技术资料

The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α,CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain,the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However,both of them have limitations in the analysis of a single cell. In this study,we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12,NPCs formed lamellipodia in the stripe assay. Furthermore,CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia,indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry,they can also generate live imaging data with comparable quality. In conclusion,stripe assay is a visual,dynamic and economical tool to study cellular mobility and its related molecule mechanisms. View Publication

H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. View Publication

Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs,voltage-gated delayed rectifier K(+) (KDR) channels are dominantly expressed. However,the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1,a high voltage-gated KDR channel,was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties,such as resting membrane potential,of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation,not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation. View Publication

Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3'-untranslated region (3' UTR) of the DMPK gene. Correcting the mutation in DM1 stem cells would be an important step toward autologous stem cell therapy. The objective of this study is to demonstrate in vitro genome editing to prevent production of toxic mutant transcripts and reverse phenotypes in DM1 stem cells. Genome editing was performed in DM1 neural stem cells (NSCs) derived from human DM1 induced pluripotent stem (iPS) cells. An editing cassette containing SV40/bGH polyA signals was integrated upstream of the CTG repeats by TALEN-mediated homologous recombination (HR). The expression of mutant CUG repeats transcript was monitored by nuclear RNA foci,the molecular hallmarks of DM1,using RNA fluorescence in situ hybridization. Alternative splicing of microtubule-associated protein tau (MAPT) and muscleblind-like (MBNL) proteins were analyzed to further monitor the phenotype reversal after genome modification. The cassette was successfully inserted into DMPK intron 9 and this genomic modification led to complete disappearance of nuclear RNA foci. MAPT and MBNL 1,2 aberrant splicing in DM1 NSCs were reversed to normal pattern in genome-modified NSCs. Genome modification by integration of exogenous polyA signals upstream of the DMPK CTG repeat expansion prevents the production of toxic RNA and leads to phenotype reversal in human DM1 iPS-cells derived stem cells. Our data provide proof-of-principle evidence that genome modification may be used to generate genetically modified progenitor cells as a first step toward autologous cell transfer therapy for DM1. View Publication

The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here,we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion,and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly,proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target,suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma. 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

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

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

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