Reaper-mediated Inhibition of DIAP1-induced DTRAF1 Degradation Results in Activation of JNK in Drosophila

Nature Cell Biology. Sep, 2002  |  Pubmed ID: 12198495

Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death is largely unknown. Here we show through a genetic screen that a mutant of Drosophila melanogaster tumour-necrosis factor receptor-associated factor 1 (DTRAF1) is a dominant suppressor of Reaper-induced cell death. We show that Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates DTRAF1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of DTRAF1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of DTRAF1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of DTRAF1-mediated JNK activation in Drosophila cells. Taken together, our results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin#150;proteasome pathway.

Isolation and Transplantation of Dopaminergic Neurons and Neural Stem Cells

Parkinsonism & Related Disorders. Oct, 2002  |  Pubmed ID: 12217619

Although transplantation of mesencephalic tissue is considered a promising therapy for Parkinson's disease (PD), its clinical use is still restricted to a very few cases. A major limiting factor of this therapy is the difficulty of obtaining sufficient quantities of viable embryonic mesencephalic tissue. To overcome this limitation, techniques to produce dopaminergic (DA) neurons in vitro have been developed. However, these cultures are likely to contain a variety of unidentified cells, which must be removed before implantation. Specific cell-surface markers to sort DA neurons or their precursors are not available. We have developed an alternative strategy, by which these cells can be labeled with green fluorescent protein and isolated with fluorescent activated cell sorter. Transplantation of the sorted cells resulted in recovery of a rat model of the PD. This strategy should be useful for developing new therapies for PD.

Tincar Encodes a Novel Transmembrane Protein Expressed in the Tinman-expressing Cardioblasts of Drosophila

Gene Expression Patterns : GEP. Dec, 2002  |  Pubmed ID: 12617821

We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up.

Tincar Encodes a Novel Transmembrane Protein Expressed in the Tinman-expressing Cardioblasts of Drosophila

Mechanisms of Development. Dec, 2002  |  Pubmed ID: 14516698

We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up.

Overexpression of Poly(ADP-ribose) Polymerase Disrupts Organization of Cytoskeletal F-actin and Tissue Polarity in Drosophila

The Journal of Biological Chemistry. Feb, 2002  |  Pubmed ID: 11744702

Poly(ADP-ribose) polymerase (PARP) may play important roles in nuclear events such as cell cycle, cell proliferation, and maintenance of chromosomal stability. However, the exact biological role played by PARP or how PARP is involved in these cellular functions is still unclear. To elucidate the biological functions of PARP in vivo, we have constructed transgenic flies that overexpress Drosophila PARP in the developing eye primordia. These flies showed mild roughening of the normally smooth ommatidial lattice and tissue polarity disruption caused by improper rotation and chirality of the ommatidia. To clarify how this phenotypical change was induced, here we analyzed transgenic flies overexpressing PARP in the developing eye, embryo, and adult in detail. PARP mRNA level and the phenotype were enhanced in flies carrying more copies of the transgene. Developing eyes from third instar larvae were analyzed by using the neural cell marker to examine the involvement of PARP in cell fate. Morphological disorder of non-neuronal accessory cells was observed in PARP transgenic flies. Interestingly, overexpression of PARP did not interfere with the cell cycle or apoptosis, but it did disrupt the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupted organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Because these phenotypes closely resembled mutants or transgenic flies of the tissue polarity genes, genetic interaction of PARP with known tissue polarity genes was examined. Transgenic flies expressing either PARP or RhoA GTPase in the eye were crossed, and co-expression of PARP suppressed the effect of RhoA GTPase. Our results indicate that PARP may play a role in cytoskeletal or cytoplasmic events in developmental processes of Drosophila.

