技术资料

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

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

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

Herein,we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors,decreased stemness,and increased proneurogenic gene expression. Importantly,this work reports for the first time a nanoparticle formulation able to modulate in vivo the subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases. View Publication

UNLABELLED A noninvasive technology that indiscriminately detects tumor tissue in the brain could substantially enhance the management of primary or metastatic brain tumors. Although the documented molecular heterogeneity of diseases that initiate or eventually deposit in the brain may preclude identifying a single smoking-gun molecular biomarker,many classes of brain tumors are generally avid for transferrin. Therefore,we reasoned that applying a radiolabeled derivative of transferrin ((89)Zr-labeled transferrin) may be an effective strategy to more thoroughly identify tumor tissue in the brain,regardless of the tumor's genetic background. METHODS Transferrin was radiolabeled with (89)Zr,and its properties with respect to human models of glioblastoma multiforme were studied in vivo. RESULTS In this report,we show proof of concept that (89)Zr-labeled transferrin ((89)Zr-transferrin) localizes to genetically diverse models of glioblastoma multiforme in vivo. Moreover,we demonstrate that (89)Zr-transferrin can detect an orthotopic lesion with exceptional contrast. Finally,the tumor-to-brain contrast conferred by (89)Zr-transferrin vastly exceeded that observed with (18)F-FDG,currently the most widely used radiotracer to assess tumor burden in the brain. CONCLUSION The results from this study suggest that (89)Zr-transferrin could be a broadly applicable tool for identifying and monitoring tumors in the brain,with realistic potential for near-term clinical translation. 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

Activation of neural stem/progenitor cells (NSPCs) is a potential therapeutic strategy of neurological disorders. In this study,NSPCs of subventricular zone were isolated and cultured from platelet-derived growth factor-β-receptor-knockout (PDGFR-β(-/-)) mice of postnatal day 1 (P1) and P28,and the roles of PDGFR-β were examined in these cells. In PDGFR-β-preserving control NSPCs,stem cell activities,such as numbers and diameters of secondary neurospheres,cell proliferation and survival rates,were significantly higher in P1 NSPCs than those in P28 NSPCs. In PDGFR-β(-/-) NSPCs,most of these parameters were decreased as compared with age-matched controls. Among them,the decrease of secondary neurosphere formation was most striking in P1 and P28 PDGFR-β(-/-) NSPCs and in P28 control NSPCs as compared with P1 control NSPCs. PCR-array and following quantitative real-time PCR (qRT-PCR) analyses demonstrated that expressions of fibroblast growth factor-2 (FGF2) and exons IV-IX of brain-derived neurotrophic factor (BDNF) were decreased,and noggin was increased in P1 PDGFR-β(-/-) as compared with P1 controls. Addition of BDNF rescued the number and diameter of secondary neurospheres in P1 PDGFR-β(-/-) NSPCs to similar levels as controls. The expressions of PDGFs and PDGFRs in control NSPCs were increased along with the differentiation-induction,where phosphorylated PDGFR-β was co-localized with neuronal and astrocyte differentiation markers. In controls,the neuronal differentiation was decreased,and the glial differentiation was increased from P1 to P28 NSPCs. Compared with P1 controls,neuronal differentiation was reduced in P1 PDGFR-β(-/-) NSPCs,whereas glial differentiation was comparable between the two genotypes. These results suggest that PDGFR-β signaling is important for the self-renewal and multipotency of NSPCs,particularly in neonatal NSPCs. BDNF,FGF2,and noggin may be involved in the effects of PDGFR-β signaling in these cells. Accordingly,the activation of PDGFR-β in NSPCs may be a novel therapeutic strategy of neurological diseases. View Publication

Huntington's disease is caused by expanded CAG repeats in HTT,conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model,and 6 exhibited activity consistent with the in vitro screening results. Among these,negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment,interferon-β lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus,we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease. View Publication