技术资料

Therapeutic modulation of phosphatidylinositol 3-kinase (PI3K)/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this,we created Olig2-cre:Pten(fl/fl) mice that showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Pten(fl/fl) animals progressively developed central nervous system white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy,chronic neurodegeneration,and death by 9 months. In contrast,during immediate postnatal development,oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Pten(fl/fl) mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor,NVP-BKM120,blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary,our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However,such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development. View Publication

Recent large-scale genomics efforts to characterize the cis-regulatory sequences that orchestrate genome-wide expression patterns have produced impressive catalogues of putative regulatory elements. Most of these sequences have not been functionally tested,and our limited understanding of the non-coding genome prevents us from predicting which sequences are bona fide cis-regulatory elements. Recently,massively parallel reporter assays (MPRAs) have been deployed to measure the activity of putative cis-regulatory sequences in several biological contexts,each with specific advantages and distinct limitations. We developed LV-MPRA,a novel lentiviral-based,massively parallel reporter gene assay,to study the function of genome-integrated regulatory elements in any mammalian cell type; thus,making it possible to apply MPRAs in more biologically relevant contexts. We measured the activity of 2,600 sequences in U87 glioblastoma cells and human neural progenitor cells (hNPCs) and explored how regulatory activity is encoded in DNA sequence. We demonstrate that LV-MPRA can be applied to estimate the effects of local DNA sequence and regional chromatin on regulatory activity. Our data reveal that primary DNA sequence features,such as GC content and dinucleotide composition,accurately distinguish sequences with high activity from sequences with low activity in a full chromosomal context,and may also function in combination with different transcription factor binding sites to determine cell type specificity. We conclude that LV-MPRA will be an important tool for identifying cis-regulatory elements and stimulating new understanding about how the non-coding genome encodes information. View Publication

Carbonic anhydrases (CAs) comprise a family of zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide. CAs contribute to a myriad of physiological processes,including pH regulation,anion transport and water balance. To date,16 known members of the mammalian alpha-CA family have been identified. Given that the catalytic family members share identical reaction chemistry,their physiologic roles are influenced greatly by their tissue and sub-cellular locations. CAVI is the lone secreted CA and exists in both saliva and the gastrointestinal mucosa. An alternative,stress-inducible isoform of CAVI (CAVI-b) has been shown to be expressed from a cryptic promoter that is activated by the CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP). The CAVI-b isoform is not secreted and is currently of unknown physiological function. Here we use neuronal models,including a model derived using Car6 and CHOP gene ablations,to delineate a role for CAVI-b in ischemic protection. Our results demonstrate that CAVI-b expression,which is increased through CHOP-signaling in response to unfolded protein stress,is also increased by oxygen-glucose deprivation (OGD). While enforced expression of CAVI-b is not sufficient to protect against ischemia,CHOP regulation of CAVI-b is necessary for adaptive changes mediated by BDNF that reduce subsequent ischemic damage. These results suggest that CAVI-b comprises a necessary component of a larger adaptive signaling pathway downstream of CHOP. View Publication

Fatty acid (FA) metabolism directly influences the functional capabilities of T cells in tumor microenvironments. Thus,developing tools to interrogate FA-uptake by T cell subsets is important for understanding tumor immunosuppression. Herein,we have generated a novel FA-Qdot 605 dye conjugate with superior sensitivity and flexibility to any of the previously commercially available alternatives. For the first time,we demonstrate that this nanoparticle can be used as a specific measure of fatty acid uptake by T cells both in-vitro and in-vivo. Flow cytometric analysis shows that both the location and activation status of T cells determines their FA uptake. Additionally,CD4+ Foxp3+ regulatory T cells (Tregs) uptake FA at a higher rate than effector T cell subsets,supporting the role of FA metabolism for Treg function. Furthermore,we are able to simultaneously detect glucose and fatty acid uptake directly within the tumor microenvironment. Cumulatively,our results suggest that this novel fluorescent probe is a powerful tool to understand FA utilization within the tumor,thereby providing an unprecedented opportunity to study T cell FA metabolism in-vivo. View Publication

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery,radiation and chemotherapy,posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma,identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet,oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent,highly disparate genomic structural variants,restricted to groups 3 and 4,resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes,GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements,including super-enhancers,instigating oncogenic activity. Our results,supported by evidence from mouse models,identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer. View Publication

Sepsis represents uncontrolled inflammation due to an infection. Cold-inducible RNA-binding protein (CIRP) is a stress-induced damage-associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM-1+ neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM-1+ neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM-1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis,we found increased expression of CIRP and higher frequencies and numbers of ICAM-1+ neutrophils in the lungs. Conversely,the CIRP-/- mice showed significant inhibition in the frequencies and numbers of ICAM-1+ neutrophils in the lungs compared to wild-type (WT) mice in sepsis. In vitro treatment of bone marrow-derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM-1+ phenotype in a time- and dose-dependent manner. The effect of rmCIRP on increasing frequencies of ICAM-1+ neutrophils was significantly attenuated in BMDN treated with anti-TLR4 Ab or NF-κB inhibitor compared,respectively,with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP-treated ICAM-1+ BMDN were significantly higher than those in ICAM-1- BMDN. Following sepsis the ICAM-1+ neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM-1- neutrophils. We further revealed that ICAM-1 and NETs were co-localized in the neutrophils treated with rmCIRP. CIRP-/- mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus,CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM-1+ neutrophils in the lungs during sepsis. View Publication

The generation of differentiated and functional neurons is a complex process,which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200,miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells,induction of miR-200 family was observed in differentiating neural stem cells,demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However,inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells,an inverse relationship was observed between expression of reprogramming transcription factors (SOX2,KLF4,NANOG,OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover,we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion,our results indicate that increased expression of the miR-200 family promotes neuronal differentiation,while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4. View Publication

Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML),we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues,and developed an algorithm to identify potential targets expressed in leukemia stem cells,but not in normal CD34+CD38- hematopoietic cells,T cells,or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19,we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML. View Publication

General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However,the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines,a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D,drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus,isoflurane destabilizes spine F-actin,resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus,a locus for anesthetic-induced amnesia,and have important implications for anesthetic effects on synaptic plasticity. View Publication

Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways,however,remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture,showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread,including localisation to epithelial cell junctions. We extended those observations here,characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation,barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally,M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology,consistent with contrasting virulence in experimentally infected mice. In summary,this study reports pericellular invasion,NHBE cytoskeletal reorganisation,and tissue remodelling with persistent infection in a human airway epithelium model,providing clear insight into the likely route for extrapulmonary spread. View Publication

Prion diseases are irreversible progressive neurodegenerative diseases,leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits,vacuolisation,astrocytosis,neuronal degeneration,and by cognitive,behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation,but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However,the development of such strategies requires a detailed knowledge of the pathology,particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade,several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However,the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly,this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease. View Publication

We studied a new method of treatment of amyotrophic lateral sclerosis with autologous mesenchymal stem cells. Autologous mesenchymal stem cells were injected intravenously (intact cells) or via lumbar puncture (cells committed to neuronal differentiation). Evaluation of the results of cell therapy after 12-month follow-up revealed slowing down of the disease progression in 10 patients in comparison with the control group consisting of 15 patients. The cell therapy was safe for the patients. View Publication