Isolation and Transplantation of Dopaminergic Neurons Generated from Mouse Embryonic Stem Cells

Neuroscience Letters. Jun, 2004  |  Pubmed ID: 15157991

Embryonic stem (ES) cells differentiate into dopamine (DA)-producing neurons when co-cultured with PA6 stromal cells, but the resulting cultures contain a variety of unidentified cells. In order to label live DA neurons in mixed populations, we introduced a GFP reporter under the control of the tyrosine hydroxylase (TH) gene promoter into ES cells. GFP expression was observed in TH-immunoreactive cells that differentiated from the ES cells that carried the TH-GFP reporter gene. DA neurons expressing GFP were sorted from the mixed cell population by fluorescence-activated cell sorting of cells exhibiting GFP fluorescence, and the sorted GFP(+) cells obtained were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a partial recovery from parkinsonian behavioral defects. This strategy of isolation and transplantation of ES-cell-derived DA neurons should be useful for cellular and molecular studies of DA neurons and for clinical application in the treatment of Parkinson's disease.

Mapping Spatio-temporal Activation of Notch Signaling During Neurogenesis and Gliogenesis in the Developing Mouse Brain

Journal of Neurochemistry. Jul, 2004  |  Pubmed ID: 15198674

Notch1 plays various important roles including the maintenance of the stem cell state as well as the promotion of glial fates in mammalian CNS development. However, because of the very low amount of the activated form of Notch1 present in vivo, its precise activation pattern has remained unknown. In this study, we mapped the active state of this signaling pathway in situ in the developing mouse brain using a specific antibody that recognizes the processed form of the intracellular domain of Notch1 cleaved by presenilin/gamma-secretase activity. By using this antibody, active state of Notch1 came to be detectable with a higher sensitivity than using conventional antibody against Notch1. We found that activated Notch1 was mainly detected in the nuclei of a subpopulation of radial glial cells, the majority of proliferating precursor cells in the ventricular zone (VZ). However, Notch1 activation was not detected in neuronal precursor cells positive for neuronal basic helix-loop-helix proteins or in differentiating neurons in the embryonic forebrain. Interestingly, we found that Notch1 was transiently activated in the astrocytic lineage during perinatal CNS development. Taken together, the present method has enabled us to determine the timing, gradients, and boundaries of the activation of Notch signaling.

Characterization of the Isoforms of MOVO Zinc Finger Protein, a Mouse Homologue of Drosophila Ovo, As Transcription Factors

Gene. Jul, 2004  |  Pubmed ID: 15225875

We previously described two isoforms (MOVO-A and -B) of a novel zinc finger protein MOVO, a mouse homologue of Drosophila Ovo protein. Here, we isolated cDNA encoding the third isoform MOVO-C, which had a transactivation domain and zinc finger domain, but lacked an N-terminal potential repression domain that was present in MOVO-A. Three isoform mRNAs were expressed highly in mouse testis and also in the ovary at lower levels. The structural analyses of the isolated Movo gene and mRNAs demonstrated that three different Movo transcripts were differentially processed to generate three isoforms. Major mRNA species encoded MOVO-B with a zinc finger domain alone, and minor mRNA species encoded MOVO-A (potential repressor) and MOVO-C (potential activator). To assign MOVO to a transcriptional factor, we characterized DNA-binding and transactivation properties. Random oligonucleotide selection, electrophoretic mobility shift assay and footprinting indicated that MOVO bound to the sequence, 5'-G(G/C/T)GGGGG-3'. These motifs were found in the 5'-flanking regions of Movo and other testis-specific genes. Nuclear proteins binding to this motif were detected in mouse testis, and the expression of MOVO mRNA was restricted in spermatocytes. The luciferase assay demonstrated that MOVO-C activated Movo promoter and MOVO-A repressed it, but MOVO-B had no effects. Mutated MOVO-binding motifs in the Movo promoter reduced the luciferase activity. All the isoforms had no effects on SV40 promoter without MOVO-binding motifs. MOVO-A partially rescued oogenesis of a Drosophila ovo mutant. These results suggest that MOVO isoforms are transcription factors to regulate genes carrying the MOVO-binding motifs in the testis.

