技术资料

Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1),we studied paired tumor samples from 41 patients,comparing higher-grade,progressed samples to their lower-grade counterparts. Integrated genomic analyses,including whole-exome sequencing and copy number,gene expression and DNA methylation profiling,demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions,as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach. View Publication

BACKGROUND Tumor cells present high levels of oxidative stress. Cancer therapeutics exploiting such biochemical changes by increasing reactive oxygen species (ROS) production or decreasing intracellular ROS scavengers could provide a powerful treatment strategy. METHODS To test the effect of our compound,obtusaquinone (OBT),we used several cell viability assays on seven different glioblastoma (GBM) cell lines and primary cells and on 12 different cell lines representing various cancer types in culture as well as on subcutaneous (n = 7 mice per group) and two intracranial GBM (n = 6-8 mice per group) and breast cancer (n = 6 mice per group) tumor models in vivo. Immunoblotting,immunostaining,flow cytometry,and biochemical assays were used to investigate the OBT mechanism of action. Histopathological analysis (n = 2 mice per group) and blood chemistry (n = 2 mice per group) were used to test for any compound-related toxicity. Statistical tests were two-sided. RESULTS OBT induced rapid increase in intracellular ROS levels,downregulation of cellular glutathione levels and increase in its oxidized form,and activation of cellular stress pathways and DNA damage,subsequently leading to apoptosis. Oxidative stress is believed to be the main mechanism through which this compounds targets cancer cells. OBT was well tolerated in mice,slowed tumor growth,and statistically prolonged survival in GBM tumor models. The ratio of median survival in U251 intracranial model in OBT vs control was 1.367 (95% confidence interval [CI] of ratio = 1.031 to 1.367,P = .008). Tumor growth inhibition was also observed in a mouse breast cancer model (average tumor volume per mouse,OBT vs control: 36.3 vs 200.4mm(3),difference = 164.1mm(3),95% CI =72.6 to 255.6mm(3),P = .005). CONCLUSIONS Given its properties and efficacy in cancer killing,our results suggest that OBT is a promising cancer therapeutic. View Publication

BACKGROUND AND PURPOSE HIV-1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo.HIV-1/Gp120 is involved in the pathogenesis of HIV-associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems,we examined the protective effects of the CB receptor agonist AM1241 on Gp120-mediated insults on neurogenesis. EXPERIMENTAL APPROACH We assessed the effects of AM1241 on survival and apoptosis in cultures of human and murine NPCs with immunohistochemical and TUNEL techniques. Neurogenesis in the hippocampus of GFAP/Gp120 transgenic mice in vivo was also assessed by immunohistochemistry. KEY RESULTS AM1241 inhibited in vitroGp120-mediated neurotoxicity and apoptosis of primary human and murine NPCs and increased their survival. AM1241 also promoted differentiation of NPCs to neuronal cells. While GFAP/Gp120 Tg mice exhibited impaired neurogenesis,as indicated by reduction in BrdU cells and doublecortin (DCX) cells,and a decrease in cells with proliferating cell nuclear antigen (PCNA),administration of AM1241 to GFAP/Gp120 Tg mice resulted in enhanced in vivo neurogenesis in the hippocampus as indicated by increase in neuroblasts,neuronal cells,BrdU cells and PCNA cells. Astrogliosis and gliogenesis were decreased in GFAP/Gp120 Tg mice treated with AM1241,compared with those treated with vehicle. CONCLUSIONS AND IMPLICATIONS The CB receptor agonist rescued impaired neurogenesis caused by HIV-1/Gp120 insult. Thus,CB receptor agonists may act as neuroprotective agents,restoring impaired neurogenesis in patients with HAD. View Publication

Human pluripotent stem cells (hPSCs) possess great value in the aspect of cellular therapies due to its self-renewal and potential to differentiate into all somatic cell types. A few defined synthetic surfaces such as polymers and adhesive biological materials conjugated substrata were established for the self-renewal of hPSCs. However,none of them was effective in the generation of human induced pluripotent stem cells (hiPSCs) and long-term maintenance of multiple hPSCs,and most of them required complicated manufacturing processes. Polydopamine has good biocompatibility,is able to form a stable film on nearly all solid substrates surface,and can immobilize adhesive biomolecules. In this manuscript,a polydopamine-mediated surface was developed,which not only supported the reprogramming of human somatic cells into hiPSCs under defined conditions,but also sustained the growth of hiPSCs on diverse substrates. Moreover,the proliferation and pluripotency of hPSCs cultured on the surface were comparable to Matrigel for more than 20 passages. Besides,hPSCs were able to differentiate to cardiomyocytes and neural cells on the surface. This polydopamine-based synthetic surface represents a chemically-defined surface extensively applicable both for fundamental research and cell therapies of hPSCs. View Publication

