技术资料

Exogenous application of neural progenitor cells (NPCs) has successful implications in treating brain disorders,and research is beginning to identify ways to mimic this exogenous application by activating endogenous stem cell compartments. The recent discovery of a functional endocannabinoid system in murine NPCs (mNPCs) represents one potential therapeutic means to influence endogenous stem cell compartments. High levels of the endogenous cannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) persist during CNS inflammation and infection. The goal of this study was to assess the influence of AEA on mNPCs to identify how the endocannabinoid system influences mNPCs in vitro,a potential model to investigate effects of endocannabinoids on endogenous stem cell compartments. Our results show that AEA affects mNPC cell fate determination. Initial glial differentiation was observed,followed by induction of neuronal differentiation with AEA treatment. Cell survival and apoptosis was not affected by AEA. These effects were coupled by an increased phosphorylation of cAMP-responsive element (CRE) binding protein (CREB). View Publication

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs,we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently,the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated,stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast,ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results. View Publication

Although a number of growth factors have been shown to be involved in neurogenesis,the role of inflammatory cytokines remains relatively unexplored in the normal brain. Here we investigated the effect of interferon gamma (IFNgamma) in the regulation of neural precursor (NP) activity in both the developing and the adult mouse brain. Exogenous IFNgamma inhibited neurosphere formation from the wild-type neonatal and adult subventricular zone (SVZ). More importantly,however,these effects were mirrored in vivo,with mutant mice lacking endogenous IFNgamma displaying enhanced neurogenesis,as demonstrated by an increase in proliferative bromodeoxyuridine-labeled cells in the SVZ and an increased percentage of newborn neurons in the olfactory bulb. Furthermore,NPs isolated from IFNgamma null mice exhibited an increase in self-renewal ability and in the capacity to produce differentiated neurons and oligodendrocytes. These effects resulted from the direct action of IFNgamma on the NPs,as determined by single-cell assays and the fact that nearly all the neurospheres were derived from cells positive for major histocompatibility complex class I antigen,a downstream marker of IFNgamma-mediated activation. Moreover,the inhibitory effect was ameliorated in the presence of SVZ-derived microglia,with their removal resulting in almost complete inhibition of NP proliferation. Interestingly,in contrast to the results obtained in the adult,exogenous IFNgamma treatment stimulated neurosphere formation from the embryonic brain,an effect that was mediated by sonic hedgehog. Together these findings provide the first direct evidence that IFNgamma acts as a regulator of the active NP pool in the non-inflammatory brain. View Publication

Reactive oxygen species (ROS) damage brain lipids,carbohydrates,proteins,as well as DNA and may contribute to neurodegeneration. We previously reported that ER- and oxidative stress cause neuronal apoptosis in infantile neuronal ceroid lipofuscinosis (INCL),a lethal neurodegenerative storage disease,caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. Polyunsaturated fatty acids (PUFA) are essential components of cell membrane phospholipids in the brain and excessive ROS may cause oxidative damage of PUFA leading to neuronal death. Using cultured neurons and neuroprogenitor cells from mice lacking Ppt1,which mimic INCL,we demonstrate that Ppt1-deficient neurons and neuroprogenitor cells contain high levels of ROS,which may cause peroxidation of PUFA and render them incapable of providing protection against oxidative stress. We tested whether treatment of these cells with omega-3 or omega-6 PUFA protects the neurons and neuroprogenitor cells from oxidative stress and suppress apoptosis. We report here that both omega-3 and omega-6 fatty acids protect the Ppt1-deficient cells from ER- as well as oxidative stress and suppress apoptosis. Our results suggest that PUFA supplementation may have neuroprotective effects in INCL. View Publication

The vast majority of pedigrees with familial Alzheimer's disease (FAD) are caused by inheritance of mutations in the PSEN1 1 gene. While genetic ablation studies have revealed a role for presenilin 1 (PS1) in embryonic neurogenesis,little information has emerged regarding the potential effects of FAD-linked PS1 variants on proliferation,self-renewal and differentiation,key events that control cell fate commitment of adult brain neural progenitors (NPCs). We used adult brain subventricular zone (SVZ)-derived NPC cultures transduced with recombinant lentivirus as a means to investigate the effects of various PS1 mutants on self-renewal and differentiation properties. We now show that viral expression of several PS1 mutants in NPCs leads to impaired self-renewal and altered differentiation toward neuronal lineage,in vitro. In line with these observations,diminished constitutive proliferation and steady-state SVZ progenitor pool size was observed in vivo in transgenic mice expressing the PS1DeltaE9 variant. Moreover,NPC cultures established from the SVZ of adult mice expressing PS1DeltaE9 exhibit reduced self-renewal capacity and premature exit toward neuronal fates. To these findings,we show that both the levels of endogenous Notch/CBF-1-transcriptional activity and transcripts encoding Notch target genes are diminished in SVZ NPCs expressing PS1DeltaE9. The deficits in self-renewal and multipotency are restored by expression of Notch1-ICD or a downstream target of the Notch pathway,Hes1. Hence,we argue that a partial reduction in PS-dependent gamma-secretase processing of the Notch,at least in part,accounts for the impairments observed in SVZ NPCs expressing the FAD-linked PS1DeltaE9 variant. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication

