技术资料

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here,we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal,proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive),neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells,as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs,an effect blocked by Notch pathway inhibition. Moreover,R-m-AEA treatment for 48 h,increased proliferation as assessed by BrdU incorporation assay,an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly,stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation),at 7 days,as shown by counting the number of NeuN-positive neurons in the cultures. Moreover,by monitoring intracellular calcium concentrations ([Ca(2+)]i) in single cells following KCl and histamine stimuli,a method that allows the functional evaluation of neuronal differentiation,we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251,for 7 days,thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation,R-m-AEA also increased neurite growth,as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together,these results demonstrate that CB1R activation induces proliferation,self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures. 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

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

Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals,they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However,little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ),we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months,the proliferative status of activated NSCs was already altered by 6 months,with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures,as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications. View Publication

Adverse effect of alcohol on neural function has been well documented. Especially,the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models,which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described,the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation,Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol's effect on JAK-STAT signaling pathway,neuroactive ligand-receptor interaction,Toll-like receptor (TLR) signaling pathway,cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3,which is associated with nociception,a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs,but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response. View Publication

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs,we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently,the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated,stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast,ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results. View Publication

Glioblastoma multiforme (GBM) is an aggressive,Grade IV astrocytoma with a poor survival rate,primarily due to the GBM tumor cells migrating away from the primary tumor site along the nanotopography of white matter tracts and blood vessels. It is unclear whether this nanotopography influences the biomechanical properties (i.e. cytoskeletal stiffness) of GBM tumor cells. Although GBM tumor cells have an innate propensity to migrate,we believe this capability is enhanced due to the influence of nanotopography on the tumor cells' biomechanical properties. In this study,we used an aligned nanofiber film that mimics the nanotopography in the tumor microenvironment to investigate the mechanical properties of GBM tumor cells in vitro. The data demonstrate that the cytoskeletal stiffness,cell traction stress,and focal adhesion area were significantly lower in the GBM tumor cells compared to healthy astrocytes. Moreover,the cytoskeletal stiffness was significantly reduced when cultured on aligned nanofiber films compared to smooth and randomly aligned nanofiber films. Gene expression analysis showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. Therefore,our data suggest that the migratory potential is elevated when GBM tumor cells are migrating along aligned nanotopographical substrates. View Publication

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain,including the variant M337V,which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool,we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions,which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA,cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells,thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS. View Publication

The understanding of the function of alpha(1)-adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant alpha(1)-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing alpha(1A)-adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult alpha(1A)-adrenergic receptor transgenic mice or normal mice given the alpha(1A)-adrenergic receptor-selective agonist,cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of alpha(1)-adrenergic receptor subtypes,and stimulation of these receptors resulted in increased expression of the alpha(1B)-adrenergic receptor subtype,proneural basic helix-loop-helix transcription factors,and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. alpha(1)-Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However,in adult normal neurospheres,alpha(1)-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo,S100-positive glial and betaIII tubulin neuronal progenitors colocalized with either alpha(1)-adrenergic receptor subtype in the olfactory bulb. Our results indicate that alpha(1)-adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases. View Publication

Slow-onset adaptive changes that arise from sustained antidepressant treatment,such as enhanced adult hippocampal neurogenesis and increased trophic factor expression,play a key role in the behavioral effects of antidepressants. alpha(2)-Adrenoceptors contribute to the modulation of mood and are potential targets for the development of faster acting antidepressants. We investigated the influence of alpha(2)-adrenoceptors on adult hippocampal neurogenesis. Our results indicate that alpha(2)-adrenoceptor agonists,clonidine and guanabenz,decrease adult hippocampal neurogenesis through a selective effect on the proliferation,but not the survival or differentiation,of progenitors. These effects persist in dopamine beta-hydroxylase knock-out (Dbh(-/-)) mice lacking norepinephrine,supporting a role for alpha(2)-heteroceptors on progenitor cells,rather than alpha(2)-autoreceptors on noradrenergic neurons that inhibit norepinephrine release. Adult hippocampal progenitors in vitro express all the alpha(2)-adrenoceptor subtypes,and decreased neurosphere frequency and BrdU incorporation indicate direct effects of alpha(2)-adrenoceptor stimulation on progenitors. Furthermore,coadministration of the alpha(2)-adrenoceptor antagonist yohimbine with the antidepressant imipramine significantly accelerates effects on hippocampal progenitor proliferation,the morphological maturation of newborn neurons,and the increase in expression of brain derived neurotrophic factor and vascular endothelial growth factor implicated in the neurogenic and behavioral effects of antidepressants. Finally,short-duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test,which normally requires 3 weeks of treatment with classical antidepressants. Our results demonstrate that alpha(2)-adrenoceptors,expressed by progenitor cells,decrease adult hippocampal neurogenesis,while their blockade speeds up antidepressant action,highlighting their importance as targets for faster acting antidepressants. View Publication

Monge's disease,also known as chronic mountain sickness (CMS),is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%,suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however,CMS subjects experience severe hypoxemia,erythrocytosis and many neurologic manifestations including migraine,headache,mental fatigue,confusion,and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS,except for phlebotomy. In the current study,we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs),then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties,but instead by a significantly lowered Na+ channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide,for the first time,evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects,hoping that such studies can pave the way to a better understanding of the neuropathology in CMS. View Publication

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup),carrying different duplication sizes,to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition,using multi-electrodes arrays,we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level,we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor,NCH-51,was validated as a potential clinical candidate. Interestingly,this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders. View Publication