技术资料

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions,such as learning and memory. To assess the behavioral importance of adult-born neurons,we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice,the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter,which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test),we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning,as well as reversal learning,but are not necessary for the retrieval of stored long-term memories. Importantly,the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together,these findings highlight the potential of stimulating neurogenesis as a means to enhance learning. View Publication

Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells,including the ability to self-renew and differentiate,commonly referred to as stemness. In addition,such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs),transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin,revealed that NSC-proximal GICs were more sensitive,corroborating the transcriptome data. Furthermore,Ca2+ drug sensitivity was reduced in GICs after differentiation,with most potent effect in the NSC-proximal GIC,supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium,sodium and Ca2+. Conversely,a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+,were expressed in NSC-distal GICs. In particular,expression of the AMPA glutamate receptor subunit GRIA1,was found to associate with Ca2+ sensitive NSC-proximal GICs,and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP,brain lipid-binding protein) in this extended analysis. In summary,NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis,providing a potential target mechanism for eradication of an immature population of malignant cells. View Publication

Hypoxia is a near-universal feature of cancer,promoting glycolysis,cellular proliferation,and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied,but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model,we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2,the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)--a critical component of hypoxic signaling and a compelling cancer drug target--is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2,but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier's principle,as well as through a steady-state kinetic model of protein interactions,both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental,thermodynamics-motivated principles. View Publication

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks,yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1,the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination. View Publication

A network of transcription factors (TFs) determines cell identity,but identity can be altered by overexpressing a combination of TFs. However,choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of 2,000 TFs. Here,we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs-Myod1,Mef2c,Esx1,Foxa1,Hnf4a,Gata2,Gata3,Myc,Elf5,Irf2,Elf1,Sfpi1,Ets1,Smad7,Nr2f1,Sox11,Dmrt1,Sox9,Foxg1,Sox2,or Ascl1-can direct efficient,specific,and rapid differentiation into myocytes,hepatocytes,blood cells,and neurons. Furthermore,transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation,and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates. View Publication

Because neurons are difficult to obtain from humans,generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells,we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation,glutamate receptor function,and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2,resulting in high responsiveness of neural cells to glutamate,N-methyl-d-aspartate (NMDA),α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA),and (S)-3,5-d... View Publication

Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact,we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG). Still,little is known regarding its effect on DG stem cell properties. Herein,we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10nM) decreased DG stem cell self-renewal,while 1nM delayed cell cycle in the G0/G1-to-S phase transition and increased the number of quiescent cells (G0 phase),which correlated with a decrease in cyclin E,pEGFR and pERK1/2 protein levels. Importantly,both drug concentrations (1 or 10nM) did not induce cell death. In accordance with the impairment of self-renewal capacity,METH (10nM) decreased Sox2(+)/Sox2(+) while increased Sox2(-)/Sox2(-) pairs of daughter cells. This effect relied on N-methyl-d-aspartate (NMDA) signaling,which was prevented by the NMDA receptor antagonist,MK-801 (10μM). Moreover,METH (10nM) increased doublecortin (DCX) protein levels consistent with neuronal differentiation. In conclusion,METH alters DG stem cell properties by delaying cell cycle and decreasing self-renewal capacities,mechanisms that may contribute to DG neurogenesis impairment followed by cognitive deficits verified in METH consumers. View Publication

Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders,including prion and Alzheimer's diseases. However,how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons,a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells,ΔCR PrP induces large,ionic currents that can be detected by patch-clamping techniques. Here,we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced,calcium-mediated death. In combination with ultrastructural and biochemical analyses,these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic,β-rich oligomers that bind to PrP(C). View Publication

OBJECTIVE Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines,suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique,here we report the characterization of GBM cell lines including GBM variants. METHODS Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. RESULTS Thirteen oncosphere lines derived from GBM and GBM variants,including a GBM with PNET features and a GBM with oligodendroglioma component,were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas,including invasion,necrosis,abnormal vascularization,and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. CONCLUSIONS We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study. View Publication

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells,and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse,we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality. View Publication

BACKGROUND In glioblastoma (GBM),Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance. METHODS Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2),a stabilized form of PGE2,to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1,a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting,quantitative real-time (qRT)-PCR,and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM. RESULTS In GBM cells,dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads,in turn,to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage,which are dependent on Id1. CONCLUSIONS In GBM,Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively,these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients. View Publication

BACKGROUND Transplantation of enteric neural stem cells (ENSC) holds promise as a potential therapy for enteric neuropathies,including Hirschsprung disease. Delivery of transplantable cells via laparotomy has been described,but we propose a novel,minimally invasive endoscopic method of cell delivery. METHODS Enteric neural stem cells for transplantation were cultured from dissociated gut of postnatal donor mice. Twelve recipient mice,including Ednrb(-/-) mice with distal colonic aganglionosis,underwent colonoscopic injection of ENSC under direct vision using a 30-gauge Hamilton needle passed through a rigid cystoureteroscope. Cell engraftment,survival,and neuroglial differentiation were studied 1-4 weeks after the procedure. KEY RESULTS All recipient mice tolerated the procedure without complications and survived to sacrifice. Transplanted cells were found within the colonic wall in 9 of 12 recipient mice with differentiation into enteric neurons and glia. CONCLUSIONS & INFERENCES Endoscopic injection of ENSC is a safe and reliable method for cell delivery,and can be used to deliver a large number of cells to a specific area of disease. This minimally invasive endoscopic approach may prove beneficial to future human applications of cell therapy for neurointestinal disease. View Publication