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Aberrant astrocyte protein secretion contributes to altered neuronal development in diverse disorders

Alison L.M. Caldwell, Jolene K. Diedrich, Maxim N. Shokhirev, Nicola J. Allen

Preprint posted on 17 February 2020 https://www.biorxiv.org/content/10.1101/2020.02.17.939991v1

Article now published in Nature Neuroscience at http://dx.doi.org/10.1038/s41593-022-01150-1

Normally astrocytes promote neuronal development; however, this is dysregulated in many neurodevelopmental disorders. Using an in vitro system combined with mass spec and RNA-seq, Caldwell et al. explore the role of secreted proteins in this process.

Selected by NYUPeerReview

Context

Glia promote neurite outgrowth and provide trophic support to neurons. Many neurodevelopmental disorders (NDs) share phenotypic neuronal alterations, including defective neurite growth, and these defects common to many NDs suggest that glial dysfunction might be an important part of their pathology. While the genetic bases of neurodevelopmental disorders like Rett syndrome (RTT), Fragile X syndrome (FXS) and Down syndrome (DS) are known, how  glia are affected by mutations that cause NDs remains poorly understood.

Astrocytes are a type of glia that are particularly important for neuronal development and survival. Neurons grown in isolation in vitro fail to thrive, but the addition of astrocytes rescues their poor survival, inhibited neurite outgrowth and defects in synapse formation. This effect of astrocytes is mediated by secreted factors; astrocyte-conditioned culture medium is sufficient to promote neuronal viability. However, astrocytes from RTT, FXS and DS mouse models and their conditioned media, fail to support normal dendritic morphology and synapse formation. In this paper, the authors aim to identify a common molecular mechanism by which ND astrocytes inhibit neuronal development.

Methods

To identify specific pathways that mediate astrocyte-neuron signaling, the authors first optimize an in vitro system for culturing astrocytes from mouse models of three NDs (RTT, FXS and DS). Immunopanning, which involves passing cells over antibody-coated plates, offers the advantage of isolating astrocytes at a later stage in development (here, postnatal day 7) compared to classical culturing methods, which use astrocytes isolated at postnatal day 0 or 1. The authors opted to use serum-free media to culture astrocytes, which maintains a gene expression profile that closely matches that of acutely isolated astrocytes. Using immunopanning, the authors cultured WT and ND mouse model-derived astrocytes to measure protein secretion and gene expression profiles in the developing cortex via mass spectrometry and RNA sequencing, respectively. To evaluate impact of particular astrocyte-secreted proteins on neurite formation, the authors co-cultured neurons with astrocyte-conditioned media (ACM) and measured the length of neurite outgrowth.

Key findings

  1. Addition of Igfbp2 to WT ACM stunted neurite outgrowth of neurons in culture, while antagonizing the activity of Igfbp2 reversed this effect

The authors identify that Igfbp2 is upregulated in ND mouse model-derived astrocytes via mass spectrometry and RNA sequencing. Via immunostaining, they observe an increase in Igfbp2 in the extracellular space around astrocytes in RTT mouse models. When Igfbp2 was added to WT ACM, neurite outgrowth was stunted. Blocking the added Igfbp2 with an Igfbp2-neutralizing antibody rescued deficits in neurite outgrowth. The authors present evidence that BMP signaling is similarly upregulated in ND mouse model-derived astrocytes. When BMP6 was added to WT ACM, neurite outgrowth was stunted, but the authors did not show evidence that blocking the added BMP6 in WT ACM restored this effect.

  1. ACM of astrocytes from an RTT mouse model stunted neurite outgrowth of WT neurons 

To test whether Igfbp2 is a protein that inhibits neuronal development in NDs, the authors added the ACM from ND model-derived astrocytes to WT neurons. For neurons cultured in ACM from an RTT mouse model (but not FXS or DS mouse models), neurite outgrowth was inhibited. When an Igfbp2 activity was blocked in RTT ACM, neurite outgrowth was moderately restored.

  1. ACM of astrocytes from an FXS mouse model moderately stunted neurite outgrowth of WT neurons

Using immunostaining for phosphorylated SMAD (a downstream BMP target), mass spectrometry and RNA sequencing, the authors identified upregulated BMP signaling in ND mouse model-derived astrocytes. The authors added the ACM from FXS model-derived astrocytes to WT neurons, which stunted neurite outgrowth. This effect was restored by blocking BMP signaling with Noggin.

