Elsevier

Neurobiology of Disease

Volume 33, Issue 2, February 2009, Pages 250-259
Neurobiology of Disease

Aberrant differentiation of glutamatergic cells in neocortex of mouse model for fragile X syndrome

https://doi.org/10.1016/j.nbd.2008.10.010Get rights and content

Abstract

The lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. Our previous studies revealed alterations in the differentiation of FMRP-deficient neural progenitors. Here, we show abnormalities in neurogenesis in the mouse and human embryonic FMRP-deficient brain as well as after in utero transfection of I304N mutated FMRP, which acts in a dominant negative manner in the wild-type mouse brain. Progenitors accumulated abnormally in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) mouse neocortex. An increased density of cells expressing sequentially an intermediate progenitor marker, T-box transcription factor (Tbr2), and a postmitotic neuron marker, T-brain 1 (Tbr1), indicated that the differentiation to glutamatergic cell lineages was particularly disturbed. These abnormalities were associated with an increased density of pyramidal cells of the layer V in the early postnatal neocortex suggesting a role for FMRP in the regulation of the differentiation of neocortical glutamatergic neurons.

Introduction

Fragile X syndrome (FXS) is a common cause of inherited intellectual and developmental disabilities. Most FXS individuals have an expanded CGG triplet repeat mutation in the fragile X mental retardation 1 (FMR1) gene that reduces FMR1 protein (FMRP) expression (Penagarikano et al., 2007). FMRP is an RNA-binding protein which associates with actively translating polysomes and plays a role in the localization, stabilization and translation of certain neuronal mRNAs (Bagni and Greenough, 2005, Zalfa et al., 2007). Several RNA-binding motifs, including the RGG box, KH domains and an amino acid sequence in N-terminus, contribute to the function of FMRP independently (Schaeffer et al., 2001, Adinolfi et al., 2003, Darnell et al., 2005a). The second KH domain (KH2) of FMRP interacts with a loop–loop pseudoknot RNA structure (Darnell et al., 2005a). An I304N mutation in the KH2 domain abrogates the ability of FMRP to bind to polysomes and act as a translational regulator of a subset of mRNAs (Feng et al., 1997, Laggerbauer et al., 2001, Schrier et al., 2004).

The mouse model generated for FXS, Fmr1-knockout (Fmr1-KO) mouse, exhibits a phenotype resembling the symptoms of the actual human disorder, including a learning defect and macro-orchidism (The Dutch-Belgian Fragile X Consortium, 1994, D'Hooge et al., 1997, Van Dam et al., 2000). Both, the human and mouse FXS cortices display pyramidal neurons with an abnormally high density of morphologically immature dendritic spines (Comery et al., 1997, Irwin et al., 2001, Nimchinsky et al., 2001, Galvez and Greenough, 2005). Recently, alterations in the organization of inhibitory interneurons in the neocortex of adult Fmr1-KO mice were reported (Selby et al., 2007); however, maturation of cells in glutamatergic lineages in FXS has not been examined extensively.

Our previous studies have shown that FMRP-deficient neural progenitor cells (NPCs) generate more cells responsive to metabotropic glutamate receptor (mGluR) activation and an increased number of neurons with morphological alterations when compared to wild-type (WT) controls (Castrén et al., 2005, Castrén, 2006). Here, we investigated how altered differentiation of NPCs lacking functional FMRP influences neocortex formation (Götz and Huttner, 2005, Molyneaux et al., 2007). The findings of this study show a significant accumulation of progenitor cells in the developing subventricular zone (SVZ) of the embryonic brain of Fmr1-KO mouse and in the embryonic WT mouse brain after in utero electroporation by FMRP carrying a missense point mutation I304N in the KH2 domain. The abnormal cell population contributed to changes in glutamatergic neurogenesis and was associated with layer specific alterations in the postnatal brain of Fmr1-KO mouse.

Section snippets

Animals

We used Fmr1-KO mice and their WT littermates with FVB background at embryonic and early postnatal age as indicated in each experiment. The Fmr1 gene was mutated by inserting a neomycin cassette into the exon 5. The Fmr1-KO mice were genotyped by PCR from tissue samples with previously described primers (The Dutch-Belgian Fragile X Consortium, 1994). Transgene delivery by in utero electroporation was performed to WT NMRI mice. All animal experiments were performed according to the guidelines of

Altered neurogenesis in FXS brain in vivo

We showed previously that NPCs lacking FMRP generate more neurons than WT NPCs and that this in vitro finding correlates with an increase in the production of new neuronal cells in the embryonic brain of the Fmr1-KO mouse (Castrén et al., 2005). Here, we examined the effects of interfering with FMRP function by transient transfection of FMRP with a substitution of Ile  Asn at the amino acid position 304 of the KH2 domain. FMRP with the I304N mutation sequesters WT FMRP, as well as its target

Discussion

The present findings show that compromised function of FMRP after transient transfection of I304N mutated FMRP to WT mouse brains as well as the absence of FMRP in the brains of Fmr1-KO mice and human FXS fetus resulted in an accumulation of newborn cells in the embryonic SVZ suggesting impairments in the differentiation and migration of neocortical cells. The data are in agreement with previous findings showing NPC alterations in cell cultures in vitro (Castrén et al., 2005; Bhattacharyya et

Acknowledgments

We are very grateful to Dr. Xufei Wu and Juha Knuuttila for their help in animal experiments, and Erja Huttu and Anna-Lisa Gidlund for their skillful technical assistance. We thank Dr. Magdalena Götz for providing the radial glial markers and Dr. Sarah Coleman for the critical comments on the manuscript. This work was supported by grants from the Arvo and Lea Ylppö Foundation, the University of Helsinki and the Academy of Finland.

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      Loss of FMRP in Fmr1 KO mice results in preferential differentiation of RG cells to intermediate progenitor cells resulting in increased presence of Tbr2 + IPCs in the embryonic cortex. The exact mechanism that drives this perturbation is unclear, but there is evidence that cultured neural progenitor cells from Fmr1 KO mice exhibit impaired interkinetic nuclear migration, which is an important determinant of differentiation and proliferation in the neural stem cell population (Achuta et al., 2014; Castrén, 2016; Tervonen et al., 2009). The perturbations to neuronal function and development result in clear behavioral abnormalities in the Fmr1 knockout mice (Santos et al., 2014).

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