Doublecortin Research Articles

Alterations in Doublecortin Expression in Human Neuronal Stem Cells in Response to Angiotensinergic Stimulation in Proliferation and Differentiation Conditions

Activation or inhibition of the renin‐angiotensin system (RAS) affects neuronal function and viability. We showed previously that AT2 selective stimulation of human neuronal stem cells (H‐9 derived, Life Technologies) increased cell numbers in both differentiation and proliferation medium compared to control and AT1 stimulated human neuronal stem cells. We now show that doublecortin expression in human neuronal stem cells which were seeded in chamber slides and cultured under proliferation (with EGF and FGF‐2) or differentiation (no added growth factors) conditions is altered with chronic exposure to AT1 and AT2 selective agonists. Conditions included 14 days treatment with once daily addition of the AT2 selective agonist CGP42112 (final concentration 100 nM); once daily treatment with the non‐selective Ang II receptor agonist Sar1 angiotensin II (100 nM) plus the AT2 receptor selective antagonist PD123319 (10 μM) for selective AT1 receptor stimulation; once daily treatment with PD123319 (10 μM) alone; or once daily treatment with vehicle. Following treatment, cells were immunostained for the neuroblast marker doublecortin. Doublecortin expression was reduced by AT1 selective stimulation in differentiation conditions relative to control (Control 70.9 ± 17% and AT1 14.0 ± 6.2%, p<0.01), AT2 stimulated (AT2 86.7 ± 0.6%, p<0.01) and AT2 antagonized with PD123319 (PD123319 64.4 ± 9.3%, p<0.05). In proliferation conditions, doublecortin expression was reduced by AT2 selective stimulation and AT2 antagonism compared to control and AT1 selectively stimulated cells (p<0.01: Control 96.5 ± 1.1% ; AT1 88.7 ± 1.6% ; AT2 9.1 ± 6.1% ; PD123319 2.6 ± 1.0%). These results suggest that angiotensinergic inhibition of doublecortin expression is dependent upon the presence of the growth factors EGF and FGF‐2.Support or Funding InformationR.C. Speth: Research Grant; Significant; NIH HL‐113905; Cardiovascular Neuroscience Research Fund, Nova Southeastern University. J. Munoz: President's Faculty Research Development Grant, Nova Southeastern University.

Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model

Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke.

The role of human umbilical cord blood-derived mesenchymal stem cells in improving the neurological function of acute ischemic stroke model of rats

Objective To investigate the role of human umbilical cord blood(UCB)-derived mesenchymal stem cells(MSCs) in improving the neurological function of acute ischemic stroke model of rats and mechanisms. Methods The middle cerebral artery occulusion(MCAO) models in 82 rats were randomly divided into UCB- MSCs group(n= 41)and control group(n= 41).Human UCB- MSCs were isolated and cultured, and identified by nestin staining. The UCB- MSCs were transplanted into the ventricles of rats 48 h after modeling and marked.At 24th h, 7th day and 14 day after transplantation, the neurological functions were assessed by using improved neurological function in rats(m NSS).The proliferation of endogenous neural stem cells and expression of vascular endothelial growth factor(VEGF) were detected by using immunofluorescence method. Results UCB-MSCs were successfully separated in 42 parts.At 7th day and 14th day after transplantation, the function scores of rats in UCB- MSCs group were 6.76±0.42 and 4.27±0.49, which were statistically significantly lower than in the control group(P< 0.05).At 14th day after transplantation, the number of 5- bromo 2'- deoxyuridine(Brd U)/doublecortin(DCX)and Brd U/glial fibrillary acidic protein(GFAP) double labeled positive cells was 74.31±3.04 and 65.38±2.43 respectively, which were significantly greater than in the control group(P< 0.05).The expression of neurotrophic factor VEGF in UCB- MSCs group was 109.53±5.91, significantly higher than in the control group(P< 0.05). Conclusion UCB-MSCs transplantation could effectively promote neurological function recovery of acute ischemic stroke model possibly by promoting the proliferation of the endogenous neural stem cells and neurotrophic factor VEGF release. Key words: Human umbilical cord blood-derived mesenchymal stem cells; Ischemic stroke; Neurological function

The study of neurogenesis contributed by the expression of survivin in hippocampus after traumatic brain injury