The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α,CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain,the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However,both of them have limitations in the analysis of a single cell. In this study,we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12,NPCs formed lamellipodia in the stripe assay. Furthermore,CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia,indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry,they can also generate live imaging data with comparable quality. In conclusion,stripe assay is a visual,dynamic and economical tool to study cellular mobility and its related molecule mechanisms. View Publication

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments,adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages,including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0,a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal,ectodermal and mesodermal cells,including CMCs with<88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets,the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood-brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay,a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053% per hour) in the cortex,consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally,brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary,our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets. View Publication

Oxidative stress as a contributor to neuronal death during prion infection is supported by the fact that various oxidative damage markers accumulate in the brain during the course of this disease. The normal cellular substrate of the causative agent,the prion protein,is also linked with protective functions against oxidative stress. Our previous work has found that,in chronic prion infection,an apoptotic subpopulation of cells exhibit oxidative stress and the accumulation of oxidised lipid and protein aggregates with caspase recruitment. Given the likely failure of antioxidant defence mechanisms within apoptotic prion-infected cells,we aimed to investigate the role of the crucial antioxidant pathway components,superoxide dismutases (SOD) 1 and 2,in an in vitro model of chronic prion infection. Increased total SOD activity,attributable to SOD1,was found in the overall population coincident with a decrease in SOD2 protein levels. When apoptotic cells were separated from the total population,the induction of SOD activity in the infected apoptotic cells was lost,with activity reduced back to levels seen in mock-infected control cells. In addition,mitochondrial superoxide production was increased and mitochondrial numbers decreased in the infected apoptotic subpopulation. Furthermore,a pan-caspase probe colocalised with SOD2 outside of mitochondria within cytosolic aggregates in infected cells and inhibition of caspase activity was able to restore cellular levels of SOD2 in the whole unseparated infected population to those of mock-infected control cells. Our results suggest that prion propagation exacerbates an apoptotic pathway whereby mitochondrial dysfunction follows mislocalisation of SOD2 to cytosolic caspases,permitting its degradation. Eventually,cellular capacity to maintain oxidative homeostasis is overwhelmed,thus resulting in cell death. View Publication

BACKGROUND Alzheimer's disease (AD) is characterized by progressive memory loss and impaired cognitive function. Early-onset familial forms of the disease (FAD) are caused by inheritance of mutant genes encoding presenilin 1 (PS1) variants. We have demonstrated that prion promoter (PrP)-driven expression of human FAD-linked PS1 variants in mice leads to impairments in environmental enrichment (EE)-induced adult hippocampal neural progenitor cell (AHNPC) proliferation and neuronal differentiation,and have provided evidence that accessory cells in the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes,in vivo. While of significant interest,these latter studies relied on transgenic mice that express human PS1 variant transgenes ubiquitously and at high levels,and the consequences of wild type or mutant PS1 expressed under physiologically relevant levels on EE-mediated AHNPC phenotypes has not yet been tested. RESULTS To assess the impact of mutant PS1 on EE-induced AHNPC phenotypes when expressed under physiological levels,we exposed adult mice that constitutively express the PSEN1 M146V mutation driven by the endogenous PSEN1 promoter (PS1 M146V knock-in" (KI) mice) to standard or EE-housed conditions. We show that in comparison to wild type PS1 mice AHNPCs in mice carrying homozygous (PS1M146V/M146V) or heterozygous (PS1M146V/+) M146V mutant alleles fail to exhibit EE-induced proliferation and commitment towards neurogenic lineages. More importantly we report that the survival of newborn progenitors are diminished in PS1 M146V KI mice exposed to EE-conditions compared to respective EE wild type controls. CONCLUSIONS Our findings reveal that expression at physiological levels achieved by a single PS1 M146V allele is sufficient to impair EE-induced AHNPC proliferation survival and neuronal differentiation in vivo. These results and our finding that microglia expressing a single PS1 M146V allele impairs the proliferation of wild type AHNPCs in vitro argue that expression of mutant PS1 in the AHNPC niche impairs AHNPCs phenotypes in a dominant non-cell autonomous manner. 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

BACKGROUND Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However,resistance inevitably emerges against these therapies,particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels,p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer,neurosphere,and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics,100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS Daoy,ONS76,UW228,and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth,induced apoptosis,and sensitized cells to SHH agents. Notably,RSK expression is correlated with SHH patients. In mice,BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly,BI-D1870 crosses the BBB,acting as a scaffold for development of more long-lived RSK inhibitors. View Publication