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BACKGROUND Neural stem cells (NSCs) display tissue trophic and immune modulatory therapeutic activities after transplantation in central nervous system disorders. The intercellular interplay between stem cells and target immune cells is increased in NSCs exposed to inflammatory cues. Here,we hypothesize that inflammatory cytokine signalling leads to metabolic reprogramming of NSCs regulating some of their immune modulatory effects. METHODS NSC lines were prepared from the subventricular zone (SVZ) of 7-12-week-old mice. Whole secretome-based screening and analysis of intracellular small metabolites was performed in NSCs exposed to cocktails of either Th1-like (IFN-γ,500 U/ml; TNF-α,200 U/ml; IL-1β,100 U/ml) or Th2-like (IL-4,IL-5 and IL-13; 10 ng/ml) inflammatory cytokines for 16 h in vitro. Isotopologues distribution of arginine and downstream metabolites was assessed by liquid chromatography/mass spectrometry in NSCs incubated with U-(13)C6 L-arginine in the presence or absence of Th1 or Th2 cocktails (Th1 NSCs or Th2 NSCs). The expression of arginase I and II was investigated in vitro in Th1 NSCs and Th2 NSCs and in vivo in the SVZ of mice with experimental autoimmune encephalomyelitis,as prototypical model of Th1 cell-driven brain inflammatory disease. The effects of the inflammatory cytokine signalling were studied in NSC-lymph node cells (LNC) co-cultures by flow cytometry-based analysis of cell proliferation following pan-arginase inhibition with N(ω)-hydroxy-nor-arginine (nor-NOHA). RESULTS Cytokine-primed NSCs showed significantly higher anti-proliferative effect in co-cultures vs. control NSCs. Metabolomic analysis of intracellular metabolites revealed alteration of arginine metabolism and increased extracellular arginase I activity in cytokine-primed NSCs. Arginase inhibition by nor-NOHA partly rescued the anti-proliferative effects of cytokine-primed NSCs. CONCLUSIONS Our work underlines the use of metabolic profiling as hypothesis-generating tools that helps unravelling how stem cell-mediated mechanisms of tissue restoration become affected by local inflammatory responses. Among different therapeutic candidates,we identify arginase signalling as novel metabolic determinant of the NSC-to-immune system communication. View Publication

The role of tumor stem cells in benign tumors such as pituitary adenomas remains unclear. In this study,we investigated whether the cells within pituitary adenomas that spontaneously develop in Rb+/- mice are hierarchically distributed with a subset being responsible for tumor growth. Cells derived directly from such tumors grew as spheres in serum-free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor. Some cells within growing pituitary tumor spheres (PTS) expressed common stem cell markers (Sca1,Sox2,Nestin,and CD133),but were devoid of hormone-positive differentiated cells. Under subsequent differentiating conditions (matrigel-coated growth surface),PTS expressed all six pituitary hormones. We next searched for specific markers of the stem cell population and isolated a Sca1(+) cell population that showed increased sphere formation potential,lower mRNA hormone expression,higher expression of stem cell markers (Notch1,Sox2,and Nestin),and increased proliferation rates. When transplanted into non-obese diabetic-severe combined immunodeficiency gamma mice brains,Sca1(+) pituitary tumor cells exhibited higher rates of tumor formation (brain tumors observed in 11/11 (100%) vs 7/12 (54%) of mice transplanted with Sca1(+) and Sca1(-) cells respectively). Magnetic resonance imaging and histological analysis of brain tumors showed that tumors derived from Sca1(+) pituitary tumor cells were also larger and plurihormonal. Our findings show that Sca1(+) cells derived from benign pituitary tumors exhibit an undifferentiated expression profile and tumor-proliferative advantages,and we propose that they could represent putative pituitary tumor stem/progenitor cells. 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

The vacuolar H+ ATPase (V-ATPase) is a proton pump responsible for acidification of cellular microenvironments,an activity exploited by tumors to survive,proliferate and resist to therapy. Despite few observations,the role of V-ATPase in human tumorigenesis remains unclear.We investigated the expression of ATP6V0C,ATP6V0A2,encoding two subunits belonging to the V-ATPase V0 sector and ATP6V1C,ATP6V1G1,ATPT6V1G2,ATP6V1G3,which are part of the V1 sector,in series of adult gliomas and in cancer stem cell-enriched neurospheres isolated from glioblastoma (GBM) patients. ATP6V1G1 expression resulted significantly upregulated in tissues of patients with GBM and correlated with shorter patients' overall survival independent of clinical variables.ATP6V1G1 knockdown in GBM neurospheres hampered sphere-forming ability,induced cell death,and decreased matrix invasion,a phenotype not observed in GBM monolayer cultures. Treating GBM organotypic cultures or neurospheres with the selective V-ATPase inhibitor bafilomycin A1 reproduced the effects of ATP6V1G1 siRNA and strongly suppressed expression of the stem cell markers Nestin,CD133 and transcription factors SALL2 and POU3F2 in neurospheres.These data point to ATP6V1G1 as a novel marker of poor prognosis in GBM patients and identify V-ATPase inhibition as an innovative therapeutic strategy for GBM. 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