Objective To investigate the expression of survivin after traumatic brain injury(TBI);and the relationship between the up- regulation of survivin and endogenous neurogenesis. Further to demonstrate the role of increased survivin to promote the adult neurogenesis, and detect the possible mechanism in this process.Finally to find the approach that can promote restoration of nervous function after TBI. Methods A-dult male C57BL/6 mice, total n= 80, were randomly divided into sham- operation group and TBI group. Mice in TBI and sham- operation group were sacrificed at 12 hours, 1, 2, 5, 7 and 14 days post TBI and sham- operation(n= 6 for each point), and hippocampus tissues from the ipsilateral hemispheres were carried out for Western blotting.Double- label immunofluorescence staining was adopted to detect survivin expression in difference kinds of cells in the hippocampus(n= 4). Results The up- regulation of survivin protein starting at day 1(42 003.15, P<0.05)and lasting to day 7, peaking at day 2(90 403.34, P<0.01). The number of survivin+, Brd U+ and doublecortin(DCX+) cells in subgranular zone(SGZ) of ipsilateral hippocampus was significantly increased after TBI by immunofluorescence staining. The results also revealed that the majority of survivin+ cells were BrdU+ cells,and a part of survivin+ cells were DCX+ cells in SGZ of dentate gyrus(DG)in the hippocampus. Conclusion Survivin gene was activated follow brain injury. The expression of survivin protein were increased in the hippocampus after TBI. Increased survivin protein is expressed in neural stem cells and immature neurons in ipsilateral hippocampus after TBI, which correlate to the proliferation of neurogenic cell in SGZ of hippocampus. Key words: Survivin; Traumatic brain injury; Hippocampus; Adult neurogenesis

Effect of neural stem cells transplantation on protein expressions of doublecortin and nestin in hippocampal regions in aPP/PS1 mice

Objective To explore the effect of neural stem cells (NSCs) transplantation on neurogenesis in an alzheimer'disease mouse model and its related mechanism. Methods A total of 16 12-month-old aPP/PS1 double transgenic aD mice were randomly divided into two groups: NSC group (receiving NSCs transplantation in the bilateral hippocampi, n=8) and PBS group as the negative control group (receiving an equal quantity of 0.01M/L phosphate buffer saline, n=8). another 8 wild type mice without any treatment were selected as the positive control group (Wt group). at five weeks after transplantation, the expressions of doublecortin (DCX) and nestin in the hippocampal dentate gyrus (DG) and subgranular zone (SGZ) were analyzed by immunofluorescence staining, the number of positive cells in these regions were counted by confocal microscopy, and Morris water maze (MWM) test was used to assess cognitive function in all mice. Results NSC group showed the enhanced spatial learning and memory ability in Morris water maze (MWM) as compared to PBS group, but it was still lower than that in Wt mice (both P 0.05). Conclusions NSCs transplantation can promote endogenous neurogenesis via the increased expression of DCX- and Nestin-positive cells in the hippocampal DG/SGZ in aD mice, which improves the cognitive abilities in aD mice in some extent. Key words: Alzheimer disease; Stem cells transplantation

Glial-like differentiation potential of human mature adipocytes.

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here, we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes, suggesting that these cells could be committed to the neural lineage. After neural induction, NeuroD1, Sox1, Double Cortin, and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties, indicating the inability of these cells to differentiate into mature neurons. In contrast, the differentiated cells expressed a high level of CLDN11, as demonstrated using molecular method, and stained positively for the glial cell markers CLDN11 and GFAP, as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production, which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion, our data demonstrate morphological, molecular, and immunocytochemical evidence of initial neural differentiation of mature adipocytes, committing to a glial lineage.

Examination of Age‐dependent effects of fetal ethanol exposure on behavior, hippocampal cell counts, and doublecortin immunoreactivity in rats

