Exosomes promote axon outgrowth by engaging the Wnt-Planar Cell Polarity pathway

  1. Samar Ahmad
  2. Melanie Pye
  3. Masahiro Narimatsu
  4. Siyuan Song
  5. Tania Christova
  6. Jeffrey L Wrana
  7. Liliana Attisano

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Abstract

In neurons, the acquisition of a polarized morphology is achieved upon the outgrowth of a single axon from one of several neurites. Exosomes or small extracellular vesicles (sEVs) from diverse sources are known to promote the neurite outgrowth and thus may have therapeutic potential. However, the effect of fibroblast-derived exosomes on axon elongation in neurons of the central nervous system under growth permissive conditions remains unclear. Here, we show that fibroblast-derived sEVs promote axon outgrowth and a polarized neuronal morphology in mouse primary embryonic cortical neurons. Mechanistically, we demonstrate that the sEV-induced increase in axon outgrowth requires endogenous Wnts and core PCP components including Prickle, Vangl, Frizzled and Dishevelled. We demonstrate that sEVs are internalized by neurons, colocalize with Wnt7b and induce relocalization of Vangl2 to the distal axon during axon outgrowth. In contrast, sEVs derived from neurons or astrocytes do not promote axon outgrowth, while sEVs from activated astrocytes inhibit elongation. Thus, our data reveals that fibroblast-derived sEVs promote axon elongation through the Wnt-PCP pathway in a manner that is dependent on endogenous Wnts.

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  1. Version published to 10.1101/2023.10.28.564542v2 on bioRxiv
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    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This manuscript examines the effects of fibroblast-derived extracellular vesicles (EV) on axon outgrowth in primary neurons, and investigates potential underlying mechanisms. The authors show that fibroblast EV increase axon outgrowth, which is dependent on components of the Wnt-PCP pathway in neurons. They further show that axonal outgrowth is not affected by neuron- or astrocyte-derived EV and that EV from activated astrocytes inhibit axon elongation. Although several experiments are performed thoroughly, major revision is required to substantiate the main claims of the manuscript.

    Major comments:

    1. Even though the authors made a good effort to characterize fibroblast-derived EV, the data so far does not indicate that strictly 'exosomes' are implicated in axon outgrowth, as it is currently virtually impossible to isolate a pure population of exosomes.
    2. In the methods the authors state that the conditioned media was stored for up to 8 weeks at 4C. As long-term storage of EV was shown to decrease their activity, the authors should specify whether they took steps to test the effect of storage time on EV concentration and activity.
    3. In Fig 3F, the authors claim that Vangl2 re-localizes from proximal to distal end of the axon. However, in the representative image the PBS-treated axon is much shorter, and Vangl2 can also be also detected in the growth cone. Therefore, it is not clear how neurons were classified in the analysis. As this is one of the main claims of the paper, the analysis should be performed in a more quantitative manner, such as quantification of intensity and volume of Vangl2 in the soma, proximal / distal axon and growth cone, while accounting for changes in axon length.
    4. The claim that exosomes mobilize neuronal Wnt to promote axon growth is unsubstantiated. Co-localization between Wnt7b and GFP-CD81 is not convincing given the low magnification and broad distribution of Wnt7b. It is also unclear how EV internalized in the soma would have this effect. Additional experiments to prove the direct influence of fibroblast EV on Wnt-PCP signaling (and optionally, how this is unique for fibroblast EV) would increase the validity of these claims.
    5. Neuronal EV were isolated at a different developmental time point (8DIV), which most likely has an effect on EV composition. Therefore, the claim that neuronal EV do not promote neurite outgrowth is not convincing. In addition, AraC or LPS used to treat neurons and astrocytes respectively, could be co-purified with EV and therefore have adverse effects on recipient neurons.
    6. The authors claim that this effect is unique to fibroblast EV. This claim is not valid without a full characterization of EV derived from multiple cell types and is therefore misleading.

    Minor comments:

    1. As in point 1 above, it is recommended to replace the term 'Exosome' in the figures and refer instead to the method of purification (eg. 100k). The term 'Exosome markers' in the text should also be replaced accordingly, as it is not currently clear whether CD81, TSG101 or Flotilin 1 are strictly on exosomes.
    2. Experiments using EV purified using gradient centrifugation (Fig S3) should be repeated at least once, such that statistical significance is calculated and results are shown as in other functional experiments. Alternatively, experiments could be performed using purified EV treated with a proteinase in the presence or absence of detergent, to verify whether it is EV cargo or extracellular proteins co-isolated with EV that mediate the observed phenotype.
    3. The authors should show that porcupine knockdown results in decreased Wnt secretion in fibroblast EV using western blotting.

    Significance

    Overall, the main take home message of this study is that fibroblast EV could have the common feature of upregulating axon outgrowth in neurons during development. While this is not relevant for CNS development per se, it could ultimately be of interest to a specialized audience investigating the translational relevance of fibroblast-derived EV.

