GLE1 dysfunction compromises cellular homeostasis, spatial organization, and peripheral axon branching

  1. T Zárybnický
  2. S Lindfors
  3. S Metso
  4. Z Szabo
  5. R Valtonen
  6. M Tulppo
  7. J Magga
  8. S Saarimäki
  9. S Bläuer
  10. I Miinalainen
  11. R Kerkelä
  12. J Väänänen
  13. R Kivelä
  14. FP Zhang
  15. P Sipilä
  16. R Hinttala
  17. S Kuure

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Abstract

The GLE1 protein is an enigmatic factor of RNA processing, associated with multiple developmental disorders including lethal congenital contracture syndrome 1 (LCCS1). Using in vivo genetic engineering to study disturbed GLE1 functions under physiological conditions we demonstrate that inactivation of Gle1 impedes cellular function and organization and causes pre-gastrulation lethality due to defects in adhesion and lineage specification. In contrast, the knock-in mice genocopying LCCS1-associated GLE1 FinMajor variant ( Gle1 PFQ/PFQ ) survive prenatal period but die suddenly at mid-adulthood. Gle1 PFQ/PFQ mice present irregular count and distribution of spinal motor neurons and impaired development of neural crest-derived tissues as demonstrated by defects in their sympathetic innervation of heart ventricles, paravertebral sympathetic ganglia volume, and adrenal chromaffin cell counts. Unlike previously reported for yeast and HeLa cells, analysis of molecular consequences of GLE1 FinMajor variant identified normal poly(A)+ RNA distribution in Gle1 PFQ/PFQ cells, which however were impaired in RNA and protein synthesis and simultaneously showed typical signs of cellular senescence. Gle1 PFQ/PFQ also induced disturbed stress responses with significant changes in G3BP1-positive stress granule count. Our results show necessity of GLE1 functions for life and indicate that LCCS1 etiology is resultant of pathogenic GLE1 FinMajor variant impinging differentiation of neural crest derivatives and leading to complex multiorgan defects.

Highlights

  • Total inactivation of GLE1 results in disorganization of blastocyst inner cell mass and early embryonic lethality.

  • The Gle1 knock-in (KI) mice, which genocopy the human GLE1 FinMajor variant causative for lethal congenital contracture syndrome 1 (LCCS1), die suddenly in mid-adulthood.

  • Normal poly(A)+ RNA distribution was observed in Gle1 KI cells, but decreased number of G3BP1-positive stress granules were detected in response to stress.

  • Abnormal sympathetic innervation of heart ventricles was detected in Gle1 KI mice.

  • Neural crest-derived tissues represent a new target of GLE1 FinMajor and GLE1-related disorders.

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    Reply to the reviewers

    The authors do not wish to provide a response at this time when we only have incorporated the reviewers' suggestions partially and are presenting here a revision plan.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Reported here is an elegant study on the role of GLE1 and its most common pathological variant through carefully constructed mouse models. GLE1 has been studied in cellular and zebrafish models as an important co-factor that regulates RNA processing and response to stress but investigations into the impact of the FinMajor mutation of GLE1 in mammalian in vivo models is lacking. Zárybnický et al. establish GLE1 KO and FinMajor variant mouse models through CRISPR/Cas9 gene editing and replicate early lethality of GLE1 KO models. The authors demonstrate this is due to augmented polarisation of blastocysts pre-gastrulation. The knock-in FinMajor mouse survives until mid-adulthood without complication but die suddenly. The rest of the study characterises the FinMajor mouse by examining known phenotypes of this model and more. Cell cycle arrest, augmented stress granule response and DNA damage repair are successfully replicated in MEFs. The authors reveal that MEFs display a prominent senescent state. Whilst polyA mRNA localisation is surprisingly unchanged, RNA and protein translation is disrupted as expected. In vivo, motor neuron number, organisation and branching is impaired, mirroring other studies, but the functional consequences of this in PFQ KI mice is unclear. The authors break ground by examining sympathetic nervous system development and identify neural crest-derived tissue as being selectively sensitive to the GLE1 mutation where increased mitotic arrest was apparent in mutant mice. Consequently, the authors identify cardiac innervation by sympathetic neurons, which are derived from neural crest tissue, to be augmented in FinMajor mice. It is unclear whether this is the cause of sudden death in mid-adulthood. The two mouse models presented here provide opportunity to study GLE1 absence or mutation in mammalian development at multiple levels. Overall, the FinMajor KI mouse model presents with milder phenotype than predicted but do display disease relevant phenotypes and the study has uncovered novel areas of research to pursue.