Ethanol is known as a potent teratogen having adverse effects on brain and behavior. However, some of the behavioral deficits caused by fetal alcohol exposure and well expressed in juveniles ameliorate with maturation may suggest some kind of functional recovery occurring during postnatal development. The aim of this study was to reexamine age-dependent behavioral impairments in fetal-alcohol rats and to investigate the changes in neurogenesis and gross morphology of the hippocampus during a protracted postnatal period searching for developmental deficits and/or delays that would correlate with behavioral impairments in juveniles and for potential compensatory processes responsible for their amelioration in adults. Ethanol was delivered to the pregnant dams by intragastric intubation throughout 7-21 gestation days at daily dose of 6 g/kg. Isocaloric intubation and intact control groups were included. Locomotor activity, anxiety, and spatial learning tasks were applied to juvenile and young-adult rats from all groups. Unbiased stereological estimates of hippocampal volumes, the total number of pyramidal and granular cells, and double cortin expressing neurons were carried out for postnatal days (PDs) PD1, PD10, PD30, and PD60. Alcohol insult during second trimester equivalent caused significant deficits in the spatial learning in juvenile rats; however, its effect on hippocampal morphology was limited to a marginally lower number of granular cells in dentate gyrus (DG) on PD30. Thus, initial behavioral deficits and the following functional recovery in fetal-alcohol subjects may be due to more subtle plastic changes within the hippocampal formation but also in other structures of the extended hippocampal circuit. Further investigation is required.

Decreased neurogenesis in the Dentate Gyrus following sensory non‐normative overstimulation.

Human infants are increasingly exposed to excessive fast‐paced television viewing leading to sensory overstimulation, which may result in significant deficits in cognition and attention. We tested this hypothesis in a mouse model in which young mice (P10) were exposed to audio and visual overstimulation for six hours per day for a total of 42 days. Overstimulation had detrimental effects resulting in diminished cognition, hyperactivity and increased risk taking (Christakis et al. 2012). Prior studies have shown that exposing mice to an enriched environment (EE) results in improved learning and memory and increased neurogenesis in the dentate gyrus. Here we demonstrate, that sensory overstimulation had the opposite effect and resulted in diminished learning abilities and decreased neurogenesis within the dentate gyrus. Decreased neurogenesis was demonstrated by staining brains of overstimulated and control mice with the antigen KI67 (Ki67), T‐Box Brain Protein 2 (Tbr2), SRY (sex determining region Y)‐box 2 (Sox2) and Double Cortin (DCX). We conclude that excessive non‐ normative stimulation has significant detrimental effects on neurogenesis. Interestingly, this effect was only seen when overstimulating mice as neonates, but not as adults.

Hippocampal plasticity after a vagus nerve injury in the rat.

Stimulation of the vagus nerve has been previously reported to promote neural plasticity and neurogenesis in the brain. Several studies also revealed plastic changes in the spinal cord after injuries to somatosensory nerves originating from both the brachial and lumbo-sacral plexuses. However, the neurogenic responses of the brain to the injury of the viscerosensory innervation are not as yet well understood. In the present study, we investigated whether cells in the dentate gyrus of the hippocampus respond to a chemical and physical damage to the vagus nerve in the adult rat. Intraperitoneal capsaicin administration was used to damage non-myelinated vagal afferents while subdiaphragmatic vagotomy was used to damage both the myelinated and non-myelinated vagal afferents. The 5-bromo-2-deoxyuridine (BrdU) incorporation together with cell-specific markers was used to study neural proliferation in subgranular zone, granule cell layer, molecular layer and hilus of the dentate gyrus. Microglia activation was determined by quantifying changes in the intensity of fluorescent staining with a primary antibody against ionizing calcium adapter-binding molecule 1. Results revealed that vagotomy decreased BrdU incorporation in the hilus 15 days after injury compared to the capsaicin group. Capsaicin administration decreased BrdU incorporation in the granular cell layer 60 days after the treatment. Capsaicin decreased the number of doublecortin-expressing cells in the dentate gyrus, whereas vagotomy did not alter the expression of doublecortin in the hippocampus. Both the capsaicin- and the vagotomy-induced damage to the vagus nerve decreased microglia activation in the hippocampus at 15 days after the injury. At 30 days post injury, capsaicin-treated and vagotomized rats revealed significantly more activated microglia. Our findings show that damage to the subdiaphragmatic vagus in adult rats is followed by microglia activation and long-lasting changes in the dentate gyrus, leading to alteration of neurogenesis.

