技术资料

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here,we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice,emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro,gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly,hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly,treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D,which,like gelsolin,produces actin disassembly,decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly,depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore,increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels,increased regional cerebral blood flow,and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together,reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and,subsequently,elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis. View Publication

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine,levels of which are modulated by stress,depression and clinical antidepressants. However,studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors,including stem cells,from the hippocampus of adult mice. Mechanistically,we provide evidence that beta(3)-adrenergic receptors,which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ),mediate this norepinephrine-dependent activation. Moreover,intrahippocampal injection of a selective beta(3)-adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly,systemic injection of the beta-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally,using a novel ex vivo slice-sphere" assay that maintains an intact neurogenic niche� View Publication

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

The tyrosine kinase Fyn plays a key role in oligodendrocyte differentiation and myelination in the central nervous system,but the molecules responsible for regulating Fyn activation in these processes remain poorly defined. Here we show that receptor-like protein-tyrosine phosphatase alpha (PTPalpha) is an important positive regulator of Fyn activation and signaling that is required for the differentiation of oligodendrocyte progenitor cells (OPCs). PTPalpha is expressed in OPCs and is up-regulated during differentiation. We used two model systems to investigate the role of PTPalpha in OPC differentiation: the rat CG4 cell line where PTPalpha expression was silenced by small interfering RNA,and oligosphere-derived primary OPCs isolated from wild-type and PTPalpha-null mouse embryos. In both cell systems,the ablation of PTPalpha inhibited differentiation and morphological changes that accompany this process. Although Fyn was activated upon induction of differentiation,the level of activation was severely reduced in cells lacking PTPalpha,as was the activation of Fyn effector molecules focal adhesion kinase,Rac1,and Cdc42,and inactivation of Rho. Interestingly,another downstream effector of Fyn,p190RhoGAP,which is responsible for Rho inactivation during differentiation,was not affected by PTPalpha ablation. In vivo studies revealed defective myelination in the PTPalpha(-/-) mouse brain. Together,our findings demonstrate that PTPalpha is a critical regulator of Fyn activation and of specific Fyn signaling events during differentiation,and is essential for promoting OPC differentiation and central nervous system myelination. View Publication

Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However,the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However,complete genetic loss,which only occurs when p53 is inactivated,mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors,which consequently impacts therapeutic sensitivity. View Publication

The understanding of the function of alpha(1)-adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant alpha(1)-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing alpha(1A)-adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult alpha(1A)-adrenergic receptor transgenic mice or normal mice given the alpha(1A)-adrenergic receptor-selective agonist,cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of alpha(1)-adrenergic receptor subtypes,and stimulation of these receptors resulted in increased expression of the alpha(1B)-adrenergic receptor subtype,proneural basic helix-loop-helix transcription factors,and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. alpha(1)-Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However,in adult normal neurospheres,alpha(1)-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo,S100-positive glial and betaIII tubulin neuronal progenitors colocalized with either alpha(1)-adrenergic receptor subtype in the olfactory bulb. Our results indicate that alpha(1)-adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases. View Publication

Glioblastoma,the most malignant type of primary brain tumor,is one of the solid cancers where cancer stem cells have been isolated,and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here,we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2,varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC),we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV,the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However,this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-),gamma34.5(-),alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly,despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs,intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs,and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover,the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis. View Publication

A commonly activated signaling cascade in many human malignancies,including glioblastoma multiforme,is the Akt pathway. This pathway can be activated via numerous upstream alterations including genomic amplification of epidermal growth factor receptor,PTEN deletion,or PIK3CA mutations. In this study,we screened phosphatidylinositol 3-kinase/Akt small-molecule inhibitors in an isogenic cell culture system with an activated Akt pathway secondary to a PIK3CA mutation. One small molecule,A-443654,showed the greatest selective inhibition of cells with the mutant phenotype. Based on these findings,this inhibitor was screened in vitro against a panel of glioblastoma multiforme cell lines. All cell lines tested were sensitive to A-443654 with a mean IC(50) of approximately 150 nmol/L. An analogue of A-443654,methylated at a region that blocks Akt binding,was on average 36-fold less active. Caspase assays and dual flow cytometric analysis showed an apoptotic mechanism of cell death. A-443654 was further tested in a rat intracranial model of glioblastoma multiforme. Animals treated intracranially with polymers containing A-443654 had significantly extended survival compared with control animals; animals survived 79% and 43% longer than controls when A-443654-containing polymers were implanted simultaneously or in a delayed fashion,respectively. This small molecule also inhibited glioblastoma multiforme stem-like cells with similar efficacy compared with traditionally cultured glioblastoma multiforme cell lines. These results suggest that local delivery of an Akt small-molecule inhibitor is effective against experimental intracranial glioma,with no observed resistance to glioblastoma multiforme cells grown in stem cell conditions. View Publication