    Major comments:

    none

    Minor comments:

    1. Based on the RNA sequencing data, there appear to be issues with high variance and data normalization that need attention. The PCA results are a little concerning and the volcano plot shows an unusual shape-with massive fold changes dominating-suggesting that low-count genes may not have been adequately filtered out, potentially skewing the analysis. It's recommended to set a minimum count threshold (e.g., 5 or 10 counts) to exclude low-expression genes and to consider log₂ fold change shrinkage methods like apeglm to adjust for variability in low-count genes. Performing exploring methods like RUVSeq could help regress out unwanted variance, especially given the inherent variability in E3.5 embryos and if increasing replicates isn't feasible.
    2. Do the gene expression changes identified in GLE1 KO blastocysts hold significance in GLE1 KI mice? Augmented function of GLE1 may induce both loss of function as well as gain of toxic function and so transcriptionally they may appear as separate disorders. However, it would be worthwhile testing by qPCR the expression levels of the most differentially regulated genes.
    3. What is the expression profile of Kcnv2 in the developing spinal cord of PFQ KI mice? Or in the heart? Is the MN organisation / cardiac innervation a feature of neurotransmitter receptor misexpression or an issue of morphogen gradient as is mentioned in the discussion.
    4. MN disorganisation is seen in LCCS1 patients and in zebrafish model of GLE1FinMajor with dramatic consequences on development. MN organisation is changed in FinMajor KI mice but the functional consequences of these changes are not addressed. Do the mice display motor impairment?
    5. It is surprising that polyA mRNA localisation is not affected in PFQ KI cells. I'm glad the authors performed oligoDT FISH on embryonic spinal cords in addition to MEFs. However, in keeping with the selective vulnerabilities of TH+ chromaffin cells to cell cycle disruption, I am curious whether these cells would demonstrate RNA dysregulation. In addition to analysis of global mRNA localisation with oligoDT, it would be good to explore selective mRNA localisation-perhaps those genes implicated in GLE1 KO eg Vimentin, or genes implicated in cell cycle arrest.
    6. Please include a description of how PFQ knock in is predicted to impact oligomerisation of GLE1? Differential attributes have been given to the various GLE1 domains (PMID: 32981894). Are the specific phenotypes observed in-keeping with predicted changes to GLE1 function?
    7. Is there a sex bias to the sudden death phenotype observed in PFQ KI mice? Given the deficit of cardiac stroke volume in female mice, does this explain the trend for premature death? Additionally, please use 'sex' instead of 'gender' when referring to male and female mice.
    8. Other minor issues:

    a. Figure 1:

    i. Typo in figure 1f (OCT3/4 not /44).

    ii. What do white arrowheads indicate?

    b. Figure 2:

    i. The padjusted heat map is from 0 to 1. Please only include GO terms that were significant.

    c. Supp figure 4:

    i. why was adult heart chosen to measure protein expression of GLE1? What are the expression differences of GLE1 between heart and SC?

