技术资料

Stem cell-based therapies have been reported in protecting cerebral infarction-induced neuronal dysfunction and death. However,most studies used rat/mouse neuron as model cell when treated with stem cell or exosomes. Whether these findings can be translated from rodent to humans has been in doubt. Here,we used human embryonic stem cell-derived neurons to detect the protective potential of exosomes against ischemia. Neurons were treated with in vitro oxygen-glucose deprivation (OGD) for 1 h. For treatment group,different exosomes were derived from neuron,embryonic stem cell,neural progenitor cell and astrocyte differentiated from H9 human embryonic stem cell and added to culture medium 30 min after OGD (100 μg/mL). Western blotting was performed 12 h after OGD,while cell counting and electrophysiological recording were performed 48 h after OGD. We found that these exosomes attenuated OGD-induced neuronal death,Mammalian target of rapamycin (mTOR),pro-inflammatory and apoptotic signaling pathway changes,as well as basal spontaneous synaptic transmission inhibition in varying degrees. The results implicate the protective effect of exosomes on OGD-induced neuronal death and dysfunction in human embryonic stem cell-derived neurons,potentially through their modulation on mTOR,pro-inflammatory and apoptotic signaling pathways. View Publication

Prion diseases are irreversible progressive neurodegenerative diseases,leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits,vacuolisation,astrocytosis,neuronal degeneration,and by cognitive,behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation,but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However,the development of such strategies requires a detailed knowledge of the pathology,particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade,several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However,the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly,this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease. View Publication

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM,peritumor tissue (brain adjacent to tumor,BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased,regardless of the tumor cell presence,whereas GD3 correlated with neoplastic infiltration. In BAT,NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM. View Publication

BACKGROUND Epigenetic modifications,particularly DNA methylation and posttranslational histone modifications regulate heritable changes in transcription without changes in the DNA sequence. Despite a number of studies showing clear links between environmental factors and DNA methylation,little is known about the effect of environmental factors on the recently identified histone lysine methylation. Since their identification numerous studies have establish critical role played by these enzymes in mammalian development. OBJECTIVES Identification of the Jumonji (Jmj) domain containing histone lysine demethylase have added a new dimension to epigenetic control of gene expression by dynamic regulation of histone methylation marks. The objective of our study was to evaluate the effect of prohexadione and trinexapac,widely used plant growth regulators of the acylcyclohexanediones class,on the enzymatic activity of histone lysine demethylases and histone modifications during the neural stem/progenitor cell differentiation. METHODS Here we show that prohexadione,but not trinexapac,directly inhibits non-heme iron (II),2-oxoglutarate-dependent histone lysine demethylase such as Jmjd2a. We used molecular modeling to show binding of prohexadione to Jmjd2a. We also performed in vitro demethylation assays to show the inhibitory effect of prohexadione on Jmjd2a. Further we tested this molecule in cell culture model of mouse hippocampal neural stem/progenitor cells to demonstrate its effect toward neuronal proliferation and differentiation. RESULTS Molecular modeling studies suggest that prohexadione binds to the 2-oxoglutarate binding site of Jmjd2a demethylase. Treatment of primary neural stem/progenitor cells with prohexadione showed a concentration dependent reduction in their proliferation. Further,the prohexadione treated neurospheres were induced toward neurogenic lineage upon differentiation. CONCLUSIONS Our results describe an important chemico-biological interaction of prohexadione,in light of critical roles played by histone lysine demethylases in human health and diseases. View Publication

In the search for ways to combat degenerative neurological disorders,neurogenesis-stimulating factors are proving to be a promising area of research. In this study,we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay,we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition,direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely,PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly,no deficit in precursor proliferation was observed in vivo,indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice,as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus,indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory. 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

Prominin-1 (CD133) is a commonly used cancer stem cell marker in central nervous system (CNS) tumors including glioblastoma (GBM). Expression of Prom1 in cancer is thought to parallel expression and function in normal stem cells. Using RNA in situ hybridization and antibody tools capable of detecting multiple isoforms of Prom1,we find evidence for two distinct Prom1 cell populations in mouse brain. Prom1 RNA is first expressed in stem/progenitor cells of the ventricular zone in embryonic brain. Conversely,in adult mouse brain Prom1 RNA is low in SVZ/SGZ stem cell zones but high in a rare but widely distributed cell population (Prom1(hi)). Lineage marker analysis reveals Prom1(hi) cells are Olig2+Sox2+ glia but Olig1/2 knockout mice lacking oligodendroglia retain Prom1(hi) cells. Bromodeoxyuridine labeling identifies Prom1(hi) as slow-dividing distributed progenitors distinct from NG2+Olig2+ oligodendrocyte progenitors. In adult human brain,PROM1 cells are rarely positive for OLIG2,but express astroglial markers GFAP and SOX2. Variability of PROM1 expression levels in human GBM and patient-derived xenografts (PDX) - from no expression to strong,uniform expression--highlights that PROM1 may not always be associated with or restricted to cancer stem cells. TCGA and PDX data show that high expression of PROM1 correlates with poor overall survival. Within proneural subclass tumors,high PROM1 expression correlates inversely with IDH1 (R132H) mutation. These findings support PROM1 as a tumor cell-intrinsic marker related to GBM survival,independent of its stem cell properties,and highlight potentially divergent roles for this protein in normal mouse and human glia. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. 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

H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. 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