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone,muscle,endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin,neurofilament,A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4,SCF,Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase,GalC,Olig2 and the late-stage marker MOSP are observed,as is the Schwann cell marker PMP22. In summary,Lin(neg) UCB cells,when appropriately cultured,are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation. View Publication

Toll-like receptors (TLRs) play important roles in innate immunity. Several TLR family members have recently been shown to be expressed by neurons and glial cells in the adult brain,and may mediate responses of these cells to injury and infection. To address the possibility that TLRs play a functional role in development of the nervous system,we analyzed the expression of TLRs during different stages of mouse brain development and assessed the role of TLRs in cell proliferation. TLR3 protein is present in brain cells in early embryonic stages of development,and in cultured neural stem/progenitor cells (NPC). NPC from TLR3-deficient embryos formed greater numbers of neurospheres compared with neurospheres from wild-type embryos. Numbers of proliferating cells,as assessed by phospho histone H3 and proliferating cell nuclear antigen labeling,were also increased in the developing cortex of TLR3-deficient mice compared with wild-type mice in vivo. Treatment of cultured embryonic cortical neurospheres with a TLR3 ligand (polyIC) significantly reduced proliferating (BrdU-labeled) cells and neurosphere formation in wild type but not TLR3(-/-)-derived NPCs. Our findings reveal a novel role for TLR3 in the negative regulation of NPC proliferation in the developing brain. View Publication

BAG-1 protein has been well characterized as necessary for proper neuronal development. However,little is known about the function of BAG-1 in the adult brain. In this work,the expression and localization of BAG-1 in the mature mouse brain was studied. The levels of both BAG-1 isoforms decrease significantly in the brain during development. BAG-1 was found preferentially expressed in Neuronal Precursor Cells (NPCs) in the two major niches of neurogenesis. Lentiviral mediated overexpression of BAG-1 increased the proliferation rate of cultured NPCs. In addition,depletion of BAG-1 from NPCs induced a decrease in NPCs proliferation in the presence of a stress hormone,corticosterone. These data suggest a role for BAG-1 in mechanisms of neurogenesis in the adult mouse brain. View Publication

The recent identification of cancer stem cells (CSCs) in multiple human cancers provides a new inroad to understanding tumorigenesis at the cellular level. CSCs are defined by their characteristics of self-renewal,multipotentiality,and tumor initiation upon transplantation. By testing for these defining characteristics,we provide evidence for the existence of CSCs in a transgenic mouse model of glioma,S100beta-verbB;Trp53. In this glioma model,CSCs are enriched in the side population (SP) cells. These SP cells have enhanced tumor-initiating capacity,self-renewal,and multipotentiality compared with non-SP cells from the same tumors. Furthermore,gene expression analysis comparing fluorescence-activated cell sorting-sorted cancer SP cells to non-SP cancer cells and normal neural SP cells identified 45 candidate genes that are differentially expressed in glioma stem cells. We validated the expression of two genes from this list (S100a4 and S100a6) in primary mouse gliomas and human glioma samples. Analyses of xenografted human glioblastoma multiforme cell lines and primary human glioma tissues show that S100A4 and S100A6 are expressed in a small subset of cancer cells and that their abundance is positively correlated to tumor grade. In conclusion,this study shows that CSCs exist in a mouse glioma model,suggesting that this model can be used to study the molecular and cellular characteristics of CSCs in vivo and to further test the CSC hypothesis. View Publication