技术资料

Since the discovery of neural stem cells (NSC) in the embryonic and adult mammalian central nervous system (CNS),there have been a growing numbers of tissue culture media and protocols to study and functionally characterize NSCs and its progeny in vitro. One of these culture systems introduced in 1992 is referred to as the Neurosphere Assay,and it has been widely used to isolate,expand,differentiate and even quantify NSC populations. Several years later because its application as a quantitative in vitro assay for measuring NSC frequency was limited,a new single-step semisolid based assay,the Neural Colony Forming Cell (NCFC) assay was developed to accurately measure NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e.,their proliferative potential). The evolution and continued improvements made to these tissue culture tools will facilitate further advances in the promising application of NSCs for therapeutic use. View Publication

Exercise has been shown to positively augment adult hippocampal neurogenesis; however,the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here,we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely,microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1,a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor,CX(3)CR1,but not control IgG,dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running,reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity,a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus,and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process. View Publication

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury,as well as neurodegenerative conditions such as Parkinson's disease,Alzheimer's disease,and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However,the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly,in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore,we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-κB-dependent mechanism. Specifically,we show that NF-κB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-κB inhibitor BAY-117082. Importantly,we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs,indicating that Puma induction is required for NPC death. Consistent with this,we demonstrate that Puma-deficient NPCs exhibit an 13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary,we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions. View Publication

Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains,microglia,the immune cells of the brain,are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties,we initially screened the most commonly used serotypes,AAV1-9 and rh10,on primary mouse microglia cultures. While these capsids were not permissive,we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed,these newly characterized rAAV6 capsid variants,specially a triply mutated Y731F/Y705F/T492V form,carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein,albeit at modest levels. We further show that CD68 promoter-driven expression of the inflammatory cytokine,interleukin-6,using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies. View Publication

The discovery of induced pluripotent stem cells (iPSCs) and their application to patient-specific disease models offers new opportunities for studying the pathophysiology of neurological disorders. However,current methods for culturing iPSC-derived neuronal cells result in clustering of neurons,which precludes the analysis of individual neurons and defined neuronal networks. To address this challenge,cultures of human neurons on micropatterned surfaces are developed that promote neuronal survival over extended periods of time. This approach facilitates studies of neuronal development,cellular trafficking,and related mechanisms that require assessment of individual neurons and specific network connections. Importantly,micropatterns support the long-term stability of cultured neurons,which enables time-dependent analysis of cellular processes in living neurons. The approach described in this paper allows mechanistic studies of human neurons,both in terms of normal neuronal development and function,as well as time-dependent pathological processes,and provides a platform for testing of new therapeutics in neuropsychiatric disorders. View Publication

Recently there has been growing interest in the differentiation of mesenchymal stem cells (MSCs) into neural lineages. Research suggests that MSCs can be differentiated into neural progenitor-like cells (NPCs) under the specific influence of paracrine factors particularly epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Our recent research has found that the addition of insulin-like growth factor 1 (IGF-1) with the combination of the EGF and bFGF could significantly improve the growth and survivability of MSC-derived NPCs. To unravel the molecular mechanism of the improved differentiation we compared the microRNA expression profiles of the differentiation under various combinations of growth factors. MSCs were differentiated into neural lineage in 3 groups; Group A (EGF + bFGF),Group B (EGF + bFGF + IGF-1),and Group C (without growth factor). Regulated microRNAs during the early differentiation were identified by detailed microRNA profiling using Affymetrix GeneChip version 2.0 at three time intervals (day 1,day 3 and day 5). The data were deposited in the Gene Expression Omnibus,series GSE60060. View Publication

Insulin-like growth factor 1 (IGF-1) enhances cellular proliferation and reduces apoptosis during the early differentiation of bone marrow derived mesenchymal stem cells (BMSCs) into neural progenitor-like cells (NPCs) in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). BMSCs were differentiated in three groups of growth factors: (A) EGF + bFGF,(B) EGF + bFGF + IGF-1,and (C) without growth factor. To unravel the molecular mechanisms of the NPCs derivation,microarray analysis using GeneChip miRNA arrays was performed. The profiles were compared among the groups. Annotated microRNA fingerprints (GSE60060) delineated 46 microRNAs temporally up-regulated or down-regulated compared to group C. The expressions of selected microRNAs were validated by real-time PCR. Among the 46 microRNAs,30 were consistently expressed for minimum of two consecutive time intervals. In Group B,only miR-496 was up-regulated and 12 microRNAs,including the let-7 family,miR-1224,miR-125a-3p,miR-214,miR-22,miR-320,miR-708,and miR-93,were down-regulated. Bioinformatics analysis reveals that some of these microRNAs (miR-22,miR-214,miR-125a-3p,miR-320 and let-7 family) are associated with reduction of apoptosis. Here,we summarize the roles of key microRNAs associated with IGF-1 in the differentiation of BMSCs into NPCs. These findings may provide clues to further our understanding of the mechanisms and roles of microRNAs as key regulators of BMSC-derived NPC maintenance. View Publication

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However,it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here,we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons,we observed rapid translocation of a subpopulation of MAP2,present in dendritic shafts,to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently,immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore,LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether,this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks. View Publication

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication

Glioblastoma (GBM) and other malignant gliomas are aggressive primary neoplasms of the brain that exhibit notable refractivity to standard treatment regimens. Recent large-scale molecular profiling has revealed distinct disease subclasses within malignant gliomas whose defining genomic features highlight dysregulated molecular networks as potential targets for therapeutic development. The proneural" designation represents the largest and most heterogeneous of these subclasses� View Publication

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs,miR-375,was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally,miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly,motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly,SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons. View Publication

Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain,but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging,in contrast to iPSC differentiation. Here,we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression,impaired axonal mitochondrial morphologies,lower mitochondrial membrane potentials,reduced energy production,and increased oxidized proteins levels. In contrast,the fibroblasts from which iNs were generated show only mild age-dependent changes,consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed,OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging. View Publication