Effect of iodine deficiency in pregnant rats on doublecortin and synaptophysin (p38) expressions of fetal brain

Objective To investigate the effects of maternal thyroid dysfunction during pregnancy caused by iodine deficiency of different degrees on doublecortin ( DCX ) and synaptophysin ( p38 ) expressions in fetal brain.Methods Wistar female rats were randomly divided into four groups:adequate iodine ( AI),mild iodine deficiency ( MiID ),moderate iodine deficiency ( MoID ),and severe iodine deficiency ( SID ),according to the total daily iodine supply( fed on an iodine deficient diet with different dosages of KI added in drinking water).Three months later the rats were mated.Serum TSH and thyroid hormones were determined in maternal rats on gestational day 20 using chemiluminescent immunoassay.The iodine contents in urine and histological changes of thyroid gland were observed in pregnant rats.The mRNA and protein levels of DCX and synaptophysin ( p38 ) were analyzed in fetal brain by using real time quantitative RT-PCR and western blotting respectively.Results ( 1 ) Iodine contents in urine of pregnant rats were reduced with the decrease of their iodine supply.Compared with group AI,serum TSH was significantly increased [ ( 2.95 ± 1.70 vs 1.31 ± 0.55 ) mU/L,P ＜ 0.05 ],and both TT4 and FT4 were significantly decreased [ ( 14.3±4.1 vs 28.4±19.3 ) nmol/L,P＜0.05 ] and [ ( 10.8±3.6 vs 20.2±8.0) pmol/L,P＜0.01 ] in pregnant rats of SID group.Whereas,a slight rise in TSH,and a mild decline in both TT4and FT4 were found in MoID and MiID groups.However,there were no significant changes in TT3 and FT3 levels among these four groups.( 2 )Histological characteristics of thyroid gland in pregnant rats showed a typical goiter with small follicular hyperplasia and lack of colloid in SID group,moderate follicular hyperplasia with decreased colloid in MoID group; but mild cellular hyperplasia without decrease in follicular size and colloid in MiID group.( 3 ) The mRNA levels of DCX were increased in fetal brains of three iodine deficiency groups compared with AI group,but a statistical significance was found in MoID group.The protein levels of DCX in all experiment groups were significantly increased.Both mRNA and protein expressions of synaptophysin ( p38 ) were significantly down-regulated in three iodine deficiency groups.Conclusions Maternal thyroid dysfunction caused by iodine deficiency,even by mild or moderate iodine deficiency,may lead to retardation of fetal neuronal and synaptic growth. Key words: Iodine deficiency; Pregnancy; Thyroid function; Doublecortin; Synaptophysin

3-[3-(3-florophenyl-2-propyn-1-ylthio)-1, 2, 5-thiadiazol-4-yl]-1, 2, 5, 6-tetrahydro-1- methylpyridine oxalate, a novel xanomeline derivative, improves neural cells proliferation and survival in adult mice.

The present study analyzed the influence of 3-[3-(3-florophenyl-2-propyn-1-ylthio)-1, 2, 5-thiadiazol-4-yl]-1, 2, 5, 6-tetrahydro-1-methylpyridine oxalate (EUK1001), a novel xanomeline derivative of the M1/M4 receptor agonist, on hippocampal neurogenesis in adult C57BL6 mice. Results showed that 15-day EUK1001 treatment via intraperitoneal injection promoted neural cell proliferation in the dentate gyrus, although cell differentiation did not change. The majority of bromodeoxyuridine-positive cells co-expressed the immature neuronal marker doublecortin. In addition, the level of neurogenesis in the subventricular zone was not altered. Brain-derived neurotrophic factor mRNA expression was up-regulated following EUK1001 treatment, but no change was observed in expression of camp-responsive element binding protein 1, paired box gene 6, vascular endothelial growth factor alpha, neurogenic differentiation factor 1, and wingless-related mouse mammary tumor virus integration site 3A mRNA. These experimental findings indicated that EUK1001 enhanced proliferation and survival of hippocampal cells, possibly by increasing brain-derived neurotrophic factor expression.

