技术资料

OBJECTIVE To develop in vitro adenoid cystic carcinoma cell line as a surrogate for functional studies. MATERIALS AND METHODS Cells obtained from a primary ACC of the base of tongue were cultivated in vitro and immortalized with h-TERT. Morphologic,cytogenetic and functional studies were performed. RESULTS Tumor cells were verified by positive reactions to keratin and smooth muscle actin and phenotypic cellular and nuclear features. In-vitro cell growth and colony formation assay supported their tumor nature. CONCLUSION We authenticated an ACC cell line with hybrid epithelial-myoepithelial feature as a resource for functional experimentation. View Publication

Inflammatory activation precedes and correlates with accumulating τ lesions in Alzheimer's disease and tauopathies. However,the relationship between neuroinflammation and etiology of pathologic τ remains elusive. To evaluate whether inflammatory signaling may promote or accelerate neurofibrillary tangle pathology,we explored the effect of recombinant adeno-associated virus (rAAV)-mediated overexpression of a master inflammatory cytokine,IFN-γ,on τ phosphorylation. In initial studies in primary neuroglial cultures,rAAV-mediated expression of IFN-γ did not alter endogenous τ production or paired helical filament τ phosphorylation. Next,we tested the effect of rAAV-mediated expression of IFN-γ in the brains of 2 mouse models of tauopathy: JNPL3 and rTg4510. In both models,IFN-γ increased 1) signal transducer and activator of transcription 1 levels and gliosis,and 2) hyperphosphorylation and conformational alterations of soluble τ compared with control cohorts. However,sarkosyl-insoluble phosphorylated τ levels and ubiquitin staining were unaltered in the IFN-γ cohorts. Notably,IFN-γ-induced τ hyperphosphorylation was associated with release of the inhibitory effect of glycogen synthase kinase 3β function by decreasing Ser9 phosphorylation. Our data suggest that type II IFN signaling can promote τ phosphorylation by modulating cellular kinase activity,though this is insufficient in accelerating neuritic tangle pathology. View Publication

A number of genes regulate regeneration of peripheral axons,but their ability to drive axon growth and regeneration in the central nervous system (CNS) remains largely untested. To address this question we overexpressed eight transcription factors and one small GTPase alone and in pairwise combinations to test whether combinatorial overexpression would have a synergistic impact on CNS neuron neurite growth. The Jun oncogene/signal transducer and activator of transcription 6 (JUN/STAT6) combination increased neurite growth in dissociated cortical neurons and in injured cortical slices. In injured cortical slices,JUN overexpression increased axon growth to a similar extent as JUN and STAT6 together. Interestingly,JUN overexpression was not associated with increased growth associated protein 43 (GAP43) or integrin alpha 7 (ITGA7) expression,though these are predicted transcriptional targets. This study demonstrates that JUN overexpression in cortical neurons stimulates axon growth,but does so independently of changes in expression of genes thought to be critical for JUNs effects on axon growth. We conclude that JUN activity underlies this CNS axonal growth response,and that it is mechanistically distinct from peripheral regeneration responses,in which increases in JUN expression coincide with increases in GAP43 expression. View Publication

The existence of a hyaluronic acid-rich node and duct system (HAR-NDS) within the lymphatic and blood vessels was demonstrated previously. The HAR-NDS was enriched with small (3.0-5.0 μm in diameter),adult stem cells with properties similar to those of the very small embryonic-like stem cells (VSELs). Sca-1(+)Lin(-)CD45(-) cells were enriched approximately 100-fold in the intravascular HAR-NDS compared with the bone marrow. We named these adult stem cells node and duct stem cells (NDSCs)." NDSCs formed colonies on C2C12 feeder layers were positive for fetal alkaline phosphatase and could be subcultured on the feeder layers. NDSCs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(+) while VSELs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(-). NDSCs had higher sphere-forming efficiency and proliferative potential than VSELs and they were found to differentiate into neuronal cells in vitro. Injection of NDSCs into mice partially repaired ischemic brain damage. Thus we report the discovery of potential adult stem cells that may be involved in tissue regeneration. The intravascular HAR-NDS may serve as a route that delivers these stem cells to their target tissues. View Publication

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors,most notably HIF2α (also known as EPAS1),are expressed in and required for maintenance of cancer stem cells (CSCs). However,the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex,displaces VHL-associated Cullin 2,and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma,ID2 positively modulates HIF2α activity. Conversely,elevated expression of DYRK1 phosphorylates Thr27 of ID2,leading to HIF2α destabilization,loss of glioma stemness,inhibition of tumour growth,and a more favourable outcome for patients with glioblastoma. View Publication

