Microglia attenuate regenerative neurogenesis via sema4ab after spinal cord injury in zebrafish

  1. Alberto Docampo-Seara
  2. M. Ilyas Cosacak
  3. Kim Heilemann
  4. Friederike Kessel
  5. Ana-Maria Oprişoreanu
  6. Markus Westphal
  7. Özge Çark
  8. Daniela Zöller
  9. Josi Arnold
  10. Anja Bretschneider
  11. Alisa Hnatiuk
  12. Nikolay Ninov
  13. Catherina G. Becker
  14. Thomas Becker

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Abstract

Zebrafish, in contrast to mammals, regenerate neurons after spinal cord injury, but little is known about the control mechanisms of this process. Here we use scRNA-seq and in vivo experiments to show that sema4ab , mainly expressed by lesion-reactive microglia, attenuates regenerative neurogenesis by changing the complex lesion environment. After spinal injury, disruption of sema4ab doubles the number of newly generated progenitor cells and neurons but attenuates axon regrowth and recovery of swimming function. Disruption of the plxnb1a/b receptors, selectively expressed by neural progenitor cells, increases regenerative neurogenesis. In addition, disruption of sema4ab alters activation state and cytokine expression of microglia, such that fibroblasts increase expression of the cytokine tgfb3 , which strongly promotes regenerative neurogenesis. Hence, sema4ab in microglia attenuates regenerative neurogenesis in multiple ways, likely directly through plxnb1a/b receptors and indirectly, by controlling the inflammatory milieu and tgfb3 levels. Targeting Sema4A-dependent signalling in non-regenerating vertebrates may be a future strategy to improve regenerative outcomes.

HIGHLIGHTS

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    Microglia suppress pro-regenerative fibroblast signalling in a spinal injury site

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    Fibroblasts promote regenerative neurogenesis via Tgfb3 signalling

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    sema4ab promotes microglia activation state after spinal injury

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    scRNA-seq reveals full complement of sema4ab -dependent changes on different cell types during repair of a spinal lesion site

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    1. PREreview

      This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14237963.

      The paper explores the function of one of the zebrafish orthologs of sema4a, sema4ab, which is highly enriched in macrophages/microglia and signal to spinal progenitors expressing the paralog receptors plxnb1a and plxnb1b after spinal cord injury.

      The main finding is the identification of the opposing pathways sema4ab/plxnb1 and tgfb3 as regulators of regenerative neurogenesis.

      These authors demonstrate that CRISPR/Cas9-mediated knockdown and germline mutants of sema4ab increases regenerative neurogenesis by 2-fold, and simultaneous knockdown of plxnb1a and plxnb1b also increases proliferation to a limited extent. Using single-cell transcriptomics, Docampo-Seara et al. identified tgfb3 as being up-regulated in fibroblasts upon sema4ab loss-of-function and demonstrated that simultaneous knockdown of sema4ab and tgfb3 impairs neurogenesis.

      Thus, regenerative proliferation would be impaired by a cellular crosstalk mediated by attenuating Sema4ab being secreted by macrophages/microglia and acting on plxnb1-expressing spinal progenitors, which would be counteracted by pro-inflammatory tgfb3 signaling from fibroblasts to macrophages.

      Major issues

      • We would like to offer a small clarification regarding the data presented on sema4ab+ macrophages and neutrophils. The authors mention that 65% of sema4ab+ cells are macrophages at 6 hours post-ligation (hpl), 85% at 24 hpl, and 75% at 48 hpl. However, when reporting that "16% of neutrophils were sema4ab+ at 6 hpl and 24 hpl," it might be helpful to explicitly state the percentage of sema4ab+ cells that are neutrophils. This would allow for a more direct comparison with the percentages of sema4ab+ macrophages and provide a clearer picture of whether the sema4ab+ cells not labeled by the macrophage marker are predominantly neutrophils. By expressing this result in such a way, the authors could enhance the clarity and comparability of the data, and it would allow readers to more easily interpret the relationship between these cell types during the regeneration process.

      • The study proposes that soluble Sema4ab secreted by macrophages regulates regenerative neurogenesis. While this hypothesis is compelling, it is important to note that it has not yet been experimentally validated. Therefore, we recommend exercising caution when drawing conclusions based on this model, as further studies are needed to confirm whether signaling occurs through soluble Sema4ab or whether it involves transmembrane ligand-receptor interactions.

        The manuscript would benefit from clarifying this distinction, as it would strengthen the conclusions by ensuring they are more robust, supported by additional experimental evidence in future work.

      • Regarding the finding that sema4ab disruption increases regenerative neurogenesis, the statement 'Moreover, this observation confirms that our sema4ab mutant carries a loss-of-function mutation' could be perceived as somewhat overstated. Although overexpression of sema4ab rescues the lost function, this does not directly confirm the presence of a loss-of-function mutation. We suggest using a more cautious phrasing to reflect the conclusion.

      • About the finding that the disruption of plxnb1a/b enhances regenerative neurogenesis, we strongly recommend providing a justification for the evaluation of receptor expression in ERGs at 6 hpl and 24 hpl, especially considering that the reported effect of the simultaneous mutation of plxnb1a and plxnb1b—specifically, an increase in the number of newly generated motor neurons—was observed at 48 hpl. A clear explanation for this discrepancy in evaluation times would strengthen the manuscript.

      Minor issues

      • The figures would benefit from the inclusion of symmetry axes for anatomical coordinates (AP, DV, LR). This addition would provide a quick reference, making the figures more accessible, particularly for readers who may not be familiar with zebrafish anatomy.

      Competing interests

      The authors declare that they have no competing interests.

    2. Version published to 10.1101/2024.10.16.618445 on bioRxiv