Impact of key findings

Stunted neurite outgrowth upon culturing WT neurons with ND model-derived ACM reinforces previous findings that glia have important developmental effects on neurons via their secreted proteins. Furthermore, the authors have generated a useful database of mass spectrometry and RNA sequencing profiles of WT and ND mouse model-derived astrocytes in vitro. Some differences between gene and protein expression between WT and ND conditions were validated to impact neuronal development. Other researchers can use this database to inform future investigations of therapeutic targets for NDs.

Open questions not addressed by this study

  1. The authors suggest that two pathways affect neuronal development in NDs. How are the activities of these two pathways linked in NDs? Are there common upstream or downstream proteins that directly inhibit neurite outgrowth in multiple NDs?
  2. The authors identified a dysregulation of other Igfbp proteins in ND mouse model-derived astrocytes. Do these also impair neurite growth? Consequently, does pharmacologic blocking of these other Igfbp proteins preserve normal neurite development?
  3. The authors identified Igfbp2 from RTT mouse model-derived astrocytes and BMP6 from FXS mouse model-derived astrocytes to inhibit neurite outgrowth in vitro. Are similar effects observed in vivo?
  4. Given that a similar immunopanning procedure could be applied to astrocytes derived from fetal, juvenile and adult human brains, which astrocyte-secreted proteins affect human neuronal development in NDs?
  5. Do different glial cell types (and the proteins they secrete) also affect neuronal development in NDs?

 

 

Posted on: 16 March 2020

doi: https://doi.org/10.1242/prelights.17672

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NYUPeerReview

The NYU preLights team was very excited to see the work by Caldwell and colleagues published recently in Nature Neuroscience – congratulations! Our discussion of the preprint focused on five interesting questions raised by the study. We revisited these questions after reading the published manuscript. Here are our postLights comments.

1)The authors suggest that two pathways affect neuronal development in NDs. How are the activities of these two pathways linked in NDs? Are there common upstream or downstream proteins that directly inhibit neurite outgrowth in multiple NDs?

>> The authors expanded their comparison of protein data in multiple NDs, which provides additional biological insights, and in keeping with the resource nature of this paper, provides additional powerful future targets for researchers in the field. While no additional in vitro/in vivo mechanistic assays were completed on these common or novel protein hits, they do provide an exciting avenue for future research.

2) The authors identified a dysregulation of other Igfbp proteins in ND mouse model-derived astrocytes. Do these also impair neurite growth? Consequently, does pharmacologic manipulation of IGF signaling preserve normal neurite development when Igfbps are overexpressed?

>> Given the clear effect of Igfbp2 overexpression and the clear link between Igfbp2 and IGF signaling it was not a high priority to measure effects mediated by dysregulation of other Igbps. Also, the data suggest that Igfbp2 is one of the more abundant Igfbps.

3) The authors identified Igfbp2 from RTT mouse model-derived astrocytes and BMP6 from FXS mouse model-derived astrocytes to inhibit neurite outgrowth in vitro. Are similar effects observed in vivo?

>> Although the authors did not directly observe a neurite outgrowth phenotype in vivo, they were able to visualize astrocytes from each of their mouse models of NDs, immunostaining for Igfbp2. This Igfbp2 staining allowed the authors to quantify differences in the intracellular and extracellular protein levels between ND and WT where an increase suggests an increase in protein secretion, similar to what was observed in vitro. The authors found similar effects in vivo for RTT and DS, where Igfbp2 level is increased. Understanding the similarity in protein levels between in vitro and in vivo models provides insightful support for the in vitro studies.

4) Given that a similar immunopanning procedure could be applied to astrocytes derived from fetal, juvenile and adult human brains, which astrocyte-secreted proteins affect human neuronal development in NDs?

>> While this was not addressed in the revised manuscript, we acknowledge that there are likely complicated methodological reasons. A recent publication from the Hoozemans group in Amsterdam and colleagues at Roche in Basel (Nolle et al., Front Cell Neurosci, 15:772011) highlighted some of these difficulties in human postmortem tissue sorting (for e.g. potential effect of the post-mortem delay and tissue treatment on gene expression and protein levels). It will be exciting to see how the advent of newer methods in future that allow such purifications of astrocytes across development and aging, will uncover additional pathways like the ones highlighted here by Caldwell and colleagues.

5) Do different glial cell types (and the proteins they secrete) also affect neuronal development in NDs?

>> Again, given the clear enrichment of Igfbp2 in astrocytes, it was likely less important to further this line of investigation of other Igfbps (as they were either not expressed in astrocytes, or more highly enriched in other CNS cells – e.g. Igfbp4 in microglia and Igfbp7 in endothelial cells).

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