技术资料

Glioblastoma multiforme (GBM) is the most common and lethal form of intracranial tumor. We have established a lentivirus-induced mouse model of malignant gliomas,which faithfully captures the pathophysiology and molecular signature of mesenchymal human GBM. RNA-Seq analysis of these tumors revealed high nuclear factor κB (NF-κB) activation showing enrichment of known NF-κB target genes. Inhibition of NF-κB by either depletion of IκB kinase 2 (IKK2),expression of a IκBαM super repressor,or using a NEMO (NF-κB essential modifier)-binding domain (NBD) peptide in tumor-derived cell lines attenuated tumor proliferation and prolonged mouse survival. Timp1,one of the NF-κB target genes significantly up-regulated in GBM,was identified to play a role in tumor proliferation and growth. Inhibition of NF-κB activity or silencing of Timp1 resulted in slower tumor growth in both mouse and human GBM models. Our results suggest that inhibition of NF-κB activity or targeting of inducible NF-κB genes is an attractive therapeutic approach for GBM. View Publication

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion,however,can be obstructed by transmembrane proteins (pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44 an abundant transmembrane protein capable of indirect association with F-actin hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages where receptor mobility was minimal. Conversely receptors were most mobile at the leading edge where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier enabling receptors to engage their targets. View Publication

Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny View Publication

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection,chemotherapy,and radiation therapy. Unfortunately,this standard therapy does not target glioma cancer stem cells (GCSCs),a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins,and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study,we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs,a large fraction of CD133-positive cells expressed HCMV-IE,and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1,Sox2,Oct4,Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres,a behavior typically displayed by GCSCs,and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor. View Publication

Glioblastomas (GBMs) are highly aggressive,invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these,cells endowed with stem properties,tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells,termed cancer stem-like cells,have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs),a family of membrane receptors,play a prominent role in cell signaling,cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here,we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs),U-87 MG cells,human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated,138 were retained for comparative studies between the different cell types. At the transcriptomic level,eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets. View Publication

NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex,GluN1 is combined primarily with GluN2A and GluN2B,which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B,the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components,in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis,revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions. View Publication

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells,which is partially associated with the induction or repression of genes that influence the ischemic response. However,the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults,we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD),an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD,total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes,whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD,confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins,such as those encoding for PICK1,GRIP1,TARPγ3,calsyntenin-2/3,SAPAP2 and SNAP-25,were down-regulated after OGD. Additionally,OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors,but increased the mRNA expression of the GluN3A subunit,thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together,our results present the expression profile elicited by in vitro ischemia in hippocampal neurons,and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins,suggesting that the synaptic proteome may change after ischemia. View Publication

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable,brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here,we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant,homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast,the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity,we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials. View Publication

Amyloid-ß (Aß) precursor protein (APP) metabolism engages neuronal endolysosomal pathways for Aß processing and secretion. In Alzheimer's disease (AD),dysregulation of APP leads to excess Aß and neuronal dysfunction; suggesting that neuronal APP/Aß trafficking can be targeted for therapeutic gain. Cathepsin B (CatB) is a lysosomal cysteine protease that can lower Aß levels. However,whether CatB-modulation of Aß improves learning and memory function deficits in AD is not known. To this end,progenitor neurons were infected with recombinant adenovirus expressing CatB and recovered cell lysates subjected to proteomic analyses. The results demonstrated Lamp1 deregulation and linkages between CatB and the neuronal phagosome network. Hippocampal injections of adeno-associated virus expressing CatB reduced Aß levels,increased Lamp1 and improved learning and memory. The findings were associated with the emergence of c-fos + cells. The results support the idea that CatB can speed Aß metabolism through lysosomal pathways and as such reduce AD-associated memory deficits. 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

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