Vascular Adventitia Generates Neuronal Progenitors in the Monkey Hippocampus After Ischemia

Hippocampus. 2004  |  Pubmed ID: 15382256

In the adult hippocampus, neurogenesis proceeds in the subgranular zone (SGZ) of the dentate gyrus (DG), but not in the cornu Ammonis (CA). Recently, we demonstrated in monkeys that transient brain ischemia induces an increase of the neuronal progenitor cells in the SGZ, but not in CA1, in the second week after the insult. To identify the origin of primary neuronal progenitors in vivo, we compared the postischemic monkey DG and CA1, using light and electron microscopy, focusing on specific phenotype markers, as well as the expression of neurotrophic factors. Laser confocal microscopy showed that 1-3% of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the SGZ after 2-96 h labeling were also positive for neuronal markers such as TUC4, betaIII tubulin, and NeuN on days 9 and 15. In contrast, despite the presence of numerous BrdU-positive cells, CA1 showed no neurogenesis at any time points, and all the progenitors were positive for glial markers: Iba1 or S-100beta on days 4, 9, and 15. Highly polysialylated neural cell adhesion molecule (PSA-NCAM)-positive cells were abundant in the SGZ, but were absent in CA1. On day 9, most of the immature neurons positive for betaIII-tubulin in SGZ showed an increase in PSA-NCAM immunoreactivity. The immunoreactivity of brain-derived neurotrophic factor (BDNF) was abundant at the vascular adventitia of the SGZ, but was absent at the adventitia of CA1. BrdU-positive progenitor cells were frequently seen in the vicinity of proliferating blood vessels. Ultrastructural analysis indicated that most of the neuronal progenitor cells and microglia originated from the pericytes of capillaries and/or adventitial cells of arterioles (called vascular adventitia). The detaching adventitial cells showed mitotic figures in the perivascular space, and the resultant neuronal progenitor cells made contact with dendritic spines associated with synaptic vesicles or boutons. These data implicate the vascular adventitia as a novel potential source of neuronal progenitor cells in the postischemic primate SGZ.

The Transmembrane Protein, Tincar, is Involved in the Development of the Compound Eye in Drosophila Melanogaster

Development Genes and Evolution. Feb, 2005  |  Pubmed ID: 15654626

We previously cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. Here, we have studied the expression pattern and functions of tinc during developmental processes. tinc mRNA is expressed in the central and peripheral nervous systems, and midgut during embryogenesis. In the third-instar larval eye disc, tinc mRNA is strongly expressed in all the differentiating ommatidial cells within and in the vicinity of the morphogenetic furrow. Loss-of-function analysis using the RNA-interference method revealed severe defects of eye morphogenesis during the late developmental stages. Our results suggested that tinc may have an indispensable role in the normal differentiation of ommatidial cells.

Activation of Cytokine Signaling Through Leukemia Inhibitory Factor Receptor (LIFR)/gp130 Attenuates Ischemic Brain Injury in Rats

Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. Jun, 2005  |  Pubmed ID: 15716858

Cytokine signaling through leukemia inhibitory factor receptor (LIFR)/gp130 is known to exert a neurotrophic action in the central nervous system, although the role of this signaling in cerebral ischemia remains unknown. We examined the effect of intracerebral injection of LIF after focal cerebral ischemia in rats. The animals underwent a sham operation (sham group) or middle cerebral artery occlusion (MCAO) followed by direct injection of either vehicle (phosphate-buffered saline, the PBS group) or recombinant LIF (10 ng in the low-LIF group and 100 ng in the high-LIF group) into the cerebral cortex adjacent to the inner boundary zone of the infarct area, and neurologic and histologic evaluations were conducted 24 h later. Expression of LIFR, gp130, and phosphorylated Stat3, Akt, and ERK1/2 was investigated by Western blot analysis and immunohistochemistry. The neurologic deficits and ischemic damage were significantly less severe in the high-LIF group than in the PBS group and the low-LIF group. Leukemia inhibitory factor receptor and gp130 were expressed in neurons, and the ischemic damage of these proteins was rescued in the high-LIF group. Early induction of phosphorylated Stat3 was significantly detected on the ischemic side in the high-LIF group after LIF injection. Exogenous LIF attenuates ischemic brain injury by activating cytokine signaling through LIFR/gp130.

