SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

  1. Pascal Forcella
  2. Niklas Ifflander
  3. Chiara Rolando
  4. Elli-Anna Balta
  5. Aikaterini Lampada
  6. Claudio Giachino
  7. Tanzila Mukhtar
  8. Thomas Bock
  9. Verdon Taylor

  • Curated by eLife

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    This manuscript addresses a non-canonical function of the Class 2 ribonuclease III Drosha in the regulation of adult neural stem cell fate, important for understanding how these cells generate neurons or oligodendrocytic cells.
    Overall, this manuscript has many strengths. The authors identify 165 proteins, several of them enriched in neural stem cells, and potentially specific for miRNA dependent or independent Drosha macromolecular complexes.
    While the authors provide systematic and convincing evidence on the biochemical interactions among the key players in this cascade, the significance of these interactions for neural stem cell fate determination in vivo remains unclear, as the in vitro cellular systems used to document most of the data reported in the paper may not (fully) represent resident neural stem cells in the adult hippocampus. The in vivo function mediated by Drosha/ Safb1 needs to be substantiated by more evidence and/or complementary approaches.

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Abstract

Neural stem cells (NSCs) are multipotent and correct fate determination is crucial to guarantee brain formation and homeostasis. How NSCs are instructed to generate neuronal or glial progeny is not well understood. Here, we addressed how murine adult hippocampal NSC fate is regulated and described how scaffold attachment factor B (SAFB) blocks oligodendrocyte production to enable neuron generation. We found that SAFB prevents NSC expression of the transcription factor nuclear factor I/B (NFIB) by binding to sequences in the Nfib mRNA and enhancing Drosha-dependent cleavage of the transcripts. We show that increasing SAFB expression prevents oligodendrocyte production by multipotent adult NSCs, and conditional deletion of Safb increases NFIB expression and oligodendrocyte formation in the adult hippocampus. Our results provide novel insights into a mechanism that controls Drosha functions for selective regulation of NSC fate by modulating the post-transcriptional destabilization of Nfib mRNA in a lineage-specific manner.

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  1. Version published to 10.7554/elife.74940 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The manuscript entitled 'Safb1 regulates cell fate determination in adult neural stem cells by enhancing Drosha cleavage of NFIB mRNA' by Iffländer et al, represents a solid piece of work addressing a non-canonical function of Drosha on NFIB mRNA processing via a newly identified Drosha partner, Safb1. The authors provide particularly systematic and convincing evidence on the biochemical interactions among the key players in this cascade. However, the significance of these interactions for NSC fate determination is not adequately supported by the data, hence, I have some remarks that would need to be addressed in order to clarify the impact of these events on NSC biology.

    1. One of my main concerns is related to the nature of the DG NSCs used in all in vitro assays. The authors refer to their previous work on how these cells are isolated using a Hes5 mouse reporter line. However, both recent scRNAseq data (http://linnarssonlab.org/dentate/ from Hochgerner et al) and the authors' own immunostainings (Fig. 7A), clearly show that Hes5 does not label only adult NSCs in the DG, but also (if not primarily) astrocytes. Considering that the initial cultures could contain a high proportion of mature astrocytes, most of the major conclusions and hypotheses should be reformulated.

    We thank the reviewer for their comment. We think that there is a misunderstanding about how the DG neural stem cells were isolated and cultured. In this manuscript we did not use the Hes5::GFP allele to isolate the stem cells. We isolated DG neural stem cells from C57Bl6 mice according to the protocol of Babu et al. (Babu et al. 2007 doi: 10.1371/journal.pone.0000388) and maintained and differentiated these according to our previous manuscripts (Ronaldo et al. 2016). This was not clear in the methods section of the original manuscript and, therefore, we have added the reference Babu et al. In order to address potential contamination with astrocytes, we have added images of the stem cells and their progeny immunostained with astrocytic markers (GFAP and S100b) in undifferentiated and differentiated states. These new data show that these neurogenic cells and their progeny do not express astrocytic markers until differentiation is induced.

