FMRP regulates neuronal migration via MAP1B

Reviewer #1 (Public Review):

This work investigated Fragile X Messenger Ribonucleoprotein (FMRP) protein impact on neuroblast tangential migration in the postnatal rostral migratory stream (RMS). Authors conducted series of live-imaging on organotypic brain slices from Fmr1-null mice. They continued their analysis silencing Fmr1 exclusively from migrating neuroblasts using electroporation-mediated RNA interference method (MiRFmr1 KD). These impressive approaches show that neuroblasts tangential migration is impaired in Fmr1-null mice RMS and these defects are mostly recapitulated in the MiRFmr1neuroblasts.This nicely supports the idea that FMRP have a cell autonomous function in tangentially migrating neuroblasts. It is an important part of this work as migrating neuroblasts are in contact with each other and surrounding glial cells while migrating towards the olfactory bulb. The authors also confirm that FMRP mRNA target Microtubule Associated Protein 1B (MAP1B) is overexpressed in the Fmr1-null mice RMS. They successfully use electroporation-mediated RNA interference method to silence Map1b in the Fmr1-null mice neuroblasts. This discreet and elaborate experiment rescues most of the migratory defects observed both in Fmr1-null and MiRFmr1neuroblasts. Altogether, these results strongly suggest that FMRP-MAP1B axis has an important role in regulation of the neuroblasts tangential migration in RMS. Neurons move forward in cyclic saltatory manner which includes repeated steps of leading process extension, migration of the cell organelles and nuclear translocation. Authors reveal by analyzing the live-imaging data that FMRP-MAP1B axis is affecting movement of centrosome and nucleus during saltatory migration. An important part of the centrosome and nucleus movement is forces mediated by microtubule dynamics. Authors propose that FMRP regulate tangential migration via microtubule dynamics regulator MAP1B. This work provides valuable new information on regulation of the neuroblasts tangential saltatory migration. These findings also increase and improve our understanding of the issues involved in Fragile X Syndrome (FXS) disorders. The conclusions of this work are mostly supported by the data. However, methods and data analysis would benefit from more careful and comprehensive scrutiny.

1.) It would be beneficial for the detail-oriented readers to have a more comprehensive section of neuronal migration analysis. It would help to understand better the details of the results and analysis. For example, percentage of pausing time. Is neuroblast migration speed and pausing time (%) separated from each other? Does this mean that migration speed is measured from time of the nucleus movement, and it excludes pausing time? Sometimes migration speed refers to the total distance that cells have moved divided by the time between images (e.g., Nam et al. 2007, J Comp Neurol 505: 190-208). This is also important as neuroblasts migration speed fluctuate during their migration in RMS (e.g., Belvindrah et al. 2017, J Cell Biol 216: 2443-2461). It could be useful, for example, to show a plot of total migration distance distribution between controls, Fmr1-null, MiRFmr1 KD and MiRMap1b KD neuroblasts.

2.) The author's claim that Fmr1 interfering RNA (MiFmr1 KD) model "recapitulates the entire migratory phenotype described in Fmr1-null mutants". This is evident from the data analysis for the migration speed, pausing time percentage and NK distance. Parallel, but slightly weaker effect is seen in, NK frequency, CK frequency and CK efficiency. However, interpretation of the directionality analysis causes some concerns.
a) Sinuosity index
Fmr1-null mice (ctr: 1.32{plus minus}0.04; Fmr1-null: 1.93{plus minus}0.16; Figure 2C) and MiFmr1 KD neurons (MiRNEG: 1.5{plus minus}0.12; MiRFmr1 KD 1.62 {plus minus}0.08; Figure S1C). The latter results are significant, but standard error of means (SEM) seems to overlap. In addition, there is only a minor difference between control and MiRFmr1 KD cells sinuosity index.
b) Directionality radar
Migration directionality radar seems to be considerably different between Fmr1-null (Figure 2D) and MiFmr1 KD results (Figure S1D).
It would be beneficial for this article to fully disclose, how these analyses were performed. For example, how sinuosity index was calculated and what does it precisely measure. It would greatly help to understand better the directionality analysis. To make these results more solid authors could have used original migration trajectories in the rose and/or trajectory plots. These plots visualize better the migration directionality results and clarify the changes in the directionality during migration.

3.) Authors claim that "Overall, our results demonstrate that MAP1B is the main FMRP mRNA target involved in the regulation of neuronal migration". Results and analysis show that migration speed, pausing time percentage, NK distance and NK frequency migratory defects are all rescued in Fmr1-null mice when MiRMap1b KD was introduced to the neuroblasts (Figure 4). These results are very interesting, linking FMRP-MAP1B axis to the microtubule dynamics.

4.) Authors could refine in discussion what is known about FRMP in neuronal migration. For example, La Fata et al. 2014 found that N-cadherin protein levels were lower in Fmr1-null mice and reintroducing N-cadherin rescued embryonic radial migration defects. N-cadherin is also expressed in the RMS and its deficiency affects negatively to the neuroblast migration (e.g., Porlan et al. 2014, Nat Cell Biol (7):629-38). This relationship of FMRP and N-cadherin could be discussed and considered in the article more closely. Overall, the article will benefit from clearer writing and more comprehensive discussion.