Annexin A2 Mediates Up-regulation of NF-κB, β-catenin, and Stem Cell in Response to Progastrin in Mice and HEK-293 Cells

Prograstrin induces proliferation in colon crypts by activating p65nuclear factor-κB (NF-κB) (p65) and β-catenin. We investigated whether Annexin A2 (AnxA2), a progastrin receptor, activates NF-κB and β-catenin in vivo. ANXA2-null (ANXA2(-/-)) and wild-type (ANXA2(+/+)) mice were studied, along with clones of progastrin-responsive HEK-293 cells that stably expressed full-length progastrin (HEK-mGAS) or an empty vector (HEK-C). Small interfering RNA was used to down-regulate AnxA2, p65NF-κB, and β-catenin in cells. Proliferation and activation of p65 and β-catenin increased significantly in HEK-mGAS compared with HEK-C clones. HEK-mGAS cells had a 2- to 4-fold increase in relative levels of c-Myc, cyclooxygenase (COX)-2, CyclinD1, double cortin CAM kinase-like 1 (DCAMKL+1), and CD44, compared with HEK-C clones. Down-regulation of AnxA2 in HEK-mGAS clones reduced activation of NF-κB and β-catenin, as well as levels of DCAMKL+1. Surprisingly, down-regulation of β-catenin had no effect on activation of p65NF-κB, whereas down-regulation of p65 significantly reduced activation of β-catenin in HEK-mGAS clones. Loss of either p65 or β-catenin significantly reduced proliferation of HEK-mGAS clones, indicating that both factors are required for the proliferative effects of progastrin. Lengths of colon crypts and levels of p65, β-catenin, DCAMKL+1, and CD44 were significantly higher in ANXA2(+/+) mice compared with either ANXA2(-/-) mice given progastrin or ANXA2(+/+) and ANXA2(-/-) mice given saline. AnxA2 expression is required for the biologic effects of progastrin in vivo and in vitro and mediates the stimulatory effect of progastrin on p65NF-κ, β-catenin, and the putative stem cell markers DCAMKL+1 and CD44. AnxA2 might therefore mediate the hyperproliferative and cocarcinogenic effects of progastrin.

Astrogliosis is temporally correlated with enhanced neurogenesis in adult rat hippocampus following a glucoprivic insult

2-Deoxy- d-glucose (2-DG) administration causes transient depletion of glucose derivates and ATP. Hence, it can be used in a model system to study the effects of a mild glycoprivic brain insult mimicking transient hypoglycemia, which often occurs when insulin or oral hypoglycemic agents are administered for diabetes control. In the present study, the effect of a single 2-DG application (500 mg/kg, a clinically applicable dose) on glial reactivity and neurogenesis in adult rat hippocampus was examined, as well as a possible temporal correlation between these two phenomena. Post-insult (PI) glial reactivity time course was assessed by immunoreaction against glial-fibrillary acidic protein (GFAP) during the following 5 consecutive days. A clear increase of GFAP immunoreactivity in hilus was observed from 48 to 96 h PI. Moreover, enhanced labeling of long radial processes in the granule cell layer adjacent to hilus was evidenced. On the other hand, a transient increase of progenitor cell proliferation was detected in the subgranular zone, prominently at 48 h PI, coinciding with the temporal peak of glial activation. This increase resulted in an augment of neuroblasts double labeled with 5-bromo-deoxyuridine (BrdU) and with double cortin (DCX) at day 7 PI. Around half of these cells survived 28 days showing matured neuronal phenotype double labeled by BrdU and a neuronal specific nuclear protein marker (NeuN). These findings suggest that a transient neuroglycoprivic state exerts a short-term effect on glial activation that possibly triggers a long-term effect on neurogenesis in hippocampus.

Identification of Linkage Disequilibrium SNPs from a Kidney-Yang Deficiency Syndrome Pedigree

In order to probe the genetic traits of Kidney-yang Deficiency Syndrome (KDS), we employed a national standard of KDS diagnosis for the collection of KDS subjects. Each candidate KDS subject from a typical family was diagnosed by 5 independent physicians of Traditional Chinese Medicine (TCM), and repeated for 3 years, all on the first Saturday of December. Fifteen samples of genomic DNA were isolated and genotyped by Affymetrix 100 K arrays of single nucleotide polymorphism (SNP). Then appropriate tools were used for the analysis of linkage disequilibrium (LD) and bioinformatic mining of LD SNPs. The results indicated that our procedure of TCM diagnosis can effectively collect KDS subjects and therefore provide substantial basis for the linkage analysis of KDS. Five SNPs (i.e. rs514207, rs1054020, rs7685923, rs10515889 and rs10516202) were identified as LD SNPs from this KDS family, representing an unprecedented set of LD SNPs derived from TCM syndrome. These SNPs demonstrate midrange linkage disequilibrium within the KDS family. Two genes with established functions were identified within 100 bp of these SNPs. One is Homo sapiens double cortin domain containing 5, which interacts selectively with mono-, di- or tri-saccharide carbohydrate and involves certain signaling cascades. Another one, leucyl-tRNA synthetase, is also a pleiotropic gene response to cysteinyl-tRNA aminoacylation and protein biosynthesis. In conclusion, KDS is involved in special SNP linkage disequilibrium in the intragenic level, and genes within the flanks of these SNPs suggest some essential symptoms of KDS. However, definitive evidence to confirm or exclude these loci and to establish their biological activities will be required.

