p75NTR prevents the onset of cerebellar granule cell migration via RhoA activation
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Evaluation Summary:
This paper will be of interest to neurobiologists and developmental biologists. Identifying novel mechanisms that prevent excessive neuronal migration is an important contribution to the field of neural development. The key conclusions of the paper are well supported by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Neuronal migration is one of the fundamental processes during brain development. Several neurodevelopmental disorders can be traced back to dysregulated migration. Although substantial efforts have been placed in identifying molecular signals that stimulate migration, little is known about potential mechanisms that restrict migration. These restrictive mechanisms are essential for proper development since it helps coordinate the timing for each neuronal population to arrive and establish proper connections. Moreover, preventing migration away from a proliferative niche is necessary in maintaining a pool of proliferating cells until the proper number of neuronal progenitors is attained. Here, using mice and rats, we identify an anti-migratory role for the p75 neurotrophin receptor (p75NTR) in cerebellar development. Our results show that granule cell precursors (GCPs) robustly express p75NTR in the external granule layer (EGL) when they are proliferating during postnatal development, however, they do not express p75NTR when they migrate either from the rhombic lip during embryonic development or from the EGL during postnatal development. We show that p75NTR prevented GCP migration by maintaining elevated levels of active RhoA. The expression of p75NTR was sufficient to prevent the migration of the granule cells even in the presence of BDNF (brain-derived neurotrophic factor), a well-established chemotactic signal for this cell population. Our findings suggest that the expression of p75NTR might be a critical signal that stops and maintains the GCPs in the proliferative niche of the EGL, by promoting the clonal expansion of cerebellar granule neurons.
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Author Response
Reviewer #1 (Public Review):
This manuscript addresses the role of the p75NTR neurotrophin receptor in the development of cerebellar granule precursor cells (CGPs). This cell type is notable for having high levels of p75NTR expression in a discrete developmental window yet the specific role of the receptor in this setting has remained obscure.
The authors show that although p75NTR expression correlates with the CGP proliferative state, expression of p75NTR is not required to maintain the proliferative state. Rather, migration CGPs in culture and within cerebellar slices is optimal only when p75NTR levels are reduced and the authors conclude that the expression of p75NTR normally reduces CGP migration. They examine signalling mechanisms that lie downstream of p75NTR to elicit this effect and show that RhoA, …
Author Response
Reviewer #1 (Public Review):
This manuscript addresses the role of the p75NTR neurotrophin receptor in the development of cerebellar granule precursor cells (CGPs). This cell type is notable for having high levels of p75NTR expression in a discrete developmental window yet the specific role of the receptor in this setting has remained obscure.
The authors show that although p75NTR expression correlates with the CGP proliferative state, expression of p75NTR is not required to maintain the proliferative state. Rather, migration CGPs in culture and within cerebellar slices is optimal only when p75NTR levels are reduced and the authors conclude that the expression of p75NTR normally reduces CGP migration. They examine signalling mechanisms that lie downstream of p75NTR to elicit this effect and show that RhoA, previously shown to be activated by p75NTR, is required to block CGP migration, that RhoA activity is lower in p75NTR-/- CGPs than in wild-type counterparts, and that RhoA inhibitors enable CGP migration, even in cells overexpressing p75NTR.
This is an important study that uses a combination of descriptive methods and chemical and genetic gain- and loss- function approaches to demonstrate that a p75NTR-RhoA signaling pathway normally functions to limit CGP migration during development. The paper is logical and well written and the data presentation is excellent.
Some points to consider:
Figure 2A introduces the CGP cultures and shows that p75NTR levels are high in cells exposed to SHH. However, these results are difficult to interpret in the absence of controls showing p75NTR levels at the time of plating - does the SHH exposure increase p75NTR expression? Or prevent its decrease?