Blockade of Interleukin-6 Signaling Aggravates Ischemic Cerebral Damage in Mice: Possible Involvement of Stat3 Activation in the Protection of Neurons

Journal of Neurochemistry. Jul, 2005  |  Pubmed ID: 15998296

Interleukin (IL)-6 expression transiently increases in the acute phase of cerebral ischemia. To investigate the physiological significance of endogenous IL-6 expression and to identify the main signal pathway for the action of IL-6, we administered anti-mouse IL-6 receptor monoclonal antibody (IL-6RA), which blocks IL-6 signaling, to mice immediately after a 45-min period of middle cerebral artery occlusion (MCAO). At 6 h after MCAO, IL-6RA administration had resulted in a significant reduction in the amount of phosphorylated signal transducer and activator of transcription-3 (Stat3) protein in the peri-infarct area of the cortex. At 24 h after MCAO, blockade of IL-6 signaling had led to an increase in number of apoptotic cells in the peri-infarct area and enlargement of the size of the infarct, and it had adversely affected neurological function. These results suggest that endogenous IL-6 plays a critical role in preventing damaged neurons from undergoing apoptosis in the acute phase of cerebral ischemia and that its role may be mediated by Stat3 activation.

Enhanced Proliferation of Progenitor Cells in the Subventricular Zone and Limited Neuronal Production in the Striatum and Neocortex of Adult Macaque Monkeys After Global Cerebral Ischemia

Journal of Neuroscience Research. Sep, 2005  |  Pubmed ID: 16047371

Cerebral ischemia in adult rodent models increases the proliferation of endogenous neural progenitor cells residing in the subventricular zone along the anterior horn of the lateral ventricle (SVZ a) and induces neurogenesis in the postischemic striatum and cortex. Whether the adult primate brain preserves a similar ability in response to an ischemic insult is uncertain. We used the DNA synthesis indicator bromodeoxyuridine (BrdU) to label newly generated cells in adult macaque monkeys and show here that the proliferation of cells with a progenitor phenotype (double positive for BrdU and the markers Musashi 1, Nestin, and beta III-tubulin) in SVZ a increased during the second week after a 20-min transient global brain ischemia. Subsequent progenitor migration seemed restricted to the rostral migratory stream toward the olfactory bulb and ischemia increased the proportion of adult-generated cells retaining their location in SVZ a with a progenitor phenotype. Despite the lack of evidence for progenitor cell migration toward the postischemic striatum or prefrontal neocortex, a small but sustained proportion of BrdU-labeled cells expressed features of postmitotic neurons (positive for the protein Neu N and the transcription factors Tbr 1 and Islet 1) in these two regions for at least 79 days after ischemia. Taken together, our data suggest an enhanced neurogenic response in the adult primate telencephalon after a cerebral ischemic insult.

Gain-of-function Screen Identifies a Role of the Sec61alpha Translocon in Drosophila Postmitotic Neurotoxicity

