技术资料

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM However,clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy,in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression,we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells,we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs,modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type,expressing pro-angiogenic factors. In contrast,phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data. View Publication

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs),cancers occurring increasingly in children to young adults,is poorly understood. We present a transplantable tumour line,maintained in immune-compromised mice,and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays,spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model,with parallel studies on 27 primary hFL-HCC tumours,provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple,normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells--newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies. View Publication

BACKGROUND The molecular mechanisms that determine social behavior are poorly understood. Pheromones play a critical role in social recognition in most animals,including mice,but how these are converted into behavioral responses is largely unknown. Here,we report that the absence of the small GTPase M-Ras affects social behavior in mice. RESULTS In their interactions with other males,Mras(-/-) males exhibited high levels of territorial aggression and social investigations,and increased fear-related behavior. They also showed increased mating behavior with females. Curiously,increased aggression and mating behaviors were only observed when Mras(-/-) males were paired with Mras(-/-) partners,but were significantly reduced when paired with wild-type (WT) mice. Since mice use pheromonal cues to identify other individuals,we explored the possibility that pheromone detection may be altered in Mras(-/-) mice. Unlike WT mice,Mras(-/-) did not show a preference for exploring unfamiliar urinary pheromones or unfamiliar isogenic mice. Although this could indicate that vomeronasal function and/or olfactory learning may be compromised in Mras(-/-) mice,these observations were not fully consistent with the differential behavioral responses to WT and Mras(-/-) interaction partners by Mras(-/-) males. In addition,induction of c-fos upon pheromone exposure or in response to mating was similar in WT and Mras (-/-) mice,as was the ex vivo expansion of neural progenitors with EGF. This indicated that acute pheromone detection and processing was likely intact. However,urinary metabolite profiles differed between Mras(-/-) and WT males. CONCLUSIONS The changes in behaviors displayed by Mras(-/-) mice are likely due to a complex combination of factors that may include an inherent predisposition to increased aggression and sexual behavior,and the production of distinct pheromones that could override the preference for unfamiliar social odors. Olfactory and/or social learning processes may thus be compromised in Mras(-/-) mice. View Publication

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

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases,for example,in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency,which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions,deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template,such as an introduced single-stranded oligo DNA nucleotide (ssODN),allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ,editing by HDR remains inefficient and can be corrupted by additional indels,preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore,targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations,and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB,we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation,whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach,we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons,which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9,facilitating study of human disease. View Publication

OBJECTIVE Oxidative stress in the brain is highly prevalent in many neurodegenerative disorders including lysosomal storage disorders,in which neurodegeneration is a devastating manifestation. Despite intense studies,a precise mechanism linking oxidative stress to neuropathology in specific neurodegenerative diseases remains largely unclear. METHODS Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease caused by mutations in the ceroid lipofuscinosis neuronal-1 (CLN1) gene encoding palmitoyl-protein thioesterase-1. Previously,we reported that in the brain of Cln1 (-/-) mice,which mimic INCL,and in postmortem brain tissues from INCL patients,increased oxidative stress is readily detectable. We used molecular,biochemical,immunohistological,and electrophysiological analyses of brain tissues of Cln1 (-/-) mice to study the role(s) of oxidative stress in mediating neuropathology. RESULTS Our results show that in Cln1 (-/-) mice oxidative stress in the brain via upregulation of the transcription factor,CCAAT/enhancer-binding protein-δ,stimulated expression of serpina1,which is an inhibitor of a serine protease,neurotrypsin. Moreover,in the Cln1 (-/-) mice,suppression of neurotrypsin activity by serpina1 inhibited the cleavage of agrin (a large proteoglycan),which substantially reduced the production of agrin-22,essential for synaptic homeostasis. Direct whole-cell recordings at the nerve terminals of Cln1 (-/-) mice showed inhibition of Ca(2+) currents attesting to synaptic dysfunction. Treatment of these mice with a thioesterase-mimetic small molecule,N-tert (Butyl) hydroxylamine (NtBuHA),increased agrin-22 levels. INTERPRETATION Our findings provide insight into a novel pathway linking oxidative stress with synaptic pathology in Cln1 (-/-) mice and suggest that NtBuHA,which increased agrin-22 levels,may ameliorate synaptic dysfunction in this devastating neurodegenerative disease. View Publication

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis,tumor maintenance and therapeutic resistance. Thus,to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse,at least in part,the gene expression signature of GBM and GSCs,this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here,we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened,thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine,we found that thioridazine induces autophagy in GBM cell lines,and upregulates AMPK activity. Moreover,LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition,thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent,but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties. View Publication