We agree with the question raised by the reviewer, and we have done this experiment. In these results, we observed an increase in p75NTR expression after 24h or 48 h of Shh exposure compared with the levels of the receptor at the time of plating. However, there are a few caveats to the interpretation of these results, that make it difficult to establish whether Shh increases or prevents the decrease of the receptor:
When quantifying the levels of p75NTR in vivo, we obtained a granule cell population that includes proliferating and differentiated granule cells, this mixed population of cells is present at the time of plating. When establishing primary culture, a large percentage of the cells do not survive, and the majority of dying cells would be differentiated cells, therefore introducing a bias toward proliferating cells for the 24 and 48 h in vitro from the time of plating. The proportion of proliferating/differentiated cells would be different between the in vivo and the in vitro after 24 or 48h.
The concentration of the mitogen most likely would be very different between the cells in vivo (the time of plating) and the exposure to Shh in vitro, introducing a second bias. It might be that the increase in p75NTR is a consequence of more cells proliferating since they respond to higher concentration of the mitogen.
We know Shh induces proliferation in CGN, and this is accompanied by an increase in p75NTR. Therefore, the increase of p75NTR might be due to more proliferating cells, but not necessarily an induction of the expression of the receptor.
I recognize the convenience of using the p75NTR-GFP construct to track migration but was surprised that the potential confounds of this approach were not examined or even mentioned. Does p75NTR-GFP activate RhoA more or less than the wild-type receptor? What experiments have been performed to ensure that this construct is an effective mimic of the wild-type receptor? Would it be possible to co-transfect p75NTR and GFP as an alternative approach?
The p75NTR/GFP construct has been used in the field for an extensive period and the biology of the construct has been well characterized (i.e. cellular localization and translocation, activation and signaling). Co-transfecting the slices with p75NTR and GFP with separate construct doesn’t necessarily mean that each cell receives both constructs, which is not the case using a fusion protein. Although we cannot completely rule out the possibility of subtle intracellular differences between the endogenous p75NTR and the construct, we are confident that this result is supported by the other experiments in the manuscript (the conditional deletion of p75, results with the p75NTR -/-, and the use of inhibitors) Paper showing that the p75-GFP construct is correctly sorted https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2139957/
The authors discuss previous findings that indicate that p75NTR can play a pro-migratory role but oddly do not place their results in other contexts where p75NTR has been shown to block migration. CGPs have been quite widely used to dissect the role of p75NTR in the Rho-dependent migration blockade induced by MAG and other myelin components and interesting insights on receptor components (e.g. LINGO1) and signalling mechanisms (e.g. RhoA) that mediate these effects. The results reported here should be discussed in the context of these previous findings.
We agree with the reviewer and we will discuss our findings in this context.
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Evaluation Summary:
This paper will be of interest to neurobiologists and developmental biologists. Identifying novel mechanisms that prevent excessive neuronal migration is an important contribution to the field of neural development. The key conclusions of the paper are well supported by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript addresses the role of the p75NTR neurotrophin receptor in the development of cerebellar granule precursor cells (CGPs). This cell type is notable for having high levels of p75NTR expression in a discrete developmental window yet the specific role of the receptor in this setting has remained obscure.
The authors show that although p75NTR expression correlates with the CGP proliferative state, expression of p75NTR is not required to maintain the proliferative state. Rather, migration CGPs in culture and within cerebellar slices is optimal only when p75NTR levels are reduced and the authors conclude that the expression of p75NTR normally reduces CGP migration. They examine signalling mechanisms that lie downstream of p75NTR to elicit this effect and show that RhoA, previously shown to be …
Reviewer #1 (Public Review):
This manuscript addresses the role of the p75NTR neurotrophin receptor in the development of cerebellar granule precursor cells (CGPs). This cell type is notable for having high levels of p75NTR expression in a discrete developmental window yet the specific role of the receptor in this setting has remained obscure.
The authors show that although p75NTR expression correlates with the CGP proliferative state, expression of p75NTR is not required to maintain the proliferative state. Rather, migration CGPs in culture and within cerebellar slices is optimal only when p75NTR levels are reduced and the authors conclude that the expression of p75NTR normally reduces CGP migration. They examine signalling mechanisms that lie downstream of p75NTR to elicit this effect and show that RhoA, previously shown to be activated by p75NTR, is required to block CGP migration, that RhoA activity is lower in p75NTR-/- CGPs than in wild-type counterparts, and that RhoA inhibitors enable CGP migration, even in cells overexpressing p75NTR.
