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FMRP regulates multipolar to bipolar transition affecting neuronal migration and cortical circuitry

Abstract

Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.

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Figure 1: Fmrp is involved in neuronal cortical positioning.
Figure 2: Fmr1−/− mice do not show differences in progenitor proliferation nor radial migration of excitatory neurons.
Figure 3: Fmr1−/− neurons have a delayed exit from the multipolar stage.
Figure 4: Fmrp regulates neuronal cortical positioning by stabilizing Cdh2 mRNA.
Figure 5: Fmrp regulates N-cadherin during multipolar-to-bipolar transition.
Figure 6: Postnatal brain networks are affected in the developing Fmr1−/− mouse.
Figure 7: Excitatory and inhibitory networks are affected in the developing Fmr1−/− mouse.
Figure 8: DTI studies show neuronal connectivity impairments in juvenile Fmr1−/− mice.

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References

  1. Jacquemont, S., Hagerman, R.J., Hagerman, P.J. & Leehey, M.A. Fragile-X syndrome and fragile X–associated tremor/ataxia syndrome: two faces of FMR1. Lancet Neurol. 6, 45–55 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Bagni, C., Tassone, F., Neri, G. & Hagerman, R. Fragile X syndrome: causes, diagnosis, mechanisms and therapeutics. J. Clin. Invest. 122, 4314–4322 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Verkerk, A.J. et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65, 905–914 (1991).

    Article  CAS  PubMed  Google Scholar 

  4. Bagni, C. & Greenough, W.T. From mRNP trafficking to spine dysmorphogenesis: the roots of fragile X syndrome. Nat. Rev. Neurosci. 6, 376–387 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Cruz-Martín, A., Crespo, M. & Portera-Cailliau, C. Delayed stabilization of dendritic spines in fragile X mice. J. Neurosci. 30, 7793–7803 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Irwin, S.A. et al. Abnormal dendritic spine characteristics in the temporal and visual cortices of patients with fragile-X syndrome: a quantitative examination. Am. J. Med. Genet. 98, 161–167 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Meguid, N.A. et al. Cognition and lobar morphology in full mutation boys with fragile X syndrome. Brain Cogn. 78, 74–84 (2012).

    PubMed  Google Scholar 

  8. Abitbol, M. et al. Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain. Nat. Genet. 4, 147–153 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Saffary, R. & Xie, Z. FMRP regulates the transition from radial glial cells to intermediate progenitor cells during neocortical development. J. Neurosci. 31, 1427–1439 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Castrén, M. et al. Altered differentiation of neural stem cells in fragile X syndrome. Proc. Natl. Acad. Sci. USA 102, 17834–17839 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bhattacharyya, A. et al. Normal neurogenesis but abnormal gene expression in human Fragile X cortical progenitor cells. Stem Cells Dev. 17, 107–117 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Tervonen, T.A. et al. Aberrant differentiation of glutamatergic cells in neocortex of mouse model for fragile X syndrome. Neurobiol. Dis. 33, 250–259 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Fernández, E., Rajan, N. & Bagni, C. The FMRP regulon: from targets to disease convergence. Front. Neurosci. 7, 191 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  14. LoTurco, J.J. & Bai, J. The multipolar stage and disruptions in neuronal migration. Trends Neurosci. 29, 407–413 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. The Dutch-Belgian Fragile X Consortium. Fmr1 knockout mice: a model to study fragile X mental retardation. Cell 78, 23–33 (1994).