    Significance

    This is a carefully constructed study with thorough examination and well-presented data. The PFQ knock-in mouse model is an elegant solution to study the FinMajor variant of GLE1 that will be a useful resource for the community. The paper is of broad interest due the breadth and strength of experimentation including characterisation of blastocysts, MEFs, developing nervous system and of cardiac functions. The mouse model phenocopies many of the known phenotypes of GLE1 dysfunction and builds upon these thereby providing an excellent platform from which to undertake further examination. However, I feel that the manuscript is disconnected in parts (how does GLE1 KO signatures relate to GLE1 KI? How do MEF phenotypes relate to in vivo phenotypes?) and does not go far enough to describe how PFQ knock-in affects GLE1 function or how disrupted GLE1 function leads to the observed phenotypes in nervous system development. These questions may be beyond the scope of this paper, which successfully establishes the first mammalian model to study GLE1 dysfunction. As such, I have made minor comments that I hope can be addressed. Furthermore, given that this is a descriptive study and that the key phenotypes used in the current title have mostly been described before, I suggest that the authors use their running title of 'Modeling LCCS1 in mouse', or similar, to reflect the scope of this paper. The paper fills a gap in our understanding of mammalian GLE1 dysfunction, demonstrating that PFQ knock-in likely leads to augmented GLE1 function rather than loss of function and provides novel areas for exploring sympathetic nervous system development and cardiac innervation in the context of LCCS1. As such, it provides an incremental and methodological advance. The paper will be of interest to a broad audience of basic and clinical researchers.

    This reviewer's expertise is based in stem cell modelling of neurodevelopment and of neurodegenerative diseases.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    In this manuscript, Zarybnicky et al characterize their two mouse models of GLE1 knockout, or knock-in of a disease-causing variant of GLE1. The authors show that knockout causes early embryonic lethality, while the GLE1 variant causes disturbed tissue and organ organization with induced mid-adulthood sudden death. In particular effects on neural crest-derived tissues, such as innervation of the heart, are detected. The authors use mainly immunocytochemistry to visualize their findings, complemented with bulk RNA sequencing, quantitative PCR, western blot, and electron microscopy. The study is extremely comprehensive and technically sound.

    Major comments:

    • Are the key conclusions convincing?

    Key conclusions and data are convincing.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The authors' fifth 'Highlight' reads "Neural crest-derived tissues represent a new target of GLE1FinMajor and GLE1-related disorders". This is not shown or clearly touched upon in the manuscript and should be removed altogether.

    On page 6 (Last part of "Gle1-/- blastocyst show transcriptional...", the authors claim "This together with altered cellular adhesion suggests...". I cannot find that cellular adhesion is addressed or experimentally investigated in the present manuscript. Hence, this needs to be shown, or text needs to be re-written.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    There are some quantifications as well as improved image quality that are needed to support their claims. - First of all, it looks like the adrenal medulla is smaller in GlePFQ/PFQ mice in Fig. 7A. Is this correct? The authors should quantify the size, since this could be of importance to understand Gle1, but also to determine if the decrease in number of chromaffin cells is due to the size of the medulla, or an actual decrease in the fraction of chromaffin cell number. Since the authors display their results as area or cell count per section, the size/total number of cells are not taken into account. - Is there a rationale for only investigating the chromaffin cells, and not the sympathoblasts in the adrenal gland? Albeit the population of sympathoblasts are profoundly smaller than that of chromaffin cells, it would be good to see if also this neural crest-derived cell population is affected. - Did the authors investigate the hearts of the mice that died suddenly during mid-adulthood? Since the sympathetic patterning of the heart is severely affected during development, analyzing the hearts to find the possible cause of death could improve the conclusions and add to the biology of this manuscript. I do understand that if hearts have not been investigated or preserved from experiments already done, it would be major work to do so, and not required.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Most edits suggested here are discussion points, image reconstructions and quantifications, hence, should not be particularly time consuming.

    • Are the data and the methods presented in such a way that they can be reproduced?

    There are a few places where some clarification is needed. However, for the majority of the manuscript, the data and methods are adequately presented.

    Figure 1E: "Individual data points generated from each embryo" - is n=4 from four different embryos? Or four blasts from one embryo? Or something else? Please clarify.

    Figure 3G: Is n=6 for both groups together? Or n=6 per group?

    • Are the experiments adequately replicated and statistical analysis adequate?