No Long‐Term Effect Two Years after Intrauterine Exposure to Dexamethasone on Dentate Gyrus Volume, Neuronal Proliferation and Differentiation in Common Marmoset Monkeys

Glucocorticoids are prenatally administered to promote the maturation of the lungs. They, however, can affect neuronal proliferation and differentiation. In newborn marmoset monkeys, intrauterine hyperexposure to dexamethasone (DEX) resulted in a significantly decreased proliferation rate in the hippocampal dentate gyrus without affecting neuronal differentiation. In this study, marmoset monkeys received 5 mg/kg body weight DEX either during early (days 42-48) or late (days 90-96) pregnancy. The volume of the dentate granule cell layer as well as the proliferation and neuronal differentiation in the dentate gyrus of their 2-year-old offspring were investigated. The density of proliferating cells (Ki-67), apoptotic cells (in situ tailing) and cells differentiating to neurons (double cortin, TUC-4 and calretinin) were determined immunohistochemically. Analysis of the dentate granule cell layer volume showed no significant differences between early or late DEX-exposed marmosets and untreated control animals. Similarly, proliferation and neuronal differentiation in DEX-treated animals was not significantly different in comparison with controls. In summary, the decreased proliferation rate observed in newborn marmosets after intrauterine exposure to DEX was no longer detectable in their 2-year-old siblings suggesting no long-lasting effect of prenatal hyperexposure to DEX on neuronal proliferation and differentiation in the dentate gyrus of marmoset monkeys.

Neurogenesis and Alterations of Neural Stem Cells in Mouse Models of Cerebral Amyloidosis

The hippocampus in Alzheimer's disease is burdened with amyloid plaques and is one of the few locations where neurogenesis continues throughout adult life. To evaluate the impact of amyloid-beta deposition on neural stem cells, hippocampal neurogenesis was assessed using bromodeoxyuridine incorporation and doublecortin staining in two amyloid precursor protein (APP) transgenic mouse models. In 5-month-old APP23 mice prior to amyloid deposition, neurogenesis showed no robust difference relative to wild-type control mice, but 25-month-old amyloid-depositing APP23 mice showed significant increases in neurogenesis compared to controls. In contrast, 8-month-old amyloid-depositing APPPS1 mice revealed decreases in neurogenesis compared to controls. To study whether alterations in neurogenesis are the result of amyloid-induced changes at the level of neural stem cells, APPPS1 mice were crossed with mice expressing green fluorescence protein (GFP) under a central nervous system-specific nestin promoter. Eight-month-old nestin-GFP x APPPS1 mice exhibited decreases in quiescent nestin-positive astrocyte-like stem cells, while transient amplifying progenitor cells did not change in number. Strikingly, both astrocyte-like and transient-amplifying progenitor cells revealed an aberrant morphologic reaction toward congophilic amyloid-deposits. A similar reaction toward the amyloid was no longer observed in doublecortin-positive immature neurons. Results provide evidence for a disruption of neural stem cell biology in an amyloidogenic environment and support findings that neurogenesis is differently affected among various transgenic mouse models of Alzheimer's disease.

Sodium Orthovanadate Enhances Proliferation of Progenitor Cells in the Adult Rat Subventricular Zone after Focal Cerebral Ischemia

