Scientific Resources

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 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

Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here,we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions,Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts,whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant,we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary,our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF. View Publication

Alzheimer's disease (AD) belongs to a category of adult neurodegenerative conditions,which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed,secreted and propagated by neurons has been the subject of intensive research,but so far no preventive or curative therapy for AD is available,and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function--accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes,and extracellular release of Aβ peptides by excessive lysosomal exocytosis. Furthermore,cerebral injection of NEU1 in an established AD mouse model substantially reduces β-amyloid plaques. Our findings identify an additional pathway for the secretion of Aβ and define NEU1 as a potential therapeutic molecule for AD. View Publication

Rotavirus,a leading cause of severe gastroenteritis and diarrhoea in young children,accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling,raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo,especially by NOD-like receptor (NLR) inflammasomes,is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that,via RNA helicase Dhx9,Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens. View Publication

The intestinal epithelium plays an essential role in nutrient absorption,hormone release,and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make-up of the tissue. The mechanism underlying the nutrient-driven changes in proliferation is not known,but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to increases in crypt proliferation. We utilized primary small intestinal epithelial crypts from C57BL/6J mice to study (1) the effect of glucose on crypt proliferation and (2) the effect of glucose on crypt metabolism using an extracellular flux analyzer for real-time metabolic measurements. We found that glucose increased both crypt proliferation and glycolysis,and the glycolytic pathway inhibitor 2-deoxy-d-glucose (2-DG) attenuated glucose-induced crypt proliferation. Glucose did not enhance glucose oxidation,but did increase the maximum mitochondrial respiratory capacity,which may contribute to glucose-induced increases in proliferation. Glucose activated Akt/HIF-1alpha signaling pathway,which might be at least in part responsible for glucose-induced glycolysis and cell proliferation. These results suggest that high glucose availability induces an increase in crypt proliferation by inducing an increase in glycolysis with no change in glucose oxidation. View Publication

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date,no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally,HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly,HH suppresses viral propagation when administered to adult mice with active ZIKV infection,highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients. View Publication

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions,Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion,notably the up-regulation of reelin (Reln),the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln,and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ. View Publication

This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs) in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation,1.21% of transplanted hNPCs survived. In these hNPCs,parvalbumin (PV)-,calretinin (CR)-,somatostatin (SS)-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-,CR-,and SS-positive cells among GFP+ cells were 35.5%,15.7%,and 17.1%,respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The amplitude,frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion,GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy. View Publication

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia,the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment,although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features,stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines,non-malignant cells,3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays,flow cytometry,colony and spheroid formation assays,Western blot analysis,antibody protein arrays,electrophoretic mobility shift assays (EMSAs),immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability,self-renewal potential,and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo,and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression,inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly,aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells,chick embryos or mice. These results highlight aspirin as an effective,inexpensive and well-tolerated co-treatment to target inflammation,desmoplasia and CSC features PDA. 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

H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. View Publication