技术资料

Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX+) cells more stringently,these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic,early postnatal,and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX(high)) in the embryonic brain coexpressed the neuronal marker betaIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro. However,the DCX+ population from the early postnatal brain and the adult subventricular zone and hippocampus,which expressed low levels of DCX (DCX(low)),was enriched for neurosphere-forming cells,with only a small subpopulation of these cells coexpressing the neuronal markers betaIII-tubulin or microtubule-associated protein 2. Similarly,the DCX(low) population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX(high) cells,which were shown to be similar to the E14 DCX(high) cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX+ cells,with some cells displaying characteristics of immature or mature neurons,whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX(high) cells,regardless of location,are restricted to the neuronal lineage or are bone fide neurons,whereas some DCX(low) cells retain their multipotentiality. View Publication

The essential functions of Polycomb Repressive Complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs),but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs which co-precipitate with PRC1 from chromatin and found candidates that impact Polycomb Group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen,CAT7,regulates expression and PcG binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain which shares high sequence similarity to a non-syntenic zebrafish analog,cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA,enhanced by interference of a PRC1 component,and suppressed by interference of a known PRC1 target gene,demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs,and that CAT7/cat7l represent convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci. View Publication

The electrophoretic pattern of the large microtubule-associated protein,MAP2,changes during rat brain development. Immunoblots of NaDodSO4 extracts obtained from the cerebral cortex,cerebellum,and thalamus at 10-15 days after birth reveal only a single electrophoretic species when probed with any of three MAP2 monoclonal antibodies. By contrast,adult MAP2 contains two immunoreactive species,MAP2a and MAP2b. The single band of MAP2 from immature brain electrophoretically comigrates with adult MAP2b. Between postnatal days 17 and 18,immature MAP2 simultaneously resolves into two species in both the cerebellum and cerebral cortex. Immunoblots of NaDodSO4 extracts from spinal cord demonstrate the adult complement of MAP2 by day 10,indicating that MAP2 does not change coordinately throughout the entire central nervous system. In vitro cAMP-dependent phosphorylation of immature MAP2 causes a band split reminiscent of that seen during brain development in vivo. The possibility that the developmentally regulated changes observed in MAP2 during brain maturation are due to timed phosphorylation events is discussed. View Publication

Down's syndrome (DS),caused by trisomy of human chromosome 21,is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons,and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally,we show that the FDA-approved antibiotic drug,minocycline,partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B,GFAP,inducible nitric oxide synthase,and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. View Publication

Human brain development exhibits several unique aspects,such as increased complexity and expansion of neuronal output,that have proven difficult to study in model organisms. As a result,in vitro approaches to model human brain development and disease are an intense area of research. Here we describe a recently established protocol for generating 3D brain tissue,so-called cerebral organoids,which closely mimics the endogenous developmental program. This method can easily be implemented in a standard tissue culture room and can give rise to developing cerebral cortex,ventral telencephalon,choroid plexus and retinal identities,among others,within 1-2 months. This straightforward protocol can be applied to developmental studies,as well as to the study of a variety of human brain diseases. Furthermore,as organoids can be maintained for more than 1 year in long-term culture,they also have the potential to model later events such as neuronal maturation and survival. View Publication

Potential teratogenic effects of alcohol on fetal development have been documented. Especially studies have demonstrated deleterious effect of ethanol exposure on neuronal development in animal models and on the maintenance and differentiation of neuronal precursor cells derived from stem cells. To better understand the molecular effect of alcohol on the process of neural differentiation,we have performed gene expression microarray analysis on human embryonic stem cells being directed to neural rosettes and neural precursor cells in the presence of ethanol treatment. Here we provide detailed experimental methods,analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE56906. Our data provide scientific insight on potential molecular effects of fetal alcohol exposure on neural differentiation of early embryo development. View Publication

OBJECTIVE To develop in vitro adenoid cystic carcinoma cell line as a surrogate for functional studies. MATERIALS AND METHODS Cells obtained from a primary ACC of the base of tongue were cultivated in vitro and immortalized with h-TERT. Morphologic,cytogenetic and functional studies were performed. RESULTS Tumor cells were verified by positive reactions to keratin and smooth muscle actin and phenotypic cellular and nuclear features. In-vitro cell growth and colony formation assay supported their tumor nature. CONCLUSION We authenticated an ACC cell line with hybrid epithelial-myoepithelial feature as a resource for functional experimentation. View Publication

In Huntington's disease (HD),whether transneuronal spreading of mutant huntingtin (mHTT) occurs and its contribution to non-cell autonomous damage in brain networks is largely unknown. We found mHTT spreading in three different neural network models: human neurons integrated in the neural network of organotypic brain slices of HD mouse model,an ex vivo corticostriatal slice model and the corticostriatal pathway in vivo. Transneuronal propagation of mHTT was blocked by two different botulinum neurotoxins,each known for specifically inactivating a single critical component of the synaptic vesicle fusion machinery. Moreover,healthy human neurons in HD mouse model brain slices displayed non-cell autonomous changes in morphological integrity that were more pronounced when these neurons bore mHTT aggregates. Altogether,our findings suggest that transneuronal propagation of mHTT might be an important and underestimated contributor to the pathophysiology of HD. View Publication