技术资料

Memantine is classified as an NMDA receptor antagonist. We recently reported that memantine promoted the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus. However,the molecular mechanism responsible for the memantine-induced promotion of cellular proliferation remains unknown. In this study we searched for a factor that mediates memantine-induced cellular proliferation,and found that pigment epithelium-derived factor (PEDF),a broad-acting neurotrophic factor,is up-regulated in the dentate gyrus of adult mice after the injection of memantine. PEDF mRNA expression increased significantly by 3.5-fold at 1 day after the injection of memantine. In addition,the expression level of PEDF protein also increased by 1.8-fold at 2 days after the injection of memantine. Immunohistochemical study using anti-PEDF antibody showed that the majority of the PEDF-expressing cells were protoplasmic and perivascular astrocytes. Using a neurosphere assay,we confirmed that PEDF enhanced cellular proliferation under the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) but was not involved in the multilineage potency of hippocampal progenitor cells. Over expression of PEDF by adeno-associated virus,however,did not stimulate cellular proliferation,suggesting PEDF per se does not promote cellular proliferation in vivo. These findings suggest that the memantine induced PEDF up-regulation is involved in increased proliferation of hippocampal progenitor cells. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor and is associated with poor survival. Recently,stem-like cell populations have been identified in numerous malignancies including GBM. To identify genes whose expression is changed with differentiation,we compared transcript profiles from a GBM oncosphere line before and after differentiation. Bioinformatic analysis of the gene expression profiles identified podocalyxin-like protein (PODXL),a protein highly expressed in human embryonic stem cells,as a potential marker of undifferentiated GBM stem-like cells. The loss of PODXL expression upon differentiation of GBM stem-like cell lines was confirmed by quantitative real-time PCR and flow cytometry. Analytical flow cytometry of numerous GBM oncosphere lines demonstrated PODXL expression in all lines examined. Knockdown studies and flow cytometric cell sorting experiments demonstrated that PODXL is involved in GBM stem-like cell proliferation and oncosphere formation. Compared to PODXL-negative cells,PODXL-positive cells had increased expression of the progenitor/stem cell markers Musashi1,SOX2,and BMI1. Finally,PODXL expression directly correlated with increasing glioma grade and was a marker for poor outcome in patients with GBM. In summary,we have demonstrated that PODXL is expressed in GBM stem-like cells and is involved in cell proliferation and oncosphere formation. Moreover,high PODXL expression correlates with increasing glioma grade and decreased overall survival in patients with GBM. View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

Nature Neuroscience 17,1180 (2014). doi:10.1038/nn.3787 View Publication

Herein,we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors,decreased stemness,and increased proneurogenic gene expression. Importantly,this work reports for the first time a nanoparticle formulation able to modulate in vivo the subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases. View Publication

INTRODUCTION Despite attempts to prevent brain injury during the hyperacute phase of stroke,most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study,we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ),and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. METHODS Pre-differentiated GABAergic neurons,undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival,cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. RESULTS Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype,showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days,suggesting an additional trophic role of these GABAergic cells. In contrast,undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. CONCLUSION Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery. View Publication

BACKGROUND Glioblastoma (GBM) is poorly responsive to current chemotherapy. The nuclear transporter exportin 1 (XPO1,CRM1) is often highly expressed in GBM,which may portend a poor prognosis. Here,we determine the efficacy of novel selective inhibitors of nuclear export (SINE) specific to XPO1 in preclinical models of GBM. METHODS Seven patient-derived GBM lines were treated with 3 SINE compounds (KPT-251,KPT-276,and Selinexor) in neurosphere culture conditions. KPT-276 and Selinexor were also evaluated in a murine orthotopic patient-derived xenograft (PDX) model of GBM. Cell cycle effects were assayed by flow cytometry in vitro and immunohistochemistry in vivo. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase 3/7 activity assays. RESULTS Treatment of GBM neurosphere cultures with KPT-276,Selinexor,and KPT-251 revealed dose-responsive growth inhibition in all 7 GBM lines [range of half-maximal inhibitory concentration (IC50),6-354 nM]. In an orthotopic PDX model,treatment with KPT-276 and Selinexor demonstrated pharmacodynamic efficacy,significantly suppressed tumor growth,and prolonged animal survival. Cellular proliferation was not altered with SINE treatment. Instead,induction of apoptosis was apparent both in vitro and in vivo with SINE treatment,without overt evidence of neurotoxicity. CONCLUSIONS SINE compounds show preclinical efficacy utilizing in vitro and in vivo models of GBM,with induction of apoptosis as the mechanism of action. Selinexor is now in early clinical trials in solid and hematological malignancies. Based on these preclinical data and excellent brain penetration,we have initiated clinical trials of Selinexor in patients with relapsed GBM. View Publication