Within the two neurogenic niches of the adult mammalian brain,i.e.,the subventricular zone lining the lateral ventricle and the subgranular zone of the hippocampus,there exist distinct populations of proliferating neural precursor cells that differentiate to generate new neurons. Numerous studies have suggested that epigenetic regulation by histone-modifying proteins is important in guiding precursor differentiation during development; however,the role of these proteins in regulating neural precursor activity in the adult neurogenic niches remains poorly understood. Here we examine the role of an NAD(+) -dependent histone deacetylase,SIRT1,in modulating the neurogenic potential of neural precursors in the neurogenic niches of the adult mouse brain. We show that SIRT1 is expressed by proliferating adult subventricular zone and hippocampal neural precursors,although its transcript and protein levels are dramatically reduced during neural precursor differentiation. Utilizing a lentiviral-mediated delivery strategy,we demonstrate that abrogation of SIRT1 signaling by RNAi does not affect neural precursor numbers or their proliferation. However,SIRT1 knock down results in a significant increase in neuronal production in both the subventricular zone and the hippocampus. In contrast,enhancing SIRT1 signaling either through lentiviral-mediated SIRT1 overexpression or through use of the SIRT1 chemical activator Resveratrol prevents adult neural precursors from differentiating into neurons. Importantly,knock down of SIRT1 in hippocampal precursors in vivo,either through RNAi or through genetic ablation,promotes their neurogenic potential. These findings highlight SIRT1 signaling as a negative regulator of neuronal differentiation of adult subventricular zone and hippocampal neural precursors. textcopyright 2013 Wiley Periodicals,Inc. View Publication

Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection,we used human neural progenitor cells (NPC),derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation,ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2 + cells was demonstrated by activation of caspases 3/7,8 and 9,and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2 + cells was prevented by incubation with the pan-caspase inhibitor,Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities,including multipolar spindle,chromosome laggards,micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy,such as monosomy,trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain,including microcephaly. View Publication

Glycolipids are amphipathic molecules which are highly expressed on cell membranes in skin and brain where they mediate several key cellular processes. Neural stem cells are defined as undifferentiated,proliferative,multipotential cells with extensive self-renewal and are responsive to brain injury. Di-rhamnolipid: α-L-rhamnopyranosyl-(1-2)α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,also referred to as di-rhamnolipid BAC-3,is a glycolipid isolated from the bacteria Pseudomonas aeruginosa. In the previous studies,di-rhamnolipid enhanced dermal tissue healing and regeneration. The present study provides the first assessment of di-rhamnolipid,and glycolipid biosurfactants in general,on the nervous system. Treatment of neural stem cells isolated from the lateral ventricle of adult mice and cultured in defined media containing growth factors at 0.5 and 1 μg/ml of di-rhamnolipid increased the number of neurospheres (2.7- and 2.8-fold,respectively) compared to controls and this effect remained even after passaging in the absence of di-rhamnolipid. In addition,neural stem cells treated with di-rhamnolipid at 50 and 100 μg/ml in defined media supplemented with fetal calf serum and without growth factors exhibited increased cell viability,indicating an interaction between di-rhamnolipid and serum components in the regulation of neural stem cells and neuroprogenitors. Intracerebroventricular administration of di-rhamnolipid at 300 and 120 ng/day increased the number of neurospheres (1.3- and 1.63-fold,respectively) that could be derived from the anterior lateral ventricles of adult mice. These results indicate that di-rhamnolipid stimulates proliferation of neural stem cells and increases their endogenous pools which may have therapeutic potential in managing neurodegenerative or neuropsychiatric disorders and promoting nervous tissue regeneration following injury. View Publication

Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs,with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1),the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms,depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans,represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES),suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II,involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein. View Publication

INTRODUCTION The α-synuclein (SNCA) gene has been implicated in the etiology of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). METHODS A computational analysis of SNCA 3' untranslated region to identify potential microRNA (miRNA) binding sites and quantitative real-time polymerase chain reaction (PCR) to determine their expression in isogenic induced pluripotent stem cell-derived dopaminergic and cholinergic neurons as a model of PD and DLB,respectively,were performed. In addition,we performed a deep sequencing analysis of the SNCA 3' untranslated region of autopsy-confirmed cases of PD,DLB,and normal controls,followed by genetic association analysis of the identified variants. RESULTS We identified four miRNA binding sites and observed a neuronal-type-specific expression profile for each miRNA in the different isogenic induced pluripotent stem cell-derived dopaminergic and cholinergic neurons. Furthermore,we found that the short structural variant rs777296100-polyT was moderately associated with DLB but not with PD. DISCUSSION We suggest that the regulation of SNCA expression through miRNAs is neuronal-type-specific and possibly plays a part in the phenotypic heterogeneity of synucleinopathies. Furthermore,genetic variability in the SNCA gene may contribute to synucleinopathies in a pathology-specific manner. View Publication