Production of new neurons throughout adulthood has been well characterized in two brain regions,the subventricular zone (SVZ) of the anterolateral ventricle and the subgranular zone (SGZ) of the hippocampus. The neurons produced from these regions arise from neural stem cells (NSCs) found in highly regulated stem cell niches. We recently showed that midline structures called circumventricular organs (CVOs) also contain NSCs capable of neurogenesis and/or astrogliogenesis in vitro and in situ (Bennett et al.). The present study demonstrates that NSCs derived from two astrogliogenic CVOs,the median eminence and organum vasculosum of the lamina terminalis of the nestin-GFP mouse,possess the potential to integrate into the SVZ and differentiate into cells with a neuronal phenotype. These NSCs,following expansion and BrdU-labeling in culture and heterotopic transplantation into a region proximal to the SVZ in adult mice,migrate caudally to the SVZ and express early neuronal markers (TUC-4,PSA-NCAM) as they migrate along the rostral migratory stream. CVO-derived BrdU(+) cells ultimately reach the olfactory bulb where they express early (PSA-NCAM) and mature (NeuN) neuronal markers. Collectively,these data suggest that although NSCs derived from the ME and OVLT CVOs are astrogliogenic in situ,they produce cells phenotypic of neurons in vivo when placed in a neurogenic environment. These findings may have implications for neural repair in the adult brain. View Publication

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here,we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice,emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro,gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly,hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly,treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D,which,like gelsolin,produces actin disassembly,decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly,depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore,increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels,increased regional cerebral blood flow,and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together,reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and,subsequently,elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis. View Publication

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor (EPOR). The presence of EPO and its receptor in the CNS suggests a different function for EPO other than erythropoiesis. The purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation of neonatal spinal cord-derived neural progenitor cells. The effect of EPO on cell cycle progression was also examined,as well as the signaling cascades involved in this process. Our results showed that EPOR was present in the neural progenitor cells and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated neural progenitor cells indicated a reduced percentage of cells in G0/G1 phase,whereas the cell proliferation index (S phase plus G2/M phase) was increased. EPO also increased the proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells. With respect to the cell cycle signaling,we examined the cyclin-dependent kinases D1,D2 and E,and cyclin-dependent kinase inhibitors,p21cip1,p27kip1 and p57kip2. No significant differences were observed in the expression of these transcripts after EPO administration. Interestingly,the anti-apoptotic factors,mcl-1 and bcl-2 were significantly increased twofold. Moreover,these specific effects of EPO were eliminated by incubation of the progenitor cells with anti-EPO neutralizing antibody. Those observations suggested that EPO may play a role in normal spinal cord development by regulating cell proliferation and apoptosis. View Publication

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine,levels of which are modulated by stress,depression and clinical antidepressants. However,studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors,including stem cells,from the hippocampus of adult mice. Mechanistically,we provide evidence that beta(3)-adrenergic receptors,which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ),mediate this norepinephrine-dependent activation. Moreover,intrahippocampal injection of a selective beta(3)-adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly,systemic injection of the beta-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally,using a novel ex vivo slice-sphere" assay that maintains an intact neurogenic niche� View Publication

Memantine is classified as an NMDA receptor antagonist. We recently reported that memantine promoted the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus. However,the molecular mechanism responsible for the memantine-induced promotion of cellular proliferation remains unknown. In this study we searched for a factor that mediates memantine-induced cellular proliferation,and found that pigment epithelium-derived factor (PEDF),a broad-acting neurotrophic factor,is up-regulated in the dentate gyrus of adult mice after the injection of memantine. PEDF mRNA expression increased significantly by 3.5-fold at 1 day after the injection of memantine. In addition,the expression level of PEDF protein also increased by 1.8-fold at 2 days after the injection of memantine. Immunohistochemical study using anti-PEDF antibody showed that the majority of the PEDF-expressing cells were protoplasmic and perivascular astrocytes. Using a neurosphere assay,we confirmed that PEDF enhanced cellular proliferation under the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) but was not involved in the multilineage potency of hippocampal progenitor cells. Over expression of PEDF by adeno-associated virus,however,did not stimulate cellular proliferation,suggesting PEDF per se does not promote cellular proliferation in vivo. These findings suggest that the memantine induced PEDF up-regulation is involved in increased proliferation of hippocampal progenitor cells. View Publication

Slow-onset adaptive changes that arise from sustained antidepressant treatment,such as enhanced adult hippocampal neurogenesis and increased trophic factor expression,play a key role in the behavioral effects of antidepressants. alpha(2)-Adrenoceptors contribute to the modulation of mood and are potential targets for the development of faster acting antidepressants. We investigated the influence of alpha(2)-adrenoceptors on adult hippocampal neurogenesis. Our results indicate that alpha(2)-adrenoceptor agonists,clonidine and guanabenz,decrease adult hippocampal neurogenesis through a selective effect on the proliferation,but not the survival or differentiation,of progenitors. These effects persist in dopamine beta-hydroxylase knock-out (Dbh(-/-)) mice lacking norepinephrine,supporting a role for alpha(2)-heteroceptors on progenitor cells,rather than alpha(2)-autoreceptors on noradrenergic neurons that inhibit norepinephrine release. Adult hippocampal progenitors in vitro express all the alpha(2)-adrenoceptor subtypes,and decreased neurosphere frequency and BrdU incorporation indicate direct effects of alpha(2)-adrenoceptor stimulation on progenitors. Furthermore,coadministration of the alpha(2)-adrenoceptor antagonist yohimbine with the antidepressant imipramine significantly accelerates effects on hippocampal progenitor proliferation,the morphological maturation of newborn neurons,and the increase in expression of brain derived neurotrophic factor and vascular endothelial growth factor implicated in the neurogenic and behavioral effects of antidepressants. Finally,short-duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test,which normally requires 3 weeks of treatment with classical antidepressants. Our results demonstrate that alpha(2)-adrenoceptors,expressed by progenitor cells,decrease adult hippocampal neurogenesis,while their blockade speeds up antidepressant action,highlighting their importance as targets for faster acting antidepressants. View Publication