Reviewer #2 (Public Review):

In this manuscript, the authors conducted a straightforward molecular approach to link FMRP and MAP1B proteins functionally. Both proteins are connected since FMRP is a translational regulator of the MAP1B protein, a microtubule-stabilizing factor.

The results combined molecular genetics (FMRP knock-out mice) with acute inactivation of FRMP and MAP1B to conclusively support the notion that FMRP-dependent regulation of MAP1B is necessary for proper neuronal migration toward the olfactory bulb using the rostral migratory stream.

Overall, these results increase our knowledge of the molecular mechanism that controls how neurons migrate in the brain to reach their final destinations and confirms that cytoskeleton regulators are key players in this process.

Reviewer #3 (Public Review):

Neuronal migration is one of the key processes for appropriate neuronal development. Defects in neuronal migration are associated with different brain disorders often accompanied by intellectual disabilities. Therefore, the study of the mechanisms involved in neuronal migration helps to understand the pathogenesis of some brain malformations and psychiatric disorders.

FMRP is an RNA-binding protein implicated in RNA metabolism regulation and mRNA local translation. FMRP loss of function causes fragile X syndrome (FXS), the most common form of inherited intellectual disability. Previous studies have shown the role of FMRP in the multipolar to bipolar transition during the radial migration in the cortex and its possible relation with periventricular heterotopia and altered synaptic communication in humans with FXS. However, the role of FMRP in neuronal tangential migration is largely unknown. In this manuscript, the authors aim to decipher the role of FMRP in the tangential migration of neuroblasts along the rostral migratory stream (RMS) in the postnatal brain. By extensive live-imaging analysis of migrating neuroblasts along the RMS, they demonstrate the requirement of FMRP for neuroblast migration and centrosomal movement. These migratory defects are cell-autonomous and mediated by the microtubule-associated protein Map1b.

Overall, the manuscript highlights the importance of FMRP in neuronal tangential migration. They performed an analysis of different aspects of migration such as nucleokinesis and cytokinesis in migrating neuroblasts from live-imaging videos.
However, the work is quite incomplete. The role of FMRP and Map1b in neuronal migration is not well introduced and discussed. In the cortex, FMRP is mainly implicated in the multipolar to bipolar transition of immature neurons, but not in the migration itself (la Fata et al., 2014). In fact, Fmr1 KO mice do not show impairment in cortical lamination. On the other hand, very less is mentioned about the role of Map1b in neuronal migration. It is not shown whether overexpression of Map1b alters neuroblast migration and recapitulates the Fmr1 KO phenotype.

Moreover, it is unclear to me which are the anatomical consequences of aberrant migration of neuroblasts in the Fmr1 KO mice. Authors mention that neuroblasts properly arrive at the OB and they refer to a previous publication (Scotto-Lomassese et al., 2011). However, this study does not show the distribution of neuroblasts in the SVZ, along the RMS or in the olfactory bulb (OB) in mutant mice. On the contrary, they said that there is no delay in the migration or maturation of granular cells arriving at the OB (Scotto-Lomassese et al., 2011). In summary, the authors do not show the functional consequences of aberrant neuroblast migration in the Fmr1 KO mice, making weaker the assumption that the study is important for the understanding of FXS pathophysiology.

Author Response:

We are grateful to the three referees for their overall positive evaluation of our work and valuable constructive suggestions. We will address their public reviews with utmost care, as well as their private recommendations.

To Reviewer #1: thanks for the positive comments and for the appreciation of our « impressive approaches »

• We will add a more comprehensive section of neuronal migration analysis in the Material and Methods section. Sorry for that regrettable lack of precision.

• We will address the comments about the sinuosity index definition and interpretations.

• We will enhance the clarity of our writing and delve deeper into the discussion. As mentioned to Reviewer #3, the brevity of the text was influenced by the Short report format.

To Reviewer #2 : thanks for the overall positive appreciation.

We will also consider the recommendations for authors with care.

To Reviewer #3 : thanks a lot for the feedback.

• We will further develop the introduction and discussion sections as suggested. Regrettably and as mentioned to Reviewer #1, we had to significantly condense them due to the space constraints imposed by the Short Report format.

• We will attempt to overexpress Map1B in order to assess the potential phenotypic similarity to the Fmr1 null condition, as suggested. However, it is important to acknowledge that this experiment may not yield a definitive answer due to potential differences in the level of Map1B expression driven by a CMV promoter compared to its endogenous expression in Fmr1 null neurons, as well as variations in the subcellular distribution of the overexpressed Map1B.

• Regarding the anatomical consequences of aberrant migration, we acknowledge that neither our present work nor our previous study by Scotto-Lomassesse et al. provide evidence in this regard, as pointed out by the reviewer. Indeed, the delayed neurons do reach the olfactory bulb based on our findings. However, other studies have demonstrated that a delay in migration can have important functional consequences (eg Bocchi et al, 2017 doi: 10.1038/s41467-017-01046-w). Accordingly, we will revise and moderate our conclusions on this specific point.