搜索结果: 'NeuroCult Proliferation'

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

The myelination of axons is a crucial step during vertebrate central nervous system (CNS) development,allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly,the differentiation of oligodendrocytes,the myelinating cells of the CNS,and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor,Myelin Regulatory Factor (Myrf),as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial,however,whether Myrf directly regulates transcription,with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly,this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins,the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination. View Publication

BACKGROUND UBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2,a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS,we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H,P497S,and P506T) in primary neuroglial cultures and in developing neonatal mouse brains. RESULTS In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread,sustained expression of ubiquilin-2 in brain. In contrast to wild type,mutant UBQLN2 expression induced significant pathology with large neuronal,cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin,p62/SQSTM,optineurin,and occasionally TDP-43,but were negative for α-synuclein,neurofilament,tau,and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3-4 months,including nonspecific clasping and rotarod deficits. CONCLUSIONS These findings demonstrate that UBQLN2 mutants (P497H,P497S,and P506T) induce proteinopathy and cause behavioral deficits,supporting a toxic" gain-of-function which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration. View Publication

Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are good candidates for cell therapy due to the accessibility of fat tissue and the abundance of AT-MSCs therein. Neurospheres are free-floating spherical condensations of cells with neural stem/progenitor cell (NSPC) characteristics that can be derived from AT-MSCs. The aims of this study were to examine the influence of oxygen (O2) tension on generation of neurospheres from canine AT-MSCs (AT-cMSCs) and to develop a hypoxic cell culture system to enhance the survival and therapeutic benefit of generated neurospheres. AT-cMSCs were cultured under varying oxygen tensions (1%,5% and 21%) in a neurosphere culture system. Neurosphere number and area were evaluated and NSPC markers were quantified using real-time quantitative PCR (qPCR). Effects of oxygen on neurosphere expression of hypoxia inducible factor 1,α subunit (HIF1A) and its target genes,erythropoietin receptor (EPOR),chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF),were quantified by qPCR. Neural differentiation potential was evaluated in 21% O2 by cell morphology and qPCR. Neurospheres were successfully generated from AT-cMSCs at all O2 tensions. Expression of nestin mRNA (NES) was significantly increased after neurosphere culture and was significantly higher in 1% O2 compared to 5% and 21% O2. Neurospheres cultured in 1% O2 had significantly increased levels of VEGF and EPOR. There was a significant increase in CXCR4 expression in neurospheres generated at all O2 tensions. Neurosphere culture under hypoxia had no negative effect on subsequent neural differentiation. This study suggests that generation of neurospheres under hypoxia could be beneficial when considering these cells for neurological cell therapies. View Publication

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36),an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly,TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand,inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. View Publication

In glioblastoma high expression of the CD133 gene,also called Prominin1,is associated with poor prognosis. The PDGF-driven proneural group represents a subset of glioblastoma in which CD133 is not overexpressed. Interestingly,this particular subset shows a relatively good prognosis. As with many other tumors,gliobastoma is believed to arise and be maintained by a restricted population of stem-like cancer cells that express the CD133 transmembrane protein. The significance of CD133(+) cells for gliomagenesis is controversial because of conflicting supporting evidence. Contributing to this inconsistency is the fact that the isolation of CD133(+) cells has largely relied on the use of antibodies against ill-defined glycosylated epitopes of CD133. To overcome this problem,we used a knock-in lacZ reporter mouse,Prom1(lacZ/+),to track Prom1(+) cells in the brain. We found that Prom1 (prominin1,murine CD133 homologue) is expressed by cells that express markers characteristic of the neuronal,glial or vascular lineages. In proneural tumors derived from injection of RCAS-PDGF into the brains of tv-a;Ink4a-Arf(-/-) Prom1(lacZ/+) mice,Prom1(+) cells expressed markers for astrocytes or endothelial cells. Mice co-transplanted with proneural tumor sphere cells and Prom1(+) endothelium had a significantly increased tumor burden and more vascular proliferation (angiogenesis) than those co-transplanted with Prom1(-) endothelium. We also identified specific genes in Prom1(+) endothelium that code for endothelial signaling modulators that were not overexpressed in Prom1(-) endothelium. These factors may support proneural tumor progression and could be potential targets for anti-angiogenic therapy. View Publication

