参考文献

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

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Within the two neurogenic niches of the adult mammalian brain,i.e.,the subventricular zone lining the lateral ventricle and the subgranular zone of the hippocampus,there exist distinct populations of proliferating neural precursor cells that differentiate to generate new neurons. Numerous studies have suggested that epigenetic regulation by histone-modifying proteins is important in guiding precursor differentiation during development; however,the role of these proteins in regulating neural precursor activity in the adult neurogenic niches remains poorly understood. Here we examine the role of an NAD(+) -dependent histone deacetylase,SIRT1,in modulating the neurogenic potential of neural precursors in the neurogenic niches of the adult mouse brain. We show that SIRT1 is expressed by proliferating adult subventricular zone and hippocampal neural precursors,although its transcript and protein levels are dramatically reduced during neural precursor differentiation. Utilizing a lentiviral-mediated delivery strategy,we demonstrate that abrogation of SIRT1 signaling by RNAi does not affect neural precursor numbers or their proliferation. However,SIRT1 knock down results in a significant increase in neuronal production in both the subventricular zone and the hippocampus. In contrast,enhancing SIRT1 signaling either through lentiviral-mediated SIRT1 overexpression or through use of the SIRT1 chemical activator Resveratrol prevents adult neural precursors from differentiating into neurons. Importantly,knock down of SIRT1 in hippocampal precursors in vivo,either through RNAi or through genetic ablation,promotes their neurogenic potential. These findings highlight SIRT1 signaling as a negative regulator of neuronal differentiation of adult subventricular zone and hippocampal neural precursors. textcopyright 2013 Wiley Periodicals,Inc. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Acyl-protein thioesterase-1 (APT1) and APT2 are cytosolic enzymes that catalyze depalmitoylation of membrane-anchored,palmitoylated H-Ras and growth-associated protein-43 (GAP-43),respectively. However,the mechanism(s) of cytosol-membrane shuttling of APT1 and APT2,required for depalmitoylating their substrates H-Ras and GAP-43,respectively,remained largely unknown. Here,we report that both APT1 and APT2 undergo palmitoylation on Cys-2. Moreover,blocking palmitoylation adversely affects membrane localization of both APT1 and APT2 and that of their substrates. We also demonstrate that APT1 not only catalyzes its own depalmitoylation but also that of APT2 promoting dynamic palmitoylation (palmitoylation-depalmitoylation) of both thioesterases. Furthermore,shRNA suppression of APT1 expression or inhibition of its thioesterase activity by palmostatin B markedly increased membrane localization of APT2,and shRNA suppression of APT2 had virtually no effect on membrane localization of APT1. In addition,mutagenesis of the active site Ser residue to Ala (S119A),which renders catalytic inactivation of APT1,also increased its membrane localization. Taken together,our findings provide insight into a novel mechanism by which dynamic palmitoylation links cytosol-membrane trafficking of APT1 and APT2 with that of their substrates,facilitating steady-state membrane localization and function of both. View Publication

Platelet-derived growth factor receptors (PDGFR) α and β have been suggested as potential targets for treatment of rhabdomyosarcoma,the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes,with PDGF-D as the solely overexpressed PDGFRβ ligand. By immunohistochemistry,PDGF-CC,PDGF-DD,and PDGFRα were found in tumor cells,whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs,PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems,where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation,vessel density,and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3α and GSK-3β. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness,differentiation,senescence,and apoptosis. In summary,the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression. View Publication

