Fragile X Mental Retardation Protein Regulates Proliferation and Differentiation of Adult Neural Stem/Progenitor Cells

Abstract

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA–binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs). We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3β. Dysregulation of GSK3β led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.

Molecular Mechanisms Regulating Impaired Neurogenesis of Fragile X Syndrome Human Embryonic Stem Cells

“…To test this hypothesis, we knocked down SOX2 expression and overexpressed SOX9 in FX-hNPCs (Fig. 5C) and measured their neural status by analyzing the relative expression of GFAP and MAP2, as previously described [17,28,44]. Our results show that SOX2 inhibition and SOX9 overexpression in FX-hNPCs resulted in a significant increase in both MAP2 and GFAP levels, reflecting a progression of these hNPCs from a primitive to a more advanced neural status.…”

“…A major finding in FXS research based on murine models is the involvement of GSK3b, a kinase that plays key roles in several molecular pathways, including the canonical Wnt/ b-catenin signaling [26,28]. Specifically, it has been found that the absence of FMRP leads to an abnormal increase in GSK3b protein levels.…”

“…A major finding in brain samples of Fmr1 -/-mice is the abnormally high levels of GSK3b, a key kinase shown to play critical roles in several molecular pathways, including MAPK, Cyclin, Akt, and the canonical Wnt/b-catenin signaling pathway [26,[45][46][47]. The role of GSK3b, as part of the canonical Wnt/bcatenin signaling pathway, was shown to be important during adult neurogenesis in the murine hippocampus [29] and in FXS pathology in Fmr1 -/-mice [27,28]. Ablation of FMRP in aNSCs leads to an increase in GSK3b protein levels, causing a phosphorylation-dependent decrease in bcatenin activation and an increase in b-catenin degradation, impairing neuronal differentiation.…”

“…We thus subjected hNPCs to 30 days of neuronal differentiation in the presence of Wnt signaling modulators: a GSK3b inhibitor (CHIR99021; ''CHIR''), which reduces GSK3b-mediated phosphorylation of b-catenin, thus increasing Wnt/b-catenin activity [48], and a specific tankyrase inhibitor (XAV939, ''XAV''), which stabilizes Axin levels in the cells, thus decreasing Wnt/b-catenin activity in a GSK3b-independent manner [49]. The MAP2/GFAP bioassay was implemented to measure the efficiency of neuronal neurogenesis, as has been done previously by us and others [17,28,44]. In control cultures (ie, no CHIR or XAV added), the efficiency of neuronal differentiation was significantly lower for FX lines than for HUES lines, as expected (Fig.…”

“…Studies conducted on Fmr1 -/-mice have consistently shown that lack of FMRP results in an abnormal increase in glycogen synthase kinase 3b (GSK3b) mRNA and protein levels [26]. Although GSK3b plays key roles in several molecular pathways, it has been proposed that its involvement in FXS neuropathology is mediated through the canonical Wnt/b-catenin signaling pathway [27,28], a critical signaling pathway for embryonic neural development as well as for adult neurogenesis [29]. These studies showed that ablation of FMRP in vivo reduced the capacity of murine adult neural stem cells (aNSCs) to differentiate into hippocampal neurons because of an increase in GSK3b and a consequent reduction in b-catenin and its downstream neuronal transcription factors, reducing neuronal yield and increasing the relative number of glia cells in the hippocampus.…”