Network activity homeostatically alters synaptic efficacy to constrain neuronal output. However,it is unclear how such compensatory adaptations coexist with synaptic information storage,especially in established networks. Here,we report that in mature hippocampal neurons in vitro,network activity preferentially regulated excitatory synapses within the proximal dendrites of CA3 neurons. These homeostatic synapses exhibited morphological,functional,and molecular signatures of the specialized contacts between mossy fibers of dentate granule cells and thorny excrescences (TEs) of CA3 pyramidal neurons. In vivo TEs were also selectively and bidirectionally altered by chronic activity changes. TE formation required presynaptic synaptoporin and was suppressed by the activity-inducible kinase,Plk2. These results implicate the mossy fiber-TE synapse as an independently tunable gain control locus that permits efficacious homeostatic adjustment of mossy fiber-CA3 synapses,while preserving synaptic weights that may encode information elsewhere within the mature hippocampal circuit. View Publication

Dendritic protein synthesis plays a critical role in several forms of synaptic plasticity,including BDNF (brain-derived neurotrophic factor)-mediated long-term synaptic potentiation (LTP). Dendritic transcripts are typically transported in a repressed state as components of large ribonucleoprotein complexes,and then translated upon stimulation at,or in the vicinity,of activated synapses. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a trans-acting factor involved in dendritic mRNA trafficking,but how the distribution of the protein in dendrites is regulated has not been characterized. Here we found that a fraction of hnRNP A2/B1 is present at the synapse under resting conditions in cultured hippocampal neurons. Accordingly,this ribonucleoprotein was detected in free mRNP,monosomal,and polyribosomal fractions obtained from synaptoneurosomes. Neuronal activity and BDNF treatment increased hnRNP A2/B1 protein levels in the cell body and dendritic compartments,and induced the delivery of this protein to synaptic sites. The activity-dependent accumulation of hnRNP A2/B1 at the synapse required,at least in part,the activation of TrkB receptors,presumably by BDNF. This neurotrophin also upregulated the hnRNP A2/B1 mRNA in the soma but was without effect on the abundance of neuritic hnRNP A2/B1 transcripts. These results show that the distribution of hnRNP A2/B1 is regulated by BDNF and by neuronal activity,an effect that may have a role in BDNF-induced synaptic plasticity events. View Publication

Defects in the control of Wnt signaling have emerged as a recurrent mechanism involved in cancer pathogenesis and acute myeloid leukaemia (AML),including the hematopoietic regeneration-associated WNT10B in AC133bright leukaemia cells,although the existence of a specific mechanism remains unproven. We have obtained evidences for a recurrent rearrangement,which involved the WNT10B locus (WNT10BR) within intron 1 (IVS1) and flanked at the 5' by non-human sequences whose origin remains to be elucidated; it also expressed a transcript variant (WNT10BIVS1) which was mainly detected in a cohort of patients with intermediate/unfavorable risk AML. We also identified in two separate cases,affected by AML and breast cancer respectively,a genomic transposable short form of human WNT10B (ht-WNT10B). The intronless ht-WNT10B resembles a long non-coding RNA (lncRNA),which suggests its involvement in a non-random microhomology-mediated recombination generating the rearranged WNT10BR. Furthermore,our studies supports an autocrine activation primed by the formation of WNT10B-FZD4/5 complexes in the breast cancer MCF7 cells that express the WNT10BIVS1. Chemical interference of WNT-ligands production by the porcupine inhibitor IWP-2 achieved a dose-dependent suppression of the WNT10B-FZD4/5 interactions. These results present the first evidence for a recurrent rearrangement promoted by a mobile ht-WNT10B oncogene,as a relevant mechanism for Wnt involvement in human cancer. View Publication

Neurotrophins and their receptors are frequently expressed in malignant gliomas,yet their functions are largely unknown. Previously,we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However,the role of Trk receptors has not been examined. In this study,we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here,we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC,not TrkA,and they also express neurotrophins NGF,BDNF,and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely,TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further,pharmacological inhibition of both ERK and Akt pathways blocked BDNF,and NT3 stimulated BTIC survival. Importantly,attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling,and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma. View Publication

Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here,we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone,while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration,which led to functional recovery as measured by sustained gait improvement. Furthermore,no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy,gastrocnemius muscles from mice exhibited substantially less muscle atrophy,an increase in muscle mass after denervation,and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies. View Publication

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM,peritumor tissue (brain adjacent to tumor,BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased,regardless of the tumor cell presence,whereas GD3 correlated with neoplastic infiltration. In BAT,NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM. View Publication

Differentiated neurons can be rapidly acquired,within days,by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons,called iNGNs,which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation,including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2,called CatCh,we could control iNGN activity with blue light stimulation. In combination with optogenetic tools,iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity,and these networks had excitatory glutamatergic synapses,which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings,whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission,along with the ability to scale-up the size of the cultures. View Publication