技术资料

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication

Overexpression of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A),encoded by a gene located in the Down syndrome (DS) critical region,is considered a major contributor to developmental abnormalities in DS. DYRK1A regulates numerous genes involved in neuronal commitment,differentiation,maturation,and apoptosis. Because alterations of neurogenesis could lead to impaired brain development and mental retardation in individuals with DS,pharmacological normalization of DYRK1A activity has been postulated as DS therapy. We tested the effect of harmine,a specific DYRK1A inhibitor,on the development of neuronal progenitor cells (NPCs) isolated from the periventricular zone of newborn mice with segmental trisomy 16 (Ts65Dn mice),a mouse model for DS that overexpresses Dyrk1A by 1.5-fold. Trisomy did not affect the ability of NPCs to expand in culture. Twenty-four hours after stimulation of migration and neuronal differentiation,NPCs showed increased expression of Dyrk1A,particularly in the trisomic cultures. After 7 days,NPCs developed into a heterogeneous population of differentiating neurons and astrocytes that expressed Dyrk1A in the nuclei. In comparison with disomic cells,NPCs with trisomy showed premature neuronal differentiation and enhanced γ-aminobutyric acid (GABA)-ergic differentiation,but astrocyte development was unchanged. Harmine prevented premature neuronal maturation of trisomic NPCs but not acceleration of GABA-ergic development. In control NPCs,harmine treatment caused altered neuronal development of NPCs,similar to that in trisomic NPCs with Dyrk1A overexpression. This study suggests that pharmacological normalization of DYRK1A activity may have a potential role in DS therapy. View Publication

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope,since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but,in contrast to over-expression systems,cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8,in agreement with the observed reduction of VAPB in sporadic ALS. View Publication

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene,whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons,but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types,including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation,affected by early onset seizure variant and X-linked epileptic encephalopathy,respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore,the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. View Publication

Interferon beta (IFN-β) is a mainline treatment for multiple sclerosis (MS); however its exact mechanism of action is not completely understood. IFN-β is known as an immunomodulator; although recent evidence suggests that IFN-β may also act directly on neural stem/progenitor cells (NPCs) in the central nervous system (CNS). NPCs can differentiate into all neural lineage cells,which could contribute to the remyelination and repair of MS lesions. Understanding how IFN-β influences NPC physiology is critical to develop more specific therapies that can better assist this repair process. In this study,we investigated the effects of IFN β-1b (Betaseron®) on human NPCs in vitro (hNPCs). Our data demonstrate a dose-dependent response of hNPCs to IFN β-1b treatment via sustained proliferation and differentiation. Furthermore,we offer insight into the signaling pathways involved in these mechanisms. Overall,this study shows a direct effect of IFN β-1b on hNPCs and highlights the need to further understand how current MS treatments can modulate endogenous NPC populations within the CNS. View Publication

Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication

The integration of microscale engineering,microfluidics,and AC electrokinetics such as dielectrophoresis has generated novel microsystems that enable quantitative analysis of cellular phenotype,function,and physiology. These systems are increasingly being used to assess diverse cell types,such as stem cells,so it becomes critical to thoroughly evaluate whether the systems themselves impact cell function. For example,engineered microsystems have been utilized to investigate neural stem/progenitor cells (NSPCs),which are of interest due to their potential to treat CNS disease and injury. Analysis by dielectrophoresis (DEP) microsystems determined that unlabeled NSPCs with distinct fate potential have previously unrecognized distinguishing electrophysiological characteristics,suggesting that NSPCs could be isolated by DEP microsystems without the use of cell type specific labels. To gauge the potential impact of DEP sorting on NSPCs,we investigated whether electric field exposure of varying times affected survival,proliferation,or fate potential of NSPCs in suspension. We found short-term DEP exposure (1 min or less) had no effect on NSPC survival,proliferation,or fate potential revealed by differentiation. Moreover,NSPC proliferation (measured by DNA synthesis and cell cycle kinetics) and fate potential were not altered by any length of DEP exposure (up to 30 min). However,lengthy exposure (textgreater5 min) to frequencies near the crossover frequency (50-100 kHz) led to decreased survival of NSPCs (maximum ∼30% cell loss after 30 min). Based on experimental observations and mathematical simulations of cells in suspension,we find that frequencies near the crossover frequency generate an induced transmembrane potential that results in cell swelling and rupture. This is in contrast to the case for adherent cells since negative DEP frequencies lower than the crossover frequency generate the highest induced transmembrane potential and damage for these cells. We clarify contrasting effects of DEP on adherent and suspended cells,which are related to the cell position within the electric field and the strength of the electric field at specific distances from the electrodes. Modeling of electrode configurations predicts optimal designs to induce cell movement by DEP while limiting the induced transmembrane potential. We find DEP electric fields are not harmful to stem cells in suspension at short exposure times,thus providing a basis for developing DEP-based applications for stem cells. View Publication

Stochastic processes and imprinting,along with genetic factors,lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system,and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition,allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates,such as A2BP1 (RBFOX1),ERBB4,NLGN4X,NRG1,NRG3,NRXN1,and NLGN1. Overall,there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square,p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance,the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications. View Publication

Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice,but their identity,origin,and potential survival in the brain are only vaguely understood. In this work,we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia,including NG2,PDGFRα,O4,NGF receptor (p75),glutamate receptor-1(AMPA),and A2B5 expression. Although these cells exhibit NG2,they do not express other pericyte markers,such as α-SMA or connexin-43,and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents,probably carried through Kir6.1 channels. Given their potential therapeutic application,we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes,we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan. View Publication

Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression,including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here,we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus,and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS(-50kbLoop) was enriched with nucleosomes epigenetically decorated with the transcriptional mark,histone H3 trimethylated at lysine 4,and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia,GAD1-TSS(-50kbLoop) was decreased compared with controls,in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS(-55kbLoop),the murine homolog to GAD1-TSS(-50kbLoop),is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture,Gad1-TSS(-55kbLoop) and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures,including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression,are conserved between the rodent and primate brain,and subject to developmental and activity-dependent regulation,and disordered in some cases with schizophrenia. More broadly,the findings presented here draw a connection between noncoding DNA,spatial genome architecture,and neuronal plasticity in development and disease. 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

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival,these regimens are highly toxic and are associated with significant morbidity. Here,we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas,independent of their subtype. Moreover,high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo,resulting in medulloblastoma regression,decreased metastasis,and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh,PlGF,and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway. View Publication