PURPOSE p53 pathway alterations are key molecular events in glioblastoma (GBM). MDM2 inhibitors increase expression and stability of p53 and are presumed to be most efficacious in patients with TP53 wild-type and MDM2-amplified cancers. However,this biomarker hypothesis has not been tested in patients or patient-derived models for GBM. EXPERIMENTAL DESIGN We performed a preclinical evaluation of RG7112 MDM2 inhibitor,across a panel of 36 patient-derived GBM cell lines (PDCL),each genetically characterized according to their P53 pathway status. We then performed a pharmacokinetic (PK) profiling of RG7112 distribution in mice and evaluated the therapeutic activity of RG7112 in orthotopic and subcutaneous GBM models. RESULTS MDM2-amplified PDCLs were 44 times more sensitive than TP53-mutated lines that showed complete resistance at therapeutically attainable concentrations (avg. IC50 of 0.52 μmol/L vs. 21.9 μmol/L). MDM4-amplified PDCLs were highly sensitive but showed intermediate response (avg. IC50 of 1.2 μmol/L),whereas response was heterogeneous in TP53 wild-type PDCLs with normal MDM2/4 levels (avg. IC50 of 7.7 μmol/L). In MDM2-amplified lines,RG7112 restored p53 activity inducing robust p21 expression and apoptosis. PK profiling of RG7112-treated PDCL intracranial xenografts demonstrated that the compound significantly crosses the blood-brain and the blood-tumor barriers. Most importantly,treatment of MDM2-amplified/TP53 wild-type PDCL-derived model (subcutaneous and orthotopic) reduced tumor growth,was cytotoxic,and significantly increased survival. CONCLUSIONS These data strongly support development of MDM2 inhibitors for clinical testing in MDM2-amplified GBM patients. Moreover,significant efficacy in a subset of non-MDM2-amplified models suggests that additional markers of response to MDM2 inhibitors must be identified. View Publication

The enteric nervous system (ENS) is composed of neural crest-derived neurons (also known as ganglion cells) the cell bodies of which are located in the submucosal and myenteric plexuses of the intestinal wall. Intramucosal ganglion cells are known to exist but are rare and often considered ectopic. Also derived from the neural crest are enteric glial cells that populate the ganglia and the associated nerves,as well as the lamina propria of the intestinal mucosa. In Hirschsprung disease (HSCR),ganglion cells are absent from the distal gut because of a failure of neural crest-derived progenitor cells to complete their rostrocaudal migration during embryogenesis. The fate of intramucosal glial cells in human HSCR is essentially unknown. We demonstrate a network of intramucosal cells that exhibit dendritic morphology typical of neurons and glial cells. These dendritic cells are present throughout the human gut and express Tuj1,S100,glial fibrillary acidic protein,CD56,synaptophysin,and calretinin,consistent with mixed or overlapping neuroglial differentiation. The cells are present in aganglionic colon from patients with HSCR,but with an altered immunophenotype. Coexpression of Tuj1 and HNK1 in this cell population supports a neural crest origin. These findings extend and challenge the current understanding of ENS microanatomy and suggest the existence of an intramucosal population of neural crest-derived cells,present in HSCR,with overlapping immunophenotype of neurons and glia. Intramucosal neuroglial cells have not been previously recognized,and their presence in HSCR poses new questions about ENS development and the pathobiology of HSCR that merit further investigation. View Publication

The vast majority of pedigrees with familial Alzheimer's disease (FAD) are caused by inheritance of mutations in the PSEN1 1 gene. While genetic ablation studies have revealed a role for presenilin 1 (PS1) in embryonic neurogenesis,little information has emerged regarding the potential effects of FAD-linked PS1 variants on proliferation,self-renewal and differentiation,key events that control cell fate commitment of adult brain neural progenitors (NPCs). We used adult brain subventricular zone (SVZ)-derived NPC cultures transduced with recombinant lentivirus as a means to investigate the effects of various PS1 mutants on self-renewal and differentiation properties. We now show that viral expression of several PS1 mutants in NPCs leads to impaired self-renewal and altered differentiation toward neuronal lineage,in vitro. In line with these observations,diminished constitutive proliferation and steady-state SVZ progenitor pool size was observed in vivo in transgenic mice expressing the PS1DeltaE9 variant. Moreover,NPC cultures established from the SVZ of adult mice expressing PS1DeltaE9 exhibit reduced self-renewal capacity and premature exit toward neuronal fates. To these findings,we show that both the levels of endogenous Notch/CBF-1-transcriptional activity and transcripts encoding Notch target genes are diminished in SVZ NPCs expressing PS1DeltaE9. The deficits in self-renewal and multipotency are restored by expression of Notch1-ICD or a downstream target of the Notch pathway,Hes1. Hence,we argue that a partial reduction in PS-dependent gamma-secretase processing of the Notch,at least in part,accounts for the impairments observed in SVZ NPCs expressing the FAD-linked PS1DeltaE9 variant. View Publication