Neuronal progenitor cells able to produce new neuron and glia persist in the adult central nervous system (CNS). Their proliferation is up-regulated by growth factors or cytokines under some pathological conditions, including ischemia. Because sodium orthovanadate (SOV), a protein tyrosine phosphatase inhibitor, can up-regulate tyrosine kinase-linked growth factor receptor signaling via the inhibition of tyrosine residue dephosphorylation, it may be capable of enhancing progenitor cells. To investigate the effect of SOV on progenitor cells in the subventricular zone (SVZ), we injected rats intraperitoneally with 50 mg/kg bromodeoxyuridine (BrdU) and 12.5 or 25 mM SOV or BrdU and saline (control) on days 1 to 7 after middle cerebral artery occlusion. The density of BrdU-positive cells in the ipsilateral SVZ showed a significant SOV dose-dependent increase. This effect was found only in the ipsilateral and not contralateral SVZ, and it was not found in nonischemic rats. Double immunolabeling with BrdU and double cortin, a marker of migrating neuroblast, revealed that the density of double-positive cells increased significantly in an SOV dose-dependent manner. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining suggested that the SOV-induced increase was not due to antiapoptotic effects. Treatment with SOV also significantly increased the density of cells positive for BrdU and phosphorylated Akt and BrdU and phosphorylated extracellular signal-regulated kinase (ERK). We postulate that ischemia triggers off the proliferation of SVZ cells by bioactive factors such as growth factors and that SOV enhances the proliferation of only triggered-off SVZ cells with Akt and ERK activation. Our findings suggest that SOV may aid in the self-repair of the postischemic CNS.

Involvement of the Phosphatidylinositol 3-Kinase/Rac1 and Cdc42 Pathways in Radial Migration of Cortical Neurons

During cortical development, newly generated neurons migrate radially toward their final positions. Although several candidate genes essential for this radial migration have been reported, the signaling pathways regulating it are largely unclear. Here we studied the role of phosphatidylinositol (PI) 3-kinase and its downstream signaling molecules in the radial migration of cortical neurons in vivo and in vitro. The expression of constitutively active and dominant-negative PI 3-kinases markedly inhibited radial migration. In the neocortical slice culture, a PI 3-kinase inhibitor suppressed the formation of GTP-bound Rac1 and Cdc42 and radial migration. Constitutively active and dominant-negative forms of Rac1 and Cdc42 but not Akt also significantly inhibited radial migration. In migrating neurons, wild-type Rac1 and Cdc42 showed different localizations; Rac1 localized to the plasma membrane and Cdc42 to the perinuclear region on the side of the leading processes. These results suggest that both the PI 3-kinase/Rac1 and Cdc42 pathways are involved in the radial migration of cortical neurons and that they have different roles.

Postnatal Neurogenesis and Gliogenesis in the Olfactory Bulb from NG2-Expressing Progenitors of the Subventricular Zone

We used a 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mouse to study postnatal subventricular zone (SVZ) progenitor fate, with a focus on the olfactory bulb (OB). The postnatal OB of the CNP-EGFP mouse contained EGFP+ interneurons and oligodendrocytes. In the anterior SVZ, the majority of EGFP+ progenitors were NG2+. These NG2+/EGFP+ progenitors expressed the OB interneuron marker Er81, the neuroblast markers doublecortin (DC) and Distalless-related homeobox (DLX), or the oligodendrocyte progenitor marker Nkx2.2. In the rostral migratory stream (RMS), EGFP+ cells displayed a migrating phenotype. A fraction of these cells were either NG2-/Er81+/DC+/DLX+ or NG2+/Nkx2.2+. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) injection into the lateral ventricle (LV) of early postnatal mice demonstrated that NG2+/EGFP+ progenitors migrate from the SVZ through the RMS into the OB. Moreover, fluorescence-activated cell-sorting-purified NG2+/CNP-EGFP+ or NG2+/beta-actin-enhanced yellow fluorescent protein-positive (EYFP+) progenitors transplanted into the early postnatal LV displayed extensive rostral and caudal migration. EYFP+ or EGFP+ graft-derived cells within the RMS were DLX+/Er81+ or Nkx2.2+, migrated to the OB, and differentiated to interneurons and oligodendrocytes. In the subcortical white matter (SCWM), grafted cells differentiated to either oligodendrocytes or astrocytes. Transplantation of NG2+/EYFP+ progenitors selectively purified from the SVZ showed that these cells were migratory and generated glia and neurons in the OB, hippocampus, and striatum. In contrast, cortical, OB, or cerebellar NG2+ cells had a very limited migratory potential and gave rise to glia in the SCWM and striatum. Our findings indicate region-specific differences between NG2+ progenitor cells and show that NG2+ cells can migrate throughout the RMS and contribute to both gliogenesis and neurogenesis in the postnatal OB.

DCX, a new mediator of the JNK pathway

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).