Azidothymidine (AZT) is a synthetic,chain-terminating nucleoside analog used to treat HIV-1 infection. While AZT is not actively transported across the blood brain barrier,it does accumulate at high levels in cerebrospinal fluid,and subsequently diffuses into the overlying parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventricular system,we hypothesize that diffusion from CSF exposes neural stem/progenitor cells and their progeny to biologically relevant levels of AZT sufficient to perturb normal cell functions. We employed in vitro and in vivo models of mouse neurogenesis in order to assess the effects of AZT on developing and adult neurogenesis. Using in vitro assays we show that AZT reduces the population expansion potential of neural stem/progenitor cells by inducing senescence. Additionally,in a model of in vitro neurogenesis AZT severely attenuates neuroblast production. These effects are mirrored in vivo by clinically-relevant animal models. We show that in utero AZT exposure perturbs both population expansion and neurogenesis among neural stem/progenitor cells. Additionally,a short-term AZT regimen in adult mice suppresses subependymal zone neurogenesis. These data reveal novel negative effects of AZT on neural stem cell biology. Given that the sequelae of HIV infection often include neurologic deficits-subsumed under AIDS Dementia Complex (Brew,1999)-it is important to determine to what extent AZT negatively affects neurological function in ways that contribute to,or exacerbate,ADC in order to avoid attributing iatrogenic drug effects to the underlying disease process,and thereby skewing the risk/benefit analysis of AZT therapy. View Publication

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique,we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition,we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells,but not in transit-amplifying cells,and directly impacts on neurogenesis. Finally,we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. View Publication

Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals,they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However,little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ),we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months,the proliferative status of activated NSCs was already altered by 6 months,with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures,as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications. View Publication

Quiescent neural stem cells (NSCs) are considered the reservoir for adult neurogenesis,generating new neurons throughout life. Until now,their isolation has not been reported,which has hampered studies of their regulatory mechanisms. We sorted by FACS quiescent NSCs and their progeny from the subventricular zone (SVZ) of adult mice according to the expression of the NSC marker LeX/CD15,the EGF receptor (EGFR) and the CD24 in combination with the vital DNA marker Hoechst 33342. Characterization of sorted cells showed that the LeX(bright)/EGFR-negative population was enriched in quiescent cells having an NSC phenotype. In contrast to proliferating NSCs and progenitors,the LeX(bright)/EGFR-negative cells,i.e. quiescent NSCs,resisted to a moderate dose of gamma-radiation (4Gy),entered the cell cycle two days after irradiation prior to EGFR acquisition and ultimately repopulated the SVZ. We further show that the GABAAR signaling regulates their cell cycle entry by using specific GABAAR agonists/antagonists and that the radiation-induced depletion of neuroblasts,the major GABA source,provoked their proliferation in the irradiated SVZ. Our study demonstrates that quiescent NSCs are specifically enriched in the LeX(bright)/EGFR-negative population,and identifies the GABAAR signaling as a regulator of the SVZ niche size by modulating the quiescence of NSCs. View Publication

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36),an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly,TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand,inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. View Publication

Adeno-associated virus (AAV) vectors expressing tumoricidal genes injected directly into brain tumors have shown some promise,however,invasive tumor cells are relatively unaffected. Systemic injection of AAV9 vectors provides widespread delivery to the brain and potentially the tumor/microenvironment. Here we assessed AAV9 for potential glioblastoma therapy using two different promoters driving the expression of the secreted anti-cancer agent sTRAIL as a transgene model; the ubiquitously active chicken β-actin (CBA) promoter and the neuron-specific enolase (NSE) promoter to restrict expression in brain. Intravenous injection of AAV9 vectors encoding a bioluminescent reporter showed similar distribution patterns,although the NSE promoter yielded 100-fold lower expression in the abdomen (liver),with the brain-to-liver expression ratio remaining the same. The main cell types targeted by the CBA promoter were astrocytes,neurons and endothelial cells,while expression by NSE promoter mostly occurred in neurons. Intravenous administration of either AAV9-CBA-sTRAIL or AAV9-NSE-sTRAIL vectors to mice bearing intracranial patient-derived glioblastoma xenografts led to a slower tumor growth and significantly increased survival,with the CBA promoter having higher efficacy. To our knowledge,this is the first report showing the potential of systemic injection of AAV9 vector encoding a therapeutic gene for the treatment of brain tumors. View Publication

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here,we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system,we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly,the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH,hormones used in therapeutic treatment in humans. Finally,we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus,we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons. View Publication