This is an important study that uses a combination of descriptive methods and chemical and genetic gain- and loss- function approaches to demonstrate that a p75NTR-RhoA signaling pathway normally functions to limit CGP migration during development. The paper is logical and well written and the data presentation is excellent.
Some points to consider:
Figure 2A introduces the CGP cultures and shows that p75NTR levels are high in cells exposed to SHH. However, these results are difficult to interpret in the absence of controls showing p75NTR levels at the time of plating - does the SHH exposure increase p75NTR expression? Or prevent its decrease?I recognize the convenience of using the p75NTR-GFP construct to track migration but was surprised that the potential confounds of this approach were not examined or even mentioned. Does p75NTR-GFP activate RhoA more or less than the wild-type receptor? What experiments have been performed to ensure that this construct is an effective mimic of the wild-type receptor? Would it be possible to co-transfect p75NTR and GFP as an alternative approach?
The authors discuss previous findings that indicate that p75NTR can play a pro-migratory role but oddly do not place their results in other contexts where p75NTR has been shown to block migration. CGPs have been quite widely used to dissect the role of p75NTR in the Rho-dependent migration blockade induced by MAG and other myelin components and interesting insights on receptor components (e.g. LINGO1) and signalling mechanisms (e.g. RhoA) that mediate these effects. The results reported here should be discussed in the context of these previous findings.
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Reviewer #2 (Public Review):
In this manuscript, the authors have set to demonstrate the function of P75NTR during the granule cell migration during cerebellar development. Authors have done a successful job in showing the link between P75NTR expression, granule cell proliferation, and migration status and have demonstrated functionally that the proliferative granule cell progenitors maintain high levels of P75NTR and the P75NTR expressing cells are not migratory due to the high RhoA levels. However, the link between the function of P75NTR and its role during cell cycle exit as the group has previously shown and the new role that they demonstrate here should be discussed further to parse the context-dependent function of P75NTR better. A wider audience would also benefit from further discussion of how these findings differ from others …
Reviewer #2 (Public Review):
In this manuscript, the authors have set to demonstrate the function of P75NTR during the granule cell migration during cerebellar development. Authors have done a successful job in showing the link between P75NTR expression, granule cell proliferation, and migration status and have demonstrated functionally that the proliferative granule cell progenitors maintain high levels of P75NTR and the P75NTR expressing cells are not migratory due to the high RhoA levels. However, the link between the function of P75NTR and its role during cell cycle exit as the group has previously shown and the new role that they demonstrate here should be discussed further to parse the context-dependent function of P75NTR better. A wider audience would also benefit from further discussion of how these findings differ from others that implicate neurotrophin signalling and specifically P75NTR in neural migration throughout development in the introduction and the discussion.
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Reviewer #3 (Public Review):
Zanin and Friedman addressed a fundamental issue in cerebellar development: how cerebellar granule cell precursors avoid excessive migration from the external granule cell layer (EGL). Although many studies focused on the mechanisms that promote neuronal migration, little is known about the mechanisms that restrict neuronal migration. By combining in vivo and in vitro experimental approaches, the authors convincingly demonstrated that in the EGL, expression of p75NTR does not promote proliferation or cell cycle re-entry. Instead, p75NTR inhibits the radial migration of granule cells via activated RhoA. These experimental results are interesting, and the conclusions of this paper are well supported by data. The paper is mostly clearly written, but some areas of experimental findings and analysis need to be …
Reviewer #3 (Public Review):
Zanin and Friedman addressed a fundamental issue in cerebellar development: how cerebellar granule cell precursors avoid excessive migration from the external granule cell layer (EGL). Although many studies focused on the mechanisms that promote neuronal migration, little is known about the mechanisms that restrict neuronal migration. By combining in vivo and in vitro experimental approaches, the authors convincingly demonstrated that in the EGL, expression of p75NTR does not promote proliferation or cell cycle re-entry. Instead, p75NTR inhibits the radial migration of granule cells via activated RhoA. These experimental results are interesting, and the conclusions of this paper are well supported by data. The paper is mostly clearly written, but some areas of experimental findings and analysis need to be clarified or extended.
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