技术资料

Although gene-gene interaction,or epistasis,plays a large role in complex traits in model organisms,genome-wide by genome-wide searches for two-way interaction have limited power in human studies. We thus used knowledge of a biological pathway in order to identify a contribution of epistasis to autism spectrum disorders (ASDs) in humans,a reverse-pathway genetic approach. Based on previous observation of increased ASD symptoms in Mendelian disorders of the Ras/MAPK pathway (RASopathies),we showed that common SNPs in RASopathy genes show enrichment for association signal in GWAS (P = 0.02). We then screened genome-wide for interactors with RASopathy gene SNPs and showed strong enrichment in ASD-affected individuals (P < 2.2 x 10-16),with a number of pairwise interactions meeting genome-wide criteria for significance. Finally,we utilized quantitative measures of ASD symptoms in RASopathy-affected individuals to perform modifier mapping via GWAS. One top region overlapped between these independent approaches,and we showed dysregulation of a gene in this region,GPR141,in a RASopathy neural cell line. We thus used orthogonal approaches to provide strong evidence for a contribution of epistasis to ASDs,confirm a role for the Ras/MAPK pathway in idiopathic ASDs,and to identify a convergent candidate gene that may interact with the Ras/MAPK pathway. View Publication

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition,patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD,we determined whether inefficient BER due to reduced DNA polymerase-β (Polβ) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild-type and 3xTgAD mice with normal or reduced Polβ levels. Compared to wild-type control mice,Polβ heterozygous (Polβ+/- ),and 3xTgAD mice,3xTgAD/Polβ+/- mice exhibited impaired performance in a buried food test of olfaction. Polβ deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However,numbers of newly generated neurons were reduced by approximately 25% in Polβ+/- and 3xTgAD mice,and by over 60% in the 3xTgAD/Polβ+/- mice compared to wild-type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polβ+/- mice compared to 3xTgAD mice. Levels of amyloid β-peptide (Aβ) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polβ+/- mice,and cultured Polβ-deficient neurons exhibited increased vulnerability to Aβ-induced death. Olfactory deficit is an early sign in human AD,but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons,and suggest a mechanism underlying early olfactory impairment in AD. View Publication

BACKGROUND Ependymoma management remains challenging because of the inherent chemoresistance of this tumor. To determine whether ependymoma stem cells (SCs) might contribute to therapy resistance,we investigated the sensitivity of ependymoma SCs to temozolomide and etoposide. METHODS The efficacies of the two DNA damaging agents were explored in two ependymoma SC lines in vitro and in vivo models. RESULTS Ependymoma SC lines were highly sensitive to temozolomide and etoposide in vitro,but only temozolomide impaired tumor-initiation properties. Consistently,temozolomide but not etoposide showed significant antitumoral activity on ependymoma SC-driven subcutaneous and orthotopic xenografts by reducing the mitotic fraction. In vitro temozolomide at the EC50 (10 µM) induced accumulation of cells in the G2/M phase that was unexpectedly accompanied by downregulation of p27 and p21 without modulation of full-length p53 (FLp53). Differentiation-committed ependymoma SCs acquired resistance to temozolomide. Inhibition of proliferation was partly due to apoptosis,that occurred earlier in differentiated cells as compared to neurospheres. The activation of apoptosis correlated with an increase in p53β/γ isoforms without modulation of FLp53 under both serum-free and differentiation-promoting media. Incubation of cells in both conditions with temozolomide resulted in increased glioneuronal differentiation exhibiting elevated glial fibrillary acidic protein,galactosylceramidase,and βIII-tubulin expression compared to untreated controls. O(6)-methylguanine DNA methyltransferase (MGMT) transcript levels were very low in SCs,and were increased by treatment and,epigenetically,by differentiation through MGMT promoter unmethylation. CONCLUSION Ependymoma growth might be impaired by temozolomide through preferential depletion of a less differentiated,more tumorigenic,MGMT-negative cell population with stem-like properties. View Publication