    To my ability to judge these points, experiments and statistical analysis are adequately replicated.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Fig. 2: It would be good with a heat map with top hits marked, to quickly visualize the core data and genes for the reader, without the need to open the supplemental file with full data list.

    Figure 4E: Why are there no p-values for the three F-action graphs, when such values are presented for all other markers?

    Figure 1E-F: For quantification, how many cells are double positive for CDX2/GATA6 and OCT3/4/GATA6/NANOG?

    Figure 5B: It is very difficult to assess the difference in perinuclear stress granules when presented in the same graph together with cytoplasmic and total. Would it be good to present separately to enable this, or is the difference so small that it is 'biologically irrelevant'?

    Figure 7C: T1-T4/SG quantification shows a big difference between the groups. This is not visible at all in the images. Are they not representative?

    Figure 6E: Are the arrowheads correct? Should they be at the exact same spot in both images? It is also extremely difficult to understand what the authors have assessed and measured. To my eye, there are no differences in appearance between the two groups/images.

    Figure 8 and corresponding text: In Fig. 8A, only results for the males are shown, however, in the text, only differences in the females are discussed. Could this be expanded/edited/clarified?

    • Are prior studies referenced appropriately?

    Yes.

    • Are the text and figures clear and accurate?

    A number of figure references are wrong. Double check and edit.

    Figure 7A: Are the SOX10+ cells quantified from a specific axis level of the embryo, i.e., is it a specific crest derivative?

    Text related to Figure 4E: The authors write "...slightly downregulated while Pai-1...". Since expression of the SASP genes are virtually absent in the mutant MEFs, the authors should remove or re-phrase from using 'slightly'.

    The authors write: "The cell morphology of Gle1PFQ/PFQ MEFs differed by eye from WT...". Clarify if this is referring to size, and/or something else?

    In Figure 3E, the authors show ZO-1. This marker is not mentioned or explained either in Figure legend or text.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Figure 4D: The images are poor and it is very difficult to see the results.

    Supplementary Fig. 11 would benefit from providing some magnified/zoomed in images as well.

    Supplementary Figure 8: It is impossible to see which cells are positive and how they look. Magnifications/zoom is necessary.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The authors create new models to study the biology of wild-type Gle1 as well as its disease-causing variant. These add to existing models and enables experiments and studies not previously possible to perform. Conceptuallt and biologically, the authors provide findings on Gle1 not previously presented. Since this gene is disease-causing, the knowledge provided in this manuscript can be used to build upon.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    To my knowledge, the authors use existing literature and data to design their work, and their results add and complement.

    • State what audience might be interested in and influenced by the reported findings.

    This work is interesting for scientists working on Gle1-related biology, the very earliest time points of embryogenesis, heart development, neural crest and LCCS1.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Neural crest biology from delamination and onwards, and disease modeling using ex vitro methods.

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    Referee #1

    Evidence, reproducibility and clarity

    Several severe diseases, in particular lethal congenital contracture syndrome type 1 (LCCS1), are caused by a mutation in the GLE1 gene that leads to the inclusion of three additional amino acids (PFQ) into the mutant gene product. GLE acts as a co-factor for RNA-dependent DEAD-box ATPases, and its function in nuclear RNA export and protein translation has been characterized by previous studies. However, the mechanisms how dysfunction of GLE1 leads to the specific cellular defects in diseases associated with GLE mutations are not well understood. The authors have generated both GLE1 full knockout and GLE1 knockin mice, in which the PFQ expansion is included. GLE1 knockout mice are embryonic lethal, and the knockin mice do not show major defects but die suddenly between 15 and 40 weeks of age.