    1. Along these lines, Safb1 expression is quite widespread in the mouse DG (Fig. 7A) and does not display any specificity towards any type of progenitor cells compared to its expression in DGCs within the GCL. The authors should discuss this and integrate this expression information into their conclusions and interpretations, highlighting all pertinent limitations.

    We appreciate and agree with the reviewer’s comment. SAFB1 is indeed broadly expressed by most if not all cells in the hippocampus. We quantified levels of SAFB1 expression across progenitors, astrocytes and neurons in the adult DG and in the SVZ, and show that SAFB1 levels differ across different neural stem cell populations and neural cells. We believe that our data show both in vitro and in vivo that the levels of SAFB1 are critical for determining the function of SAFB1 in regulating neural stem cell fate. We also showed that elevating SAFB1 levels in SVZ-derived neural stem cells suppresses their differentiation into oligodendrocytes, This we have made clearer in the text. However, how cells sense the levels of SAFB1 remains to be shown and it is difficult to speculate on the mechanism.

  3. eLife

    eLife assessment

    This manuscript addresses a non-canonical function of the Class 2 ribonuclease III Drosha in the regulation of adult neural stem cell fate, important for understanding how these cells generate neurons or oligodendrocytic cells.
    Overall, this manuscript has many strengths. The authors identify 165 proteins, several of them enriched in neural stem cells, and potentially specific for miRNA dependent or independent Drosha macromolecular complexes.
    While the authors provide systematic and convincing evidence on the biochemical interactions among the key players in this cascade, the significance of these interactions for neural stem cell fate determination in vivo remains unclear, as the in vitro cellular systems used to document most of the data reported in the paper may not (fully) represent resident neural stem cells in the adult hippocampus. The in vivo function mediated by Drosha/ Safb1 needs to be substantiated by more evidence and/or complementary approaches.

  4. eLife

    Reviewer #1 (Public Review):

    The manuscript entitled 'Safb1 regulates cell fate determination in adult neural stem cells by enhancing Drosha cleavage of NFIB mRNA' by Iffländer et al, represents a solid piece of work addressing a non-canonical function of Drosha on NFIB mRNA processing via a newly identified Drosha partner, Safb1. The authors provide particularly systematic and convincing evidence on the biochemical interactions among the key players in this cascade. However, the significance of these interactions for NSC fate determination is not adequately supported by the data, hence, I have some remarks that would need to be addressed in order to clarify the impact of these events on NSC biology.

    1. One of my main concerns is related to the nature of the DG NSCs used in all in vitro assays. The authors refer to their previous work on how these cells are isolated using a Hes5 mouse reporter line. However, both recent scRNAseq data (http://linnarssonlab.org/dentate/ from Hochgerner et al) and the authors' own immunostainings (Fig. 7A), clearly show that Hes5 does not label only adult NSCs in the DG, but also (if not primarily) astrocytes. Considering that the initial cultures could contain a high proportion of mature astrocytes, most of the major conclusions and hypotheses should be reformulated.

    2. Along these lines, Safb1 expression is quite widespread in the mouse DG (Fig. 7A) and does not display any specificity towards any type of progenitor cells compared to its expression in DGCs within the GCL. The authors should discuss this and integrate this expression information into their conclusions and interpretations, highlighting all pertinent limitations.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript, the authors uncover a variety of macromolecular Drosha complexes in NSCs and propose that they might exert specific functions in adult neurogenesis. This is an interesting and important area of research, the proteomics data are very useful, and the manuscript is well written and easy to understand. Overall, this manuscript has many strengths. The authors identify 165 proteins, several of them enriched in NSCs, and potentially specific for miRNA dependent or independent Drosha macromolecular complexes. Moreover, the authors convincingly show that Safb1 binds and post-transcriptionally destabilizes NFIB transcript in complex with Drosha, in vitro. With that said, most of the functional evidence are based on Safb1 overexpression in vitro, and in some cases with immortalized cell lines. This is a major limitation of the study. Further experiments should be done to convincingly demonstrate that Safb1 regulates cell fate determination in adult neural stem cells by enhancing Drosha cleavage of NFIB mRNA.

  6. Version published to 10.1101/2021.10.22.465432v1 on bioRxiv