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

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

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

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

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

Fatty-acid-binding proteins (FABPs) are key intracellular molecules involved in the uptake,transportation and storage of fatty acids and in the mediation of signal transduction and gene transcription. However,little is known regarding their expression and function in the oligodendrocyte lineage. We evaluate the in vivo and in vitro expression of FABP5 and FABP7 in oligodendrocyte lineage cells in the cortex and corpus callosum of adult mice,mixed cortical culture and oligosphere culture by immunofluorescent counter-staining with major oligodendrocyte lineage markers. In all settings,FABP7 expression was detected in NG2(+)/PDGFRα(+) oligodendrocyte progenitor cells (OPCs) that did not express FABP5. FABP5 was detected in mature CC1(+)/MBP(+) oligodendrocytes that did not express FABP7. Analysis of cultured OPCs showed a significant decrease in the population of FABP7-knockout (KO) OPCs and their BrdU uptake compared with wild-type (WT) OPCs. Upon incubation of OPCs in oligodendrocyte differentiation medium,a significantly lower percentage of FABP7-KO OPCs differentiated into O4(+) oligodendrocytes. The percentage of mature MBP(+) oligodendrocytes relative to whole O4(+)/MBP(+) oligodendrocytes was significantly lower in FABP7-KO and FABP5-KO than in WT cell populations. The percentage of terminally mature oligodendrocytes with membrane sheet morphology was significantly lower in FABP5-KO compared with WT cell populations. Thus,FABP7 and FABP5 are differentially expressed in oligodendrocyte lineage cells and regulate their proliferation and/or differentiation. Our findings suggest the involvement of FABP7 and FABP5 in the pathophysiology of demyelinating disorders,neuropsychiatric disorder and glioma,conditions in which OPCs/oligodendrocytes play central roles. View Publication

Evidence is presented that bone marrow (BM) in addition to CD45(positive) hematopoietic stem cells contains a rare population of heterogenous CD45(negative) nonhematopoietic tissue committed stem cells (TCSC). These nonhematopoietic TCSC (i) are enriched in population of CXCR4(+) CD34(+) AC133(+) lin(-) CD45(-) and CXCR4(+) Sca-1(+) lin(-) CD45(-) in humans and mice,respectively,(ii) display several markers of pluripotent stem cells (PSC) and (iii) as we envision are deposited in BM early in development. Thus,since BM contains versatile nonhematopoietic stem cells,previous studies on plasticity trans-dedifferentiation of BM-derived hematopoietic stem cells (HSC) that did not include proper controls to exclude this possibility could lead to wrong interpretations. Therefore,in this spotlight review we present this alternative explanation of 'plasticity' of BM-derived stem cells based on the assumption that BM stem cells are heterogenous. We also discuss a potential relationship of TCSC/PSC identified by us with other BM-derived CD45(negative) nonhematopoietic stem cells that were recently identified by other investigators (eg MSC,MAPC,USSC and MIAMI cells). Finally,we discuss perspectives and pitfalls in potential application of these cells in regenerative medicine. View Publication

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells,including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells,known as brain tumor initiating cells,likely contribute to glioma recurrence,as they are highly invasive,mobile,resistant to radiation and chemotherapy,and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate,which may be a key process in the death of peritumoral neurons,formation of necrosis,local inflammation,and glioma-related seizures. Moreover,elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells,resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells,which express the stem cell markers nestin and SOX-2,we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors,compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting,immunocytochemistry,and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells. View Publication

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID,celecoxib,to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. View Publication

The mechanisms by which sex differences in the mammalian brain arise are poorly understood,but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development,we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR%=%0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes,causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts,there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes,a transmissible effect that was maintained in cellular progeny. Additionally,we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS,and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained. View Publication