Most neurodegenerative diseases are linked to aberrant accumulation of aggregation-prone proteins. Among them,Huntington's disease (HD) is caused by an expanded polyglutamine repeat stretch in the N terminus of the mutant huntingtin protein (mHTT),which gets cleaved and aggregates in the brain. Recently established human induced pluripotent stem cell-derived HD neurons exhibit some disease-relevant phenotypes and provide tools for HD research. However,they have limitations such as genetic heterogeneity and an absence of mHTT aggregates and lack a robust neurodegeneration phenotype. In addition,the relationship between the phenotype and mHTT levels has not been elucidated. Herein,we present a human embryonic stem cell (hESC)-derived HD neuronal model expressing HTTexon1 fragments,which addresses the deficiencies enumerated above. The wild-type and HD lines are derived from an isogenic background and exhibit insoluble mHTT aggregates and neurodegeneration. We also demonstrate a quantitative relationship between neurodegeneration and soluble monomeric (but not oligomeric or aggregated) mHTT levels. Reduction of ∼10% of mHTT is sufficient to prevent toxicity,whereas ∼90% reduction of wild-type HTT is safe and well-tolerated in these cells. A known HD toxicity modifier (Rhes) showed expected rescue of neurodegeneration. Therefore,the hESC-derived neuronal models complement existing induced pluripotent stem cell-derived neuronal models and provide valuable tools for HD research.—Lu,B.,Palacino,J. A novel human embryonic stem cell-derived Huntington's disease neuronal model exhibits mutant huntingtin (mHTT) aggregates and soluble mHTT-dependent neurodegeneration. View Publication

Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However,conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here,primary macrophages isolated from Atrx(f/f) mice were infected with adenovirus expressing Cre recombinase or β-galactosidase,and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal,anti-Fas antibody,C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally,we demonstrate that multiple primary cell types (myoblasts,embryonic fibroblasts and neurospheres) were sensitive to 5-FU,cisplatin,and UV light treatment. Together,our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover,it identifies potential treatment options for cancers associated with ATRX mutations,including glioblastoma and pancreatic neuroendocrine tumors. View Publication

Microtubules (MTs),key cytoskeletal elements in living cells,are critical for axonal transport,synaptic transmission,and maintenance of neuronal morphology. NAP (NAPVSIPQ) is a neuroprotective peptide derived from the essential activity-dependent neuroprotective protein (ADNP). In Alzheimer's disease models,NAP protects against tauopathy and cognitive decline. Here,we show that NAP treatment significantly affected the alpha tubulin tyrosination cycle in the neuronal differentiation model,rat pheochromocytoma (PC12) and in rat cortical astrocytes. The effect on tubulin tyrosination/detyrosination was coupled to increased MT network area (measured in PC12 cells),which is directly related to neurite outgrowth. Tubulin beta3,a marker for neurite outgrowth/neuronal differentiation significantly increased after NAP treatment. In rat cortical neurons,NAP doubled the area of dynamic MT invasion (Tyr-tubulin) into the neuronal growth cone periphery. NAP was previously shown to protect against zinc-induced MT/neurite destruction and neuronal death,here,in PC12 cells,NAP treatment reversed zinc-decreased tau-tubulin-MT interaction and protected against death. NAP effects on the MT pool,coupled with increased tau engagement on compromised MTs imply an important role in neuronal plasticity,protecting against free tau accumulation leading to tauopathy. With tauopathy representing a major pathological hallmark in Alzheimer's disease and related disorders,the current findings provide a mechanistic basis for further development. NAP (davunetide) is in phase 2/3 clinical trial in progressive supranuclear palsy,a disease presenting MT deficiency and tau pathology. View Publication

Skin-derived precursor (SKP) cells are a valuable resource for tissue engineering and regenerative medicine,because they represent multipotent stem cells that differentiate into neural and mesodermal progenies. Previous studies suggest that the stem cell pool decreases with age. Here,we show that human multipotent SKP cells can be efficiently collected from adult cheek/chin skin,even in aged individuals of 70-78years. SKP cells were isolated from 38 skin samples by serum-free sphere culture and examined for the ability to differentiate into neural and mesodermal lineages. The number of spheres obtained from adult facial skin was significantly higher than that of trunk or extremity skin. SKP cells derived from cheek/chin skin exhibited a high ability to differentiate into neural and mesodermal cells relative to those derived from eyelid,trunk,or extremity skin. Furthermore,cheek/chin skin SKP cells were shown to express markers for undifferentiated stem cells,including a high expression level of the Sox9 gene. These results indicate that cheek/chin skin is useful for the recovery of multipotent stem cells for tissue engineering and regenerative therapy. View Publication