    Specific comments:

    1. The data shown in Fig. 4D are not convincing. SA-β-Gal-expression is hardly detectable, the number of investigated cultures is low and it is unclear whether these data allow conclusions on pathological cellular senescence. This should be based on additional parameters such as altered proteasome function and altered protein turnover.
    2. Most of the cellular assays for investigating cellular defects in the GLE-PFQ/PFQ cells have been done with mouse embryonic fibroblasts, and unfortunately not with primary neuronal cells such as motoneurons or sympathetic neurons that seem to be prominently affected in human patients with the same mutation. Since the GLE1-PFQ/PFQ mice do not show defects in body size or any severe symptoms pointing to defects in connective tissue, it is unclear whether the alterations observed in MEFs are representative on the disease phenotype. In Fig. 5, the authors show that there are apparently no defects in nuclear export of mRNAs in the MEFs. This does not exclude the possibility that such defects occur in primary neurons. This should be adequately addressed, ideally with additional analyses using primary spinal motoneurons derived from this mouse model.
    3. The authors show by puromycinilation that protein synthesis is altered (Fig. S6A and B). The gel shown in Fig. S6B is not convincing, it appears as if differential blotting efficacies contribute to the appearance of the blot. This needs to be performed in a more convincing manner with higher n. Moreover, such analyses should also be done with primary neuronal cultures, in order to test whether neuronal cells are more severe or diffentially affected in comparison to fibroblasts by the GLE1 mutation.
    4. Previous studies, for example the manuscript by Bresson et al., Molecular Cell 80: 470-484, 2020, have shown that stress-induced translation inhibition involves DEAD-box translation initiation factor, in particular DED1, which interacts with eIF4A. The translation inhibition phenotype observed in this study appears very similar to that observed in these previous studies, and the question arises whether GLE1 can also modify the interaction with DED1 and its role in modulating translation via eIF4A. A clear analysis of the mechanisms how GLE1 is involved in modulating protein synthesis in fibroblasts and also in cells that seem to be more severely affected, such as neurons, would highly strengthen the impact of this paper.
    5. The data shown on the quantification of motoneurons and sympathetic neurons are not convincing. In Fig. S7B, a minor reduction of ISL1/2 positive cells is observed at E11.5, before the period of physiological cell death starts in mice. Since these mice day at postnatal stages, it appears essential that the authors quantify motoneuron numbers in the adult spinal cord in 20 week old mutant mice. Previous studies have shown that mice can live normally with loss of more than 50% of spinal motoneurons (for example Jablonka et al., HMG 9, 341-46, 2000), and it is unclear whether the mild loss of motoneurons is a cause of death in these animals.
    6. The same is true for the loss of the sympathetic fibers. Changes shown in Fig. 7C and D appear very low, and clear conclusions can only be drawn when the number of neurons are quantitated. Moreover, the data shown in Fig. 7 are from embryonic stages, whereas death of these mice occurs at postnatal stages between 15 and 40 weeks. Therefore, the morphology and cell numbers in sympathetic neurons at these critical postnatal stage might be much more relevant than the number of embryonic neurons before the stage of physiological cell death.
    7. The data shown on altered arborization in the periphery are not convincing. Such data have to be based on highly systematic 3D analyses, which have not been performed in this study. In addition, denervation of skeletal muscle or loss of sympathetic terminals in the heart ventricles appear much more relevant. These analyses need to be expanded. It would also be interesting to know how these mice at a stage when sudden death occurs, react to sympathomimetic drugs or inhibitors of noradrenergic receptors. I also do not understand which the authors have not measured adrenaline levels in the circulation of these mice, in order to find out whether the morphological changes in the adrenal gland, as shown in Fig. 7A, are of functional significance.

    Minor point: The data shown in Fig. S6A and B are highly important and should be shifted to the main part of the manuscript.

    Referees cross-commenting

    I agree with all comments that have been made by reviewer 2 and 3.

    Significance

    These mouse models will certainly be very useful for this research field, and the characterization of the knockout mouse and, at least in part, also of the knockin mouse model is well done and a significant contribution to understand the physiological function of GLE1. However, the manuscript falls short in explaining the cell type specificity of the disease mechanisms for several types of neurons. Moreover, some of the analyses on the losses of specific cell populations, in particular motoneurons and sympathetic neurons, are technically not sound and not convincing. The paper would highly benefit from revisions.

  5. Version published to 10.1101/2024.10.14.618161v1 on bioRxiv