技术资料

Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins,chromosomal instability,or DNA damage is associated with p53/p21 activation,culminating in either senescence or apoptosis,depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH,in contrast to circulating pituitary-derived endocrine GH,has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway,with subsequent marked induction of intracellular pituitary GH in vitro. In contrast,GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo,treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland,as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence,likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary,in nonpituitary cells GH exerts antiapoptotic properties. Thus,the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence. View Publication

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice,while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover,gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice,resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells,and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline. View Publication

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70,HSP90,GRP94,GRP58,HSP27,HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1,receptors for Ebola/Marburg/Hepatitis A,Lassa fever,and Hepatitis B viruses,respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya,Mumps,Measles,Rubella,RSV,CMV,and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus,Dna K and bacterial phosphodiesterases are novel antibiotic targets,and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. View Publication

Huntington's disease (HD) is a fatal neurodegenerative disease,caused by expansion of polyglutamine repeats in the Huntingtin gene,with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however,the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines,which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity,as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed,blocking glutamate signaling,not just through the NMDA but also mGlu and AMPA/Kainate receptors,completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together,these results provide important insight into HD mechanisms at early developmental time points,which may suggest novel approaches to HD therapeutics. View Publication

Medulloblastoma (MB) is a highly malignant pediatric brain tumor. Despite aggressive therapy,many patients succumb to the disease,and survivors experience severe side effects from treatment. MYC-driven MB has a particularly poor prognosis and would greatly benefit from more effective therapies. We used an animal model of MYC-driven MB to screen for drugs that decrease viability of tumor cells. Among the most effective compounds were histone deacetylase inhibitors (HDACIs). HDACIs potently inhibit survival of MYC-driven MB cells in vitro,in part by inducing expression of the FOXO1 tumor suppressor gene. HDACIs also synergize with phosphatidylinositol 3-kinase inhibitors to inhibit tumor growth in vivo. These studies identify an effective combination therapy for the most aggressive form of MB. View Publication

The mammalian central nervous system (CNS) undergoes significant expansion postnatally,producing astrocytes,oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy,a condition commonly treated with valproate/valproic acid (VPA),a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs,however,may be essential for the differentiation of distinct subpopulations of neurons and glia. Here,we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7-P14,combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG,rostral migratory stream (RMS),and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed,however,that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA,OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity,we found that short term treatment with VPA in vivo reduced neurosphere numbers and size,a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor,Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively,these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered,especially in individuals whose brains are actively undergoing key postnatal time windows of development. View Publication

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo,SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via α6β1 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEC) regulate SVZ cells neurogenic capacities,cocultures of SVZ neurospheres and primary BEC,both obtained from C57BL/6 mice,were performed. The involvement of laminin-integrin interactions in SVZ homeostasis was tested in three ways. Firstly,SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (CHX) prior to coculture,a treatment expected to decrease membrane proteins. Secondly,SVZ cells were cocultured with BEC in the presence of an anti-α6 integrin neutralizing antibody. Thirdly,BEC were cultured with β1-/- SVZ cells. We showed that contact with BEC supports,at least in part,proliferation and stemness of SVZ cells,as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEC. These effects are dependent on BEC-derived laminin binding to α6β1 integrin and are decreased in cocultures incubated with anti-α6 integrin neutralizing antibody and in cocultures with SVZ β1-/- cells. Moreover,BEC-derived laminin sustains stemness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving α6β1 integrin binding to laminin,contribute to SVZ cell proliferation and stemness. View Publication

The electrophoretic pattern of the large microtubule-associated protein,MAP2,changes during rat brain development. Immunoblots of NaDodSO4 extracts obtained from the cerebral cortex,cerebellum,and thalamus at 10-15 days after birth reveal only a single electrophoretic species when probed with any of three MAP2 monoclonal antibodies. By contrast,adult MAP2 contains two immunoreactive species,MAP2a and MAP2b. The single band of MAP2 from immature brain electrophoretically comigrates with adult MAP2b. Between postnatal days 17 and 18,immature MAP2 simultaneously resolves into two species in both the cerebellum and cerebral cortex. Immunoblots of NaDodSO4 extracts from spinal cord demonstrate the adult complement of MAP2 by day 10,indicating that MAP2 does not change coordinately throughout the entire central nervous system. In vitro cAMP-dependent phosphorylation of immature MAP2 causes a band split reminiscent of that seen during brain development in vivo. The possibility that the developmentally regulated changes observed in MAP2 during brain maturation are due to timed phosphorylation events is discussed. 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

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development,modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (˜70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (textgreater250%) MN production in chemically defined adherent cultures. Furthermore,we show that Islet-1 is essential for formation of mature and functional human MNs,but,unlike its mouse counterpart,does not regulate cell survival or suppress the V2a interneuron fate. Together,our discoveries improve the strategy for MN derivation,advance our understanding of human neural specification and MN development,and provide invaluable tools for human developmental studies,drug discovery and regenerative medicine. View Publication

Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development,which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently,representative DNA biomarkers become equally important in pre-clinical studies. However,it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here,we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres,adherent cell cultures,and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes,and new actionable mutations such as the loss of PIK3R1,and reveals clear patient-to-patient differences. In contrast,for each patient,we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly,we observe 96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles,we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests,at a remarkable resolution,the genome-wide conservation of a patient's tumor genetics in various pre-clinical models,and therefore supports their use for the development and testing of personalized targeted therapies. View Publication

Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind,we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation,of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin,deguelin,patulin and phenethyl caffeate) exhibited high cytotoxicity,with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular,the FDA approved drug for the treatment of alcoholism,disulfiram (DSF),was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu),which markedly increased GSC death. DSF-Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs,consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier,we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. View Publication