技术资料

In vitro exposure of neural progenitor cell (NPC) populations to reduced O(2) (e.g. 3% versus 20%) can increase their proliferation,survival and neuronal differentiation. Our objective was to determine if an acute (textless1hr),in vivo exposure to intermittent hypoxia (AIH) alters expansion and/or differentiation of subsequent in vitro cultures of NPC from the subventricular zone (SVZ). Neonatal C57BL/6 mice (postnatal day 4) were exposed to an AIH paradigm (20×1 minute; alternating 21% and 10% O(2)). Immediately after AIH,SVZ tissue was isolated and NPC populations were cultured and assayed either as neurospheres (NS) or as adherent monolayer cells (MASC). AIH markedly increased the capacity for expansion of cultured NS and MASC,and this was accompanied by increases in a proliferation maker (Ki67),MTT activity and hypoxia-inducible factor-1α (HIF-1α) signaling in NS cultures. Peptide blockade experiments confirmed that proteins downstream of HIF-1α are important for both proliferation and morphological changes associated with terminal differentiation in NS cultures. Finally,immunocytochemistry and Western blotting experiments demonstrated that AIH increased expression of the neuronal fate determination transcription factor Pax6 in SVZ tissue,and this was associated with increased neuronal differentiation in cultured NS and MASC. We conclude that in vivo AIH exposure can enhance the viability of subsequent in vitro SVZ-derived NPC cultures. AIH protocols may therefore provide a means to prime" NPC prior to transplantation into the injured central nervous system." View Publication

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice,while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover,gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice,resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells,and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline. View Publication

The identification of succinate dehydrogenase (SDH),fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate,respectively,which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes,including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2,which catalyse the interconversion of isocitrate and 2-OG,are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG,but not (S)-2HG,stimulates EGLN activity,leading to diminished HIF levels,which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy. View Publication

Within high-grade gliomas,the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche,ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis,we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity,while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly,Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further,eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival,while knockdown of Olig2 within Id1(low) cells has a significant survival benefit,underscoring the importance of non-self-renewing lineages in disease progression. View Publication

BACKGROUND: Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo,where activity of inputs can be differentially regulated,but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here,we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. METHODOLOGY/PRINCIPAL FINDINGS: GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons,both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures,synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. CONCLUSIONS/SIGNIFICANCE: The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde 'reward' signal generated by WT neurons,although in this paradigm there was no 'punishment' signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system. 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

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

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

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

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients,the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg,which are involved in the activation of Fanconi pathway,in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors,but not that of postmitotic neurons,was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice,we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition,embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis. View Publication

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei,suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs,cumulus cells,Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However,the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%),but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g.,85% in Sertoli cells and 75% in cumulus cells),the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g.,50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type. View Publication

Slow-onset adaptive changes that arise from sustained antidepressant treatment,such as enhanced adult hippocampal neurogenesis and increased trophic factor expression,play a key role in the behavioral effects of antidepressants. alpha(2)-Adrenoceptors contribute to the modulation of mood and are potential targets for the development of faster acting antidepressants. We investigated the influence of alpha(2)-adrenoceptors on adult hippocampal neurogenesis. Our results indicate that alpha(2)-adrenoceptor agonists,clonidine and guanabenz,decrease adult hippocampal neurogenesis through a selective effect on the proliferation,but not the survival or differentiation,of progenitors. These effects persist in dopamine beta-hydroxylase knock-out (Dbh(-/-)) mice lacking norepinephrine,supporting a role for alpha(2)-heteroceptors on progenitor cells,rather than alpha(2)-autoreceptors on noradrenergic neurons that inhibit norepinephrine release. Adult hippocampal progenitors in vitro express all the alpha(2)-adrenoceptor subtypes,and decreased neurosphere frequency and BrdU incorporation indicate direct effects of alpha(2)-adrenoceptor stimulation on progenitors. Furthermore,coadministration of the alpha(2)-adrenoceptor antagonist yohimbine with the antidepressant imipramine significantly accelerates effects on hippocampal progenitor proliferation,the morphological maturation of newborn neurons,and the increase in expression of brain derived neurotrophic factor and vascular endothelial growth factor implicated in the neurogenic and behavioral effects of antidepressants. Finally,short-duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test,which normally requires 3 weeks of treatment with classical antidepressants. Our results demonstrate that alpha(2)-adrenoceptors,expressed by progenitor cells,decrease adult hippocampal neurogenesis,while their blockade speeds up antidepressant action,highlighting their importance as targets for faster acting antidepressants. View Publication