the formation of tight junctions and the blood–testis barrier. Furthermore, adult-like Leydig cells emerge around this stage and express Hsd3b6, a marker associated with steroidogenic function. Therefore, P10 represents a biologically important stage for evaluating the developmental and functional abnormalities of seminiferous tubules, Sertoli cells, Leydig cells, and spermatogonial progenitors in the mutant mice. We have added this explanation to the revised manuscript (p. 7, lines 256–259).

3)  Why compare the results with mice at P60, which corresponds rather to a period of reduced functionality?

We thank the reviewer for this important comment. The analyses were performed at different developmental stages depending on the biological process examined. Testicular morphology was analyzed at both P10 and P60 to evaluate developmental progression and adult phenotypes. In contrast, analyses of spermatogonial progenitors and mitotic abnormalities were performed at P10, when proliferative activity in the seminiferous tubules is more prominent. Hematological analyses were conducted at P60 to assess stable systemic phenotypes in adult mice. We have clarified this rationale in the revised manuscript (p. 11, lines 342–348).

4) Figure 3 : For ease of reading, it would be more logical to place figure 3 after the text.

As suggested by the reviewer, we have moved Figure 3 to appear after the relevant text in the revised manuscript.

5)  Discussion session ″ two knock-in mouse models″ : typo error ?

Please accept our apologies for this typographical error. We have corrected it in the revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This study extends previous work of the authors about the description of the phenotype of Seckel-syndrome model mice. I have the following remarks.

1. The reasoning why spermatogenesis and blood cell maturation was studied remains to the critical reader unclear and should be explained in greater detail. What about other biological processes requiring high mitotic rates? A table summarizing the general abnormalities in both mouse models would be very helpful.
2. The reported abnormalities in oligodendrocyte differentiation in one of the mouse models are difficult to understand without presenting the information about the other mouse model. Here, also a table showing the data for both models could clarify, why these effects were studied and how they are linked to the phenotype of the mice.
3. Why the phenotype of the knock-in mice is more severe than of the knock-out mice?

Author Response

Reviewer #3

1) The reasoning why spermatogenesis and blood cell maturation was studied remains to the critical reader unclear and should be explained in greater detail. What about other biological processes requiring high mitotic rates? A table summarizing the general abnormalities in both mouse models would be very helpful.

We thank the reviewer for this important suggestion. We focused on spermatogenesis and hematopoiesis because both processes involve active cell proliferation and continuous mitotic progression even in adult mice, making them particularly suitable for detecting pathological consequences caused by defects in CEP152. In addition, reproductive and hematological abnormalities have been reported in patients with Seckel syndrome, further supporting the biological and clinical relevance of investigating these tissues.

According to the reviewer’s question regarding other highly proliferative tissues, we had examined the femoral bone marrow, intestinal epithelium, and skin in Cep152^W105*/K897* mice. However, no obvious histological abnormalities were detected in these tissues compared with wild-type controls. Representative images have been added as Supplementary Figure S1.

These findings suggest that the consequences of CEP152 dysfunction may differ among proliferative tissues and support the possibility of tissue-specific vulnerability and/or compensatory mechanisms. Regarding the reviewer’s suggestion of a summary table, we agree that such a comparison would be informative. However, equivalent analyses were not performed in Cep152^Q32P/Q32P mice, and therefore a comprehensive comparison between the two models is currently not possible. We have clarified these points in the Discussion section of the revised manuscript (p. 11, 362 - 367).

2) The reported abnormalities in oligodendrocyte differentiation in one of the mouse models are difficult to understand without presenting the information about the other mouse model. Here, also a table showing the data for both models could clarify, why these effects were studied and how they are linked to the phenotype of the mice.

We thank the reviewer for this important comment. In our previous study, RNA-seq analyses were performed in both mouse models (Hamada et al., EMBO Mol. Med. 2026, Ref 26). In Cep152^Q32P/Q32P mice, we identified altered expression of multiple genes associated with synaptic formation and function, dendritic morphology, and axon development, which was consistent with the abnormalities observed in dendritic architecture, reduced spine density, and altered synaptic activity in this model. In contrast, no significantly altered genes were identified by RNA-seq analysis in Cep152^W105*/K897* mice. Therefore, in the present study, we focused on Cep152^W105*/K897* mice and performed additional gene expression analyses using alternative approaches. Because Olig2-positive cells were significantly reduced in this model (Fig. 5A and B), we specifically examined genes related to oligodendrocyte-associated cellular processes. We have explained this background more clearly in the revised manuscript (p. 10, line 309 - p. 11, line 330).

We also thank the reviewer for the suggestion regarding the table. However, identical oligodendrocyte-related analyses were not performed in both mouse models, and therefore a direct comparative table could not be generated. We hope that this explanation clarifies our rationale.　

3) Why the phenotype of the knock-in mice is more severe than of the knock-out mice?

We thank the reviewer for this important comment. Complete loss of CEP152 function causes embryonic lethality in knockout mice, indicating that total absence of CEP152 is incompatible with normal development. In contrast, the patient-derived variants analyzed in this study likely retain partial protein function, allowing survival to birth despite severe developmental abnormalities. In addition, these pathogenic variants may exert effects beyond simple loss of function. CEP152-Q32P retained centrosomal localization despite impaired interaction with its binding partner PLK4 kinase, raising the possibility that the mutant protein interferes with normal centrosome organization or centriole assembly. In contrast, the CEP152-W105* and -K897* variants exhibited marked protein instability or abnormal cytoplasmic localization, respectively, suggesting variant-specific disruption of centrosomal function rather than complete absence of CEP152 activity. Such residual but abnormal CEP152 function may contribute to the severe phenotypes observed in the knock-in mice. We hope that this explanation addresses the reviewer’s concern and clarifies our interpretation.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

My comments 1 and 2 have been adequately addressed. However, despite the provided explanation of authors regarding my comment 3, nothing has been changed in the revised manuscript. I strongly recommend to provide this explanation in the discussion section. 

Author Response

Comment 1: My comments 1 and 2 have been adequately addressed. However, despite the provided explanation of authors regarding my comment 3, nothing has been changed in the revised manuscript. I strongly recommend to provide this explanation in the discussion section. 

Response: We thank the reviewer for this important comment. We agree with the reviewer and have incorporated this explanation into the Discussion section (p. 12, lines 368–382).