  16. Calderon de Anda, F., Gartner, A., Tsai, L.H. & Dotti, C.G. Pyramidal neuron polarity axis is defined at the bipolar stage. J. Cell Sci. 121, 178–185 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Nelson, D.L., Orr, H.T. & Warren, S.T. The unstable repeats: three evolving faces of neurological disease. Neuron 77, 825–843 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Jossin, Y. & Cooper, J.A. Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex. Nat. Neurosci. 14, 697–703 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gärtner, A. et al. N-cadherin specifies first asymmetry in developing neurons. EMBO J. 31, 1893–1903 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Miyashiro, K.Y. et al. RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice. Neuron 37, 417–431 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Lucá, R. et al. The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation. EMBO Mol. Med. 5, 1523–1536 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zalfa, F. et al. A new function for the fragile X mental retardation protein in regulation of PSD-95 mRNA stability. Nat. Neurosci. 10, 578–587 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. D'Hulst, C. et al. Decreased expression of the GABAA receptor in fragile X syndrome. Brain Res. 1121, 238–245 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Kadowaki, M. et al. N-cadherin mediates cortical organization in the mouse brain. Dev. Biol. 304, 22–33 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Bartolini, G., Ciceri, G. & Marin, O. Integration of GABAergic interneurons into cortical cell assemblies: lessons from embryos and adults. Neuron 79, 849–864 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Rubenstein, J.L. & Merzenich, M.M. Model of autism: increased ratio of excitation/inhibition in key neural systems. Genes Brain Behav. 2, 255–267 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. D'Hulst, C. & Kooy, R.F. The GABAA receptor: a novel target for treatment of fragile X? Trends Neurosci. 30, 425–431 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Gonçalves, J.T., Anstey, J.E., Golshani, P. & Portera-Cailliau, C. Circuit level defects in the developing neocortex of Fragile X mice. Nat. Neurosci. 16, 903–909 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Blankenship, A.G. & Feller, M.B. Mechanisms underlying spontaneous patterned activity in developing neural circuits. Nat. Rev. Neurosci. 11, 18–29 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Garaschuk, O., Linn, J., Eilers, J. & Konnerth, A. Large-scale oscillatory calcium waves in the immature cortex. Nat. Neurosci. 3, 452–459 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Lodato, S. et al. Excitatory projection neuron subtypes control the distribution of local inhibitory interneurons in the cerebral cortex. Neuron 69, 763–779 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Alexander, A.L., Lee, J.E., Lazar, M. & Field, A.S. Diffusion tensor imaging of the brain. Neurotherapeutics 4, 316–329 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kwon, S.H., Vasung, L., Ment, L.R. & Huppi, P.S. The role of neuroimaging in predicting neurodevelopmental outcomes of preterm neonates. Clin. Perinatol. 41, 257–283 (2014).

    Article  PubMed  Google Scholar 

  34. Maximo, J.O., Cadena, E.J. & Kana, R.K. The implications of brain connectivity in the neuropsychology of autism. Neuropsychol. Rev. 24, 16–31 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Nadarajah, B., Brunstrom, J.E., Grutzendler, J., Wong, R.O. & Pearlman, A.L. Two modes of radial migration in early development of the cerebral cortex. Nat. Neurosci. 4, 143–150 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Englund, C. et al. Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells and postmitotic neurons in developing neocortex. J. Neurosci. 25, 247–251 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ohshima, T. et al. Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex. Development 134, 2273–2282 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Sakakibara, A. et al. Dynamics of centrosome translocation and microtubule organization in neocortical neurons during distinct modes of polarization. Cereb. Cortex 24, 1301–1310 (2013).

    Article  PubMed  Google Scholar 

  39. Tabata, H. & Nakajima, K. Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex. J. Neurosci. 23, 9996–10001 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Harlow, E.G. et al. Critical period plasticity is disrupted in the barrel cortex of FMR1 knockout mice. Neuron 65, 385–398 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Meredith, R.M., Dawitz, J. & Kramvis, I. Sensitive time-windows for susceptibility in neurodevelopmental disorders. Trends Neurosci. 35, 335–344 (2012).

    Article  CAS  PubMed  Google Scholar 

  42. De Felipe, J., Marco, P., Fairen, A. & Jones, E.G. Inhibitory synaptogenesis in mouse somatosensory cortex. Cereb. Cortex 7, 619–634 (1997).

    Article  CAS  PubMed  Google Scholar 

  43. Moro, F. et al. Periventricular heterotopia in fragile X syndrome. Neurology 67, 713–715 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Peñagarikano, O. et al. Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities and core autism-related deficits. Cell 147, 235–246 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. De Rubeis, S. et al. CYFIP1 coordinates mRNA translation and cytoskeleton remodeling to ensure proper dendritic spine formation. Neuron 79, 1169–1182 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hall, S.S., Jiang, H., Reiss, A.L. & Greicius, M.D. Identifying large-scale brain networks in fragile X syndrome. JAMA Psychiatry 70, 1215–1223 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Baribeau, D.A. & Anagnostou, E. A comparison of neuroimaging findings in childhood onset schizophrenia and autism spectrum disorder: a review of the literature. Front. Psychiatry 4, 175 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Zhang, J., Aggarwal, M. & Mori, S. Structural insights into the rodent CNS via diffusion tensor imaging. Trends Neurosci. 35, 412–421 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Heard, T.T. et al. EEG abnormalities and seizures in genetically diagnosed Fragile X syndrome. Int. J. Dev. Neurosci. 38C, 155–160 (2014).

    Article  Google Scholar 

  50. Yeh, F.C., Verstynen, T.D., Wang, Y., Fernandez-Miranda, J.C. & Tseng, W.Y. Deterministic diffusion fiber tracking improved by quantitative anisotropy. PLoS ONE 8, e80713 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bakker, C.E. et al. FMR1 knockout mice: a model to study fragile X mental retardation. Cell 78, 23–33 (1994).