Biochimica Et Biophysica Acta. Nov, 2005  |  Pubmed ID: 16243437

To elucidate the intrinsic mechanisms of neurotoxicity induction, including those underlying neural cell death and neurodegeneration, we developed a gain-of-function screen for gene products causing neural cell loss. To identify novel genes with a cell-death-related function in neurons, we screened 4,964 Drosophila GS lines, in which one or two genes from much of the Drosophila genome can be overexpressed. Approximately 0.68% of the GS lines produced phenotypes involving a loss of postmitotic neurons. Of these, we identified and characterized the endd2 gene, which encodes the Drosophila ortholog of Sec61alpha (DSec61alpha), an endoplasmic reticulum protein with protein translocation activity. Ectopic expression of DSec61alpha caused neural cell death accompanied by the accumulation of ubiquitinated proteins, which was mediated by DSec61alpha's translocon activity. This supported our previous observation that the DSec61alpha translocon contributes to expanded polyglutamine-mediated neuronal toxicity, which is also associated with ubiquitinated protein accumulation. These data suggest that the translocon may be a novel component of neural cell death and degeneration pathways. Our approach can be used to identify potential neurotoxic factors within the whole genome, which will increase our understanding of the molecular mechanisms of various types of cell death, including those associated with human neurodegenerative diseases.

New Neurons Follow the Flow of Cerebrospinal Fluid in the Adult Brain

Science (New York, N.Y.). Feb, 2006  |  Pubmed ID: 16410488

In the adult brain, neuroblasts born in the subventricular zone migrate from the walls of the lateral ventricles to the olfactory bulb. How do these cells orient over such a long distance and through complex territories? Here we show that neuroblast migration parallels cerebrospinal fluid (CSF) flow. Beating of ependymal cilia is required for normal CSF flow, concentration gradient formation of CSF guidance molecules, and directional migration of neuroblasts. Results suggest that polarized epithelial cells contribute important vectorial information for guidance of young, migrating neurons.

Distinct Functions of Human Numb Isoforms Revealed by Misexpression in the Neural Stem Cell Lineage in the Drosophila Larval Brain

Developmental Neuroscience. 2006  |  Pubmed ID: 16508311

Mammalian Numb (mNumb) has multiple functions and plays important roles in the regulation of neural development, including maintenance of neural progenitor cells and promotion of neuronal differentiation in the central nervous system (CNS). However, the molecular bases underlying the distinct functions of Numb have not yet been elucidated. mNumb, which has four splicing isoforms, can be divided into two types based on the presence or absence of an amino acid insert in the proline-rich region (PRR) in the C-terminus. It has been proposed that the distinct functions of mNumb may be attributable to these two different types of isoforms. In this study, we used the outer optic anlage (OOA) of the Drosophila larval brain as an assay system to analyze the functions of these two types of isoforms in the neural stem cells, since the proliferation pattern of neuroepithelial (NE) stem cells in the OOA closely resembles that of the vertebrate neural stem/progenitor cells. They divide to expand the progenitor cell pool during early neurogenesis and to produce neural precursors/neurons during late neurogenesis. Clonal analysis in the OOA allows one to discriminate between the NE stem cells, which divide symmetrically to expand the progenitor pool, and the postembryonic neuroblasts (pNBs), which divide asymmetrically to produce neural precursors (ganglion mother cells), each of which divides once to produce two neurons. We found that in the OOA, the human Numb isoform with a long PRR domain (hNumb-PRRL), which is mainly expressed during early neurogenesis in the mouse CNS, promotes proliferation of both NE cells and pNBs without affecting neuronal differentiation, while the other type of hNumb isoform with a short PRR domain (hNumb-PRRS), which is expressed throughout neurogenesis in the mouse embryonic CNS, inhibits proliferation of the stem cells and promotes neuronal differentiation. We also found that hNumb-PRRS, a functional homologue of Drosophila Numb, more strongly decreases the amount of nuclear Notch than hNumb-PRRL, and could antagonize Notch functions probably through endocytic degradation, suggesting that the two distinct types of hNumb isoforms could contribute to different phases of neurogenesis in the mouse embryonic CNS.

