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

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

Proliferation of cultured mouse choroid plexus epithelial cells.

The choroid plexus (ChP) epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF) that bathes and nourishes the central nervous system (CNS). In addition to the CSF, ChP epithelial cells (CPECs) produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration

CPECs proliferate in response to scratch-wound injury.

<p><b>(A-B)</b> Cell proliferation assay (EdU incorporation) with (B) and without (A) administering a scratch to a monolayer of mouse primary CPECs stained with Claudin 1 (Cldn1, green) and pulsed with EdU (red) for 48 hours. Nuclear stain = Hoechst (blue). Scale bar = 100 μm. <b>(C)</b> Percentage of EdU⁺ CPECs quantified from (A-B). Values represent mean±s.e.m. (n = 3 biological replicates each; ***<i>P</i><0.001, Student´s t-test). <b>(D)</b> High magnification phase image of a CPEC in mitosis (anaphase stage) after a scratch (at 1 hr and 12 min from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121738#pone.0121738.s002" target="_blank">S1 Video</a>, demonstrated by two arrowheads). Arrowheads designate the chromosomes and nuclei following sister chromatid separation.</p

Proliferation of Cultured Mouse Choroid Plexus Epithelial Cells

<div><p>The choroid plexus (ChP) epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF) that bathes and nourishes the central nervous system (CNS). In addition to the CSF, ChP epithelial cells (CPECs) produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.</p></div

IGF-1 and EGF promotes CPEC proliferation in whole ChP explant cultures.

<p><b>(A-D)</b> Sequential images of intact ChP tissue derived from Fucci mice treated with IGF-1 and EGF (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121738#pone.0121738.s007" target="_blank">S6 Video</a>). White arrow identifies the same cell across panels. S phase (mAG1 and mKO, orange nuclei) is detected at ~12 hours (A) and G₂/M phases (mAG1, green nuclei) at ~14 hours (B). mAG1 expression is lost after 18 hours (C). After 32 hours, two cells are detected (mKO, two red nuclei) (D).</p

IGF-1 and EGF stimulation drives CPEC proliferation.

<p><b>(A-B)</b> Mouse primary CPECs plated at 150,000 cell/well were treated with EGF alone (A) or IGF-1 and EGF together (B) and pulsed with EdU (red) for 48 hours. Right panels merged with nuclear Hoechst counterstain (blue). <b>(C)</b> Quantification of (A-B) showing percentage of EdU⁺ CPECs treated with IGF-1 and/or EGF. Values represent mean±s.e.m. (n = 3; **<i>P</i><0.01, Student´s t-test). <b>(D)</b> Percentage of EdU⁺ CPECs plated at different cell densities with EGF +/- IGF-1. Values represent mean±s.e.m. (n = 3; black vs. blue bars—***<i>P</i><0.001, Student´s t-test; no significance between black bars).</p

Scratch-wound injury increases CPEC proliferation.

<p>Sequential images (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121738#pone.0121738.s003" target="_blank">S2 Video</a>) of scratch assay on primary CPEC from Fucci mice. <b>(A)</b> White arrow (adjacent to scratch) and yellow arrowhead (within the scratch) show the nuclei of two cells in either S phase (mAG1 and mKO, orange nuclei) or G₂/M phase (mAG1, green nuclei). By 24 hours, the nuclei have divided (mKO, paired red nuclei). Blue lines indicate scratch borders. Scale bar = 100 μm. <b>(B)</b> Magnified images (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121738#pone.0121738.s004" target="_blank">S3 Video</a>) of the cell designated by white arrow in (A), merged with phage contrast images. Scale bar = 50 μm.</p

Identification of astrocyte-expressed factors that modulate neural stem/progenitor cell differentiation.

Multipotent neural stem/progenitor cells (NSPCs) can be isolated from many regions of the adult central nervous system (CNS), yet neurogenesis is restricted to the hippocampus and subventricular zone in vivo. Identification of the molecular cues that modulate NSPC fate choice is a prerequisite for their therapeutic applications. Previously, we demonstrated that primary astrocytes isolated from regions with higher neuroplasticity, such as newborn and adult hippocampus and newborn spinal cord, promoted neuronal differentiation of adult NSPCs, whereas astrocytes isolated from the nonneurogenic region of the adult spinal cord inhibited neural differentiation. To identify the factors expressed by these astrocytes that could modulate NSPC differentiation, we performed gene expression profiling analysis using Affymetrix rat genome arrays. Our results demonstrated that these astrocytes had distinct gene expression profiles. We further tested the functional effects of candidate factors that were differentially expressed in neurogenesis-promoting and -inhibiting astrocytes using in vitro NSPC differentiation assays. Our results indicated that two interleukins, IL-1beta and IL-6, and a combination of factors that included these two interleukins could promote NSPC neuronal differentiation, whereas insulin-like growth factor binding protein 6 (IGFBP6) and decorin inhibited neuronal differentiation of adult NSPCs. Our results have provided further evidence to support the ongoing hypothesis that, in adult mammalian brains, astrocytes play critical roles in modulating NSPC differentiation. The finding that cytokines and chemokines expressed by astrocytes could promote NSPC neuronal differentiation may help us to understand how injuries induce neurogenesis in adult brains