    Google Scholar 

  52. Shariati, S.A. et al. APLP2 regulates neuronal stem cell differentiation during cortical development. J. Cell Sci. 126, 1268–1277 (2013).

    Article  CAS  PubMed  Google Scholar 

  53. Shoval, I., Ludwig, A. & Kalcheim, C. Antagonistic roles of full-length N-cadherin and its soluble BMP cleavage product in neural crest delamination. Development 134, 491–501 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. Ferrari, F. et al. The fragile X mental retardation protein-RNP granules show an mGluR-dependent localization in the post-synaptic spines. Mol. Cell. Neurosci. 34, 343–354 (2007).

    Article  CAS  PubMed  Google Scholar 

  55. Calderon de Anda, F., Gartner, A., Tsai, L.H. & Dotti, C.G. Pyramidal neuron polarity axis is defined at the bipolar stage. J. Cell Sci. 121, 178–185 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. Yeh, F.C., Verstynen, T.D., Wang, Y., Fernandez-Miranda, J.C. & Tseng, W.Y. Deterministic diffusion fiber tracking improved by quantitative anisotropy. PLoS ONE 8, e80713 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Gärtner, A. et al. N-cadherin specifies first asymmetry in developing neurons. EMBO J. 31, 1893–1903 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Jossin, Y. & Cooper, J.A. Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex. Nat. Neurosci. 14, 697–703 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are very grateful to E. Lemmens for administrative support, K. Jonckers, J. Royaert and I. Beheydt for their help and assistance with primary neurons and mouse colonies. We are thankful to T. Voets and S. Munck (coordinator of LiMoNe) for providing us excellent suggestions on the calcium imaging, P. Fazzari for helping with the synapses quantification, and M. Regoli for suggestions on the statistical analysis. G.L.F., N.D.-I. and M.A. were supported by grants FWO-G.0705.11 and FWO-G.0667.09 to C.B. T.A. was supported by a VIB grant to C.B. A.G. was partially supported by a VIB grant to C.B. and C.G.D. as well by an FWO grant (ZKC6058-00-W01) to A.G. U.H. and T.D. received financial support from the KUL-program financing IMIR (PF10/017). T.D. was supported by FWO-ZKC5858. R.M.M. J.D. and R.B.P. were supported by grants from FRAXA and Dutch Medical Research Council NWO VIDI (#917.10.372). G.L.F. is part of the Brain Train network (http://www.brain-train.nl/braintrain-program/partners/). This work was supported by grants from VIB, SAO, FWO-G.0705.11 and FWO-G.0667.09, and Associazione Italiana Sindrome X Fragile to C.B., and FWO-G0.666.10N, NEUROBRAINNET IAP 7/16, Flemish Methusalem grant, and Innovation Ingenio-Consolider, CSD2010-00045 to C.G.D.

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G.L.F., A.G., C.G.D., T.A. and C.B. designed the experiments and wrote the manuscript. A.G. and C.B. supervised the project. G.L.F. and A.G. performed a major part of the experiments and analyzed the data. N.D.-I. contributed substantially to the experiments and data analysis during the entire review process. M.A. contributed to some experiments. T.D. and U.H. performed and analyzed the MRI scans. R.M.M., J.D. and R.B.P. performed and analyzed the initial calcium imaging experiments and A.G., N.D.-I. and T.A. performed and analyzed the electroporation with calcium imaging experiments.

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Correspondence to Claudia Bagni.

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Integrated supplementary information

Supplementary Figure 1 Expression of Fmrp in early cortical development.

a) Representative western blot showing Fmrp expression during two developmental stages (E14 and E17) in WT and Fmr1−/− embryonic somatosensory cortex. Ribosomal protein S6 (rpS6) was used as loading control b) Confocal images showing the distribution of WT and Fmr1−/− neuronal cells, upon IUE with the pCAG-EGFP plasmid at E14.5, in mouse cortices at P0. EGFP fluorescence (green) and DAPI staining (blue). Right histogram shows the frequency distribution and quantification of EGFP positive cells in ten equal bins (VZ 1 to CP 10) in WT (n = 6), Fmr1−/− (n = 5), Two-way ANOVA; F (10, 165) = 51.97, Values: mean ± SEM. Scale bars = 50 μm.