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    Referee #2

    Evidence, reproducibility and clarity

    The definition of axonal fate has been under a lot of scrutiny. However nowadays it is not clear how the axonal fate could be distinguished from axon elongation. In other words, how one the neurite is selected to eventually grow as an axon versus the mechanism sustaining axonal growth once the fate is established. In addition, it is not clear, once the axon is growing, what is the mechanism that precludes the other neurites (future dendrites) from growing.

    The authors of this manuscript claim that fibroblast-derived exosomes promote axon growth; meanwhile, exosomes from activated astrocytes inhibit axon elongation. Moreover, the authors claim that exosomes mediate axon elongation throughout the PCP and Wnts pathways.

    I have several concerns regarding the data and concept presented in this manuscript that I consider precluding its publication in the current form.

    First, the claim that axon growth is promoted by exosomes is not well supported by the quantifications. It will be more convincing to show the frequency distribution for each experiment rather than the average growth per experiment (e.g., Figure 1F). In the way the data is presented now, we do not learn the real effect of the treatment on axonal growth. For instance, how is the variability in axon length in each experiment? the way the data is presented now might mask variability that could reduce the effect of their treatment

    Second, I find it difficult to explain this concept in neurons differentiating in the developing cortex. Which cell type is the source of exosomes mediating axon elongation? Is this cellular mechanism an artifact of the culture condition? In other words, are exosomes relevant for axonal growth in situ?

    In the model presented in Figure 7, authors show that fibroblast might mediate axon extension meanwhile activated astrocytes preclude axon extension. Authors do not consider that in the developing cortex, neurons are formed and elongating an axon (while they migrate to the cortical plate) before astrocytes are produced (Noctor et al Nature Neurosci. 2004). How do the authors reconcile this conceptual discrepancy with her in vitro studies? How do the authors reconcile this discrepancy with their studies in situ?

    Significance

    The authors of this manuscript claim that fibroblast-derived exosomes promote axon growth; meanwhile, exosomes from activated astrocytes inhibit axon elongation. Moreover, the authors claim that exosomes mediate axon elongation throughout the PCP and Wnts pathways.

    I have several concerns regarding the data and concept presented in this manuscript that I consider precluding its publication in the current form.

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    Referee #1

    Evidence, reproducibility and clarity

    Ahmad et al. report that exosomes isolated from fibroblast cell lines stimulate neurite extension in cultured neurons while those from neurons or astrocytes do not. They investigate the role of different components from the planar cell polarity (PCP) pathway and conclude that exosomes stimulate autocrine signaling by Wnts in a PCP pathway-dependent manner. However, the inactivation of most PCP components severely reduces neurite length already in untreated controls suggesting that they are required for neurite growth in general rather than specifically for the response to exosomes. The manuscript reports an extensive set of interesting results but does not provide sufficient evidence for the proposed mechanism.

    Major points:

    1. Knockout, knockdown, pharmacological inhibition or blocking antibodies were used to inactivate multiple components of the PCP pathway in cultured neurons. In most cases, the inactivation resulted in a reduction of neurite length both in control cultures and in cultures with exosomes. Because neurons lack the capacity to extend longer neurites after the inactivation of PCP components, it is not possible to determine if they are required for the response to exosomes. Only Fzd7 appears to be specifically required for the effect of exosomes since its knockdown does not reduce neurite length in controls.
    2. The physiological relevance of the stimulation of neurite growth by exosomes is unclear. It is not explained how cortical neurons come into contact with fibroblasts or the exosomes produced by them as the authors acknowledge at the end of the discussion.
    3. The mechanism how exosomes stimulate neurite growth remains unclear. The authors suggest that exosomes modulate autocrine Wnt signaling but do not provide sufficient evidence for this. The knockdown of Wntless or Wnts and blocking Wnt secretion by inhibiting Porcupine suppress neurite extension. This phenotype could results from a defect in autocrine signaling but also from a reduced secretion of Wnts into the medium.
    4. The authors claim that the cell body takes up exosomes that then acquire endogenous Wnt7b. The co-localization of the signals in Fig. 5H is not informative because Wnt7b shows uniform distribution while the CD81-EYFP signal is present in distinct structures that do not show a stronger EYFP signal.
    5. The specificity of the siRNAs has to be verified by rescue experiments in neurons.

    Minor points:

    It is not explained why the authors tested exosomes from fibroblasts.

    Fig. 7: The graphical summary shows that exosomes somehow enter the soma of neurons. It is not clear if this happens by endocytosis or fusion with the membrane. In either case, the topology of exosomes in the soma is incorrect.

    Significance

    Ahmad et al. report that exosomes isolated from fibroblast cell lines stimulate neurite extension in cultured neurons while those from neurons or astrocytes do not. They investigate the role of different components from the planar cell polarity (PCP) pathway and conclude that exosomes stimulate autocrine signaling by Wnts in a PCP pathway-dependent manner. However, the inactivation of most PCP components severely reduces neurite length already in untreated controls suggesting that they are required for neurite growth in general rather than specifically for the response to exosomes. The manuscript reports an extensive set of interesting results but does not provide sufficient evidence for the proposed mechanism.

  10. Version published to 10.1101/2023.10.28.564542v1 on bioRxiv