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells,which is partially associated with the induction or repression of genes that influence the ischemic response. However,the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults,we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD),an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD,total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes,whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD,confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins,such as those encoding for PICK1,GRIP1,TARPγ3,calsyntenin-2/3,SAPAP2 and SNAP-25,were down-regulated after OGD. Additionally,OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors,but increased the mRNA expression of the GluN3A subunit,thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together,our results present the expression profile elicited by in vitro ischemia in hippocampal neurons,and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins,suggesting that the synaptic proteome may change after ischemia. View Publication

We describe here a novel method for the determination of cytotoxicity in cell cultures using Fluoro-Jade C (FJ-C). FJ-C has been previously used for the assessment of neurodegeneration in fixed brain tissue samples,and has never been utilized in live cell cultures or in different types of cells other than neurons. In the present study we examined the utility of FJ-C for the determination of cytotoxicity in vitro. Various cell cultures were evaluated including neural stem cells,brain microvessel endothelial cells,and SH-SY5Y,PC12 and MDCK cells. Cytotoxicities induced by toxicants in cell cultures,as determined by the FJ-C labeling,were further confirmed by commonly used cytotoxicity assays. This in vitro approach is simple,fast,and sensitive and,thus,has the potential to augment if not replace currently used cell-based cytotoxicity assays. View Publication

Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system,including a model driven by PDGF-B,H3.3K27M,and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study,an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R,IR,MET,TRKA,TRKB,AURKA,AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM,significant inhibition of proliferation at a concentration of 1.5 μM,as well as inhibition of AKT activation. Interestingly,IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo,systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50,suggesting that inadequate drug delivery may limit in vivo efficacy. In summary,an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG,but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice. View Publication

The present study examined whether nestin+ neural-like stem cells detected in the scar tissue of rats 1 week after myocardial infarction (MI) were derived from bone marrow and/or were resident cells of the normal myocardium. Irradiated male Wistar rats transplanted with beta-actin promoter-driven,green fluorescent protein (GFP)-labeled,unfractionated bone marrow cells were subjected to coronary artery ligation. Three weeks after MI,GFP-labeled bone marrow cells were detected in the infarct region,and a modest number were associated with nestin immunoreactivity. The paucity of GFP+/nestin+ cells in the scar tissue provided the impetus to explore whether neural-like stem cells were derived from cardiac tissue. Nestin mRNA and immunoreactivity were detected in normal rat myocardium,and transcript levels were increased in the damaged heart after MI. In primary-passage,cardiac tissue-derived neural cells,filamentous nestin staining was associated with a diffuse,cytoplasmic glial fibrillary acidic protein signal. Unexpectedly,in viable myocardium,numerous nestin+/glial fibrillary acidic protein+ fiberlike structures of varying length were detected and observed in close proximity to neurofilament-M+ fibers. The infarct region was likewise innervated,and the preponderance of neurofilament-M+ fibers appeared to be physically associated with nestin+ fiberlike structures. These data highlight the novel observation that the normal rat heart contained resident nestin+/glial fibrillary acidic protein+ neural-like stem cells,fiberlike structures,and nestin mRNA levels that were increased in response to myocardial ischemia. Cardiac tissue-derived neural stem cell migration to the infarct region and concomitant nestin+ fiberlike innervation represent obligatory events of reparative fibrosis in the damaged rat myocardium. View Publication

Leptin is detected in the sera,and leptin receptors are expressed in the cerebrum of mouse embryos,suggesting that leptin plays a role in cerebral development. Compared with the wild type,leptin-deficient (ob/ob) mice had fewer cells at embryonic day (E) 16 and E18 and had fewer 5-bromo-2'-deoxyuridine(+) cells at E14 and E16 in the neuroepithelium. Intracerebroventricular leptin injection in E14 ob/ob embryos increased the number of neuroepithelium cells at E16. In cultured neurosphere cells,leptin treatment increased Hes1 mRNA expression and maintained neural progenitors. Astrocyte differentiation was induced by low-dose (0.1 microg/ml) but not high-dose (1 microg/ml) leptin. High-dose leptin decreased Id mRNA and increased Ngn1 mRNA in neurosphere cells. The neuropeptide Y mRNA level in the cortical plate was lower in ob/ob than the wild type at E16 and E18. These results suggest that leptin maintains neural progenitors and is related to glial and neuronal development in embryos. View Publication

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin,a member of the DCC receptor family,has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S,Deitinghoff L,Davis D,Conrad S,Skutella T,Chedotal A,Mueller B,Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors,including neurogenic radial glia,in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast,neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition,we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences,suggesting a role for neogenin in tangential migration. Therefore,neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain. View Publication