Supplementary Figure 2 Absence of Fmrp does not affect proliferation of precursor cells at E14.5.

a) Left, confocal images from WT and Fmr1−/− E14.5 mouse cortices stained with markers for the intermediate (Tbr2-positive) and radial (Sox2-positive) progenitor populations (anti-Tbr2, green; anti-Sox2, red). Right, the graph represents the ratio of Tbr2-positive to Sox2-positive in WT and Fmr1−/− cortices. Scale bars = 20 μm. (Values are mean ± SEM; two tailed unpaired t-test: P = 0.1503, t=1.776, df = 4, n = 3 WT animals and n = 3 Fmr1−/− animals). b) Histogram represents the percentage of cells transfected at E14.5 with pCAG-EGFP that are Ki67-positive in the VZ/SVZ/IZ of WT and Fmr1−/− cortices. (Values are mean ± SEM; two tailed unpaired t-test: P = 0.423, t = 0.86, df = 6, n = 3 WT animals and n = 3 Fmr1−/− animals).

Supplementary Figure 3 Morphology and positioning of radial glia cells are not affected in Fmr1−/− cortices.

a) Individual radial glia cells in E15.5 WT and Fmr1−/− embryos were labeled, by IUE, using a BLBP-EGFP (Brain Lipid Binding Protein promoter specific for radial glia cells) construct. Brain sections were analysed at E16.5. Fmr1−/− radial glia cells display normal morphology spanning the entire cortex (green arrow-heads) and have apical (yellow arrow-heads) and basal (red arrow-heads) endfeets as observed in WT. b) Coronal section from E17.5 WT and Fmr1−/− littermates labeled with anti β-catenin (upper panel) and γ-tubulin antibodies (lower panels). Scale bars = 50 μm.

Supplementary Figure 4 Migration in vitro of the Fmr1−/− neurons is not affected.

WT and Fmr1−/− cells were isolated from E14.5 embryonic cortices, left to form aggregates and embedded in Matrigel 18 h later. Sequential images show four time points of WT and Fmr1−/− neurons migrating away from the in vitro explants. Both average velocity and directionality (total path length from time 0 to time x/ net distance time 0 to time x) were not different between WT and Fmr1−/−­­ neurons: Average velocity: WT = 0.1537 μm/min ± 0.0226, n = 6 (71 neurons); Fmr1−/− = 0.1609 μm/min ± 0.01687, n = 5 (59 neurons); unpaired two tailed t-test: P = 0.8122, t = 0.2447, df = 9, n = number of explants. Directionality: WT 0.6629 ± 0.03070, arbitrary units; Fmr1−/− 0.6642 ± 0.06615 arbitrary units, P = 0.9857, t=0.01844, df=9. Scale bar = 10 μm

Supplementary Figure 5 Absence of Fmrp does not alter cortical layering.

Left, immunohistochemistry showing E17.5 confocal images of coronal sections from WT and Fmr1−/− cortices labeled with anti-COUP-TF-interacting protein 2 antibody (Ctip2, transcription factor) for lower layer neurons and anti-Cut homeobox 1 gene antibody (Cux1, transcription factor) for differentiated neurons in the upper layer. Scale bars = 50 μm. Right, quantification of the average line profiles throughout the cortex (n = 3). Normalization was performed taking into account the slice thickness. Displayed is the average plot profile.

Supplementary Figure 6 Activity-driven calcium events were correlated to action potential firing of individual neurons.

a) Cell numbers within networks in WT and postnatal (P0-P7) Fmr1−/− brains (values are mean ± SEM; ANOVA: age 0 (P < 0,0001), genotype (p = 0.64), genotype*age interaction (p = 0.504) F(3,88)=8,618, n = 21 pus and 53 slices from WT and n = 21 pups and 44 slices from Fmr1−/− animals) b) Individual events from sample traces during aCSF baseline and following TTX application. Grey bar indicates events in aCSF condition. c) Cell-attached patch recording showing correlated synchronization of events to AP spiking in cell and neighbor. d) Raster plot for corresponding slice showing network activity across all cells for baseline and TTX perfusion in 4 min recording blocks. e) TTX blockage effect on cell activity across the network.

Supplementary Figure 7 Representative Western blotting.

a) Representative Western blotting (entire membrane) showing N-cadherin expression in E14.5 embryonic somatosensory cortex lysates from WT and Fmr1−/− mice. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been used as the internal control for normalization. This entire gel refers to the experiment described in Figure 4a. b) Representative Western blotting (entire membrane) showing Fmrp immunoprecipitation performed from E17 WT and Fmr1−/− cortical extracts. This entire gel refers to the experiment described in Figure 4b.

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La Fata, G., Gärtner, A., Domínguez-Iturza, N. et al. FMRP regulates multipolar to bipolar transition affecting neuronal migration and cortical circuitry. Nat Neurosci 17, 1693–1700 (2014). https://doi.org/10.1038/nn.3870

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