UNLABELLED This pilot feasibility study aimed to determine the outcome of canine epidermal neural crest stem cell (cEPI-NCSC) grafts in the normal spinal cords of healthy bred-for-research dogs. This included developing novel protocols for (a) the ex vivo expansion of cEPI-NCSCs,(b) the delivery of cEPI-NCSCs into the spinal cord,and (c) the labeling of the cells and subsequent tracing of the graft in the live animal by magnetic resonance imaging. A total of four million cEPI-NCSCs were injected into the spinal cord divided in two locations. Differences in locomotion at baseline and post-treatment were evaluated by gait analysis and compared with neurological outcome and behavioral exams. Histopathological analyses of the spinal cords and cEPI-NCSC grafts were performed at 3 weeks post-transplantation. Neurological and gait parameters were minimally affected by the stem cell injection. cEPI-NCSCs survived in the canine spinal cord for the entire period of investigation and did not migrate or proliferate. Subsets of cEPI-NCSCs expressed the neural crest stem cell marker Sox10. There was no detectable expression of markers for glial cells or neurons. The tissue reaction to the cell graft was predominantly vascular in addition to a degree of reactive astrogliosis and microglial activation. In the present study,we demonstrated that cEPI-NCSC grafts survive in the spinal cords of healthy dogs without major adverse effects. They persist locally in the normal spinal cord,may promote angiogenesis and tissue remodeling,and elicit a tissue response that may be beneficial in patients with spinal cord injury. SIGNIFICANCE It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities,clinically and pathologically,to the syndrome in people. For this reason,dogs provide a readily accessible,clinically realistic,spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI. View Publication

Despite the global prevalence of gastric disease,there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/β-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium,and that β-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types,including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology,and also represent a new platform for drug discovery. View Publication

Aging and oxidative stress are two prominent pathological mechanisms for Parkinson's disease (PD) that are strongly associated with the degeneration of dopamine (DA) neurons in the midbrain. DA and other catechols readily oxidize into highly reactive o-quinone species that are precursors of neuromelanin (NM) pigment and under pathological conditions can modify and damage macromolecules. The role of DA oxidation in PD pathogenesis remains unclear in part due to the lack of appropriate disease models and the absence of a simple method for the quantification of DA-derived oxidants. Here,we describe a rapid,simple,and reproducible method for the quantification of o-quinones in cells and tissues that relies on the near-infrared fluorescent properties of these species. Importantly,we demonstrate that catechol-derived oxidants can be quantified in human neuroblastoma cells and midbrain dopamine neurons derived from induced pluripotent stem cells,providing a novel model to study the downstream actions of o-quinones. This method should facilitate further study of oxidative stress and DA oxidation in PD and related diseases that affect the dopaminergic system. 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

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

Aberrant neural progenitor cell (NPC) proliferation and self-renewal have been linked to age-related neurodegeneration and neurodegenerative disorders including Alzheimer's disease (AD). Rhizoma Acori tatarinowii is a traditional Chinese herbal medicine against cognitive decline. In this study,we found that the extract of Rhizoma Acori tatarinowii (AT) and its active constituents,asarones,promote NPC proliferation. Oral administration of AT enhanced NPC proliferation and neurogenesis in the hippocampi of adult and aged mice as well as that of transgenic AD model mice. AT and its fractions also enhanced the proliferation of NPCs cultured in vitro. Further analysis identified α-asarone and β-asarone as the two active constituents of AT in promoting neurogenesis. Our mechanistic study revealed that AT and asarones activated extracellular signal-regulated kinase (ERK) but not Akt,two critical kinase cascades for neurogenesis. Consistently,the inhibition of ERK activities effectively blocked the enhancement of NPC proliferation by AT or asarones. Our findings suggest that AT and asarones,which can be orally administrated,could serve as preventive and regenerative therapeutic agents to promote neurogenesis against age-related neurodegeneration and neurodegenerative disorders. View Publication

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

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples,while requiring low cell numbers. To date,a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative,EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris,doublets and dead cells from the analysis. For validation,the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers,HBEC recovered from BAL (2.3% of live cells),BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed,validated,and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. View Publication

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions,a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as thunder god vine" has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity migratory potential and a switch in morphology to a fibroblastoid phenotype as well as stem cell- and epithelial-mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial-mesenchymal transition (EMT) and CSC features in established highly malignant cell lines whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors triptolide downregulated markers of CSCs proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA. View Publication