The Sox2 Regulatory Region 2 Functions As a Neural Stem Cell-specific Enhancer in the Telencephalon

The Journal of Biological Chemistry. May, 2006  |  Pubmed ID: 16547000

Sox2 is expressed at high levels in neuroepithelial stem cells and persists in neural stem/progenitor cells throughout adulthood. We showed previously that the Sox2 regulatory region 2 (SRR2) drives strong expression in these cells. Here we generated transgenic mouse strains with the beta-geo reporter gene under the control of the SRR2 in order to examine the spatiotemporal function of this regulatory region. We show that the SRR2 functions specifically in neural stem/progenitor cells. However, unlike Nestin 2nd intronic enhancer, the SRR2 shows strong regional specificity functioning only in restricted areas of the telencephalon but not in any other portions of the central nervous system such as the spinal cord. We also show by in vitro clonogenic assay that at least some of these SRR2-functioning cells possess the hallmark properties of neural stem cells. In adult brains, we could detect strong beta-geo expression in the subventricular zone of the lateral ventricle and along the rostral migrating stream where actively dividing cells reside. Chromatin immunoprecipitation assays reveal interactions of POU and Sox factors with SRR2 in neural stem/progenitor cells. Our data also suggest that the specific recruitment of these proteins to the SRR2 in the telencephalon defines the spatiotemporal activity of the enhancer in the developing nervous system.

A Carbohydrate-binding Protein, Galectin-1, Promotes Proliferation of Adult Neural Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. May, 2006  |  Pubmed ID: 16636291

In the subventricular zone of the adult mammalian forebrain, neural stem cells (NSCs) reside and proliferate to generate young neurons. We screened factors that promoted the proliferation of NSCs in vitro by a recently developed proteomics technique, the ProteinChip system. In this screen, we identified a soluble carbohydrate-binding protein, Galectin-1, as a candidate. We show herein that Galectin-1 is expressed in a subset of slowly dividing subventricular zone astrocytes, which includes the NSCs. Based on results from intraventricular infusion experiments and phenotypic analyses of knockout mice, we demonstrate that Galectin-1 is an endogenous factor that promotes the proliferation of NSCs in the adult brain.

Enhanced Neurogenesis in the Ischemic Striatum Following EGF-induced Expansion of Transit-amplifying Cells in the Subventricular Zone

Neuroscience Letters. Jul, 2006  |  Pubmed ID: 16701951

In the subventricular zone (SVZ) of the adult mammalian brain, neural stem cells continually produce transit-amplifying precursors, which generate neuroblasts migrating into the olfactory bulb. Previous studies have suggested that SVZ cells also have the capacity to generate some striatal neurons after cerebral ischemia. The infusion of epidermal growth factor (EGF) has been demonstrated to increase the number of these regenerated neurons. However, which cell types in the SVZ are stimulated to proliferate or differentiate after EGF infusion remains unknown. In this paper, we demonstrated that cerebral ischemia results in an increase in the number of EGF receptor (EGFR)-positive transit-amplifying cells in the SVZ. EGF infusion into the ischemic brain caused the number of transit-amplifying cells to increase and the number of neuroblasts to decrease. On the other hand, after an interval of 6 days after the discontinuation of EGF infusion, a significant increase in the number of neuroblasts was found, both in the striatum and the SVZ. These results suggest that the replacement of neurons in injured striatum can be enhanced by an EGF-induced expansion of transit-amplifying cells in the SVZ.

Subventricular Zone-derived Neuroblasts Migrate and Differentiate into Mature Neurons in the Post-stroke Adult Striatum

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2006  |  Pubmed ID: 16775151

Recent studies have revealed that the adult mammalian brain has the capacity to regenerate some neurons after various insults. However, the precise mechanism of insult-induced neurogenesis has not been demonstrated. In the normal brain, GFAP-expressing cells in the subventricular zone (SVZ) of the lateral ventricles include a neurogenic cell population that gives rise to olfactory bulb neurons only. Herein, we report evidence that, after a stroke, these cells are capable of producing new neurons outside the olfactory bulbs. SVZ GFAP-expressing cells labeled by a cell-type-specific viral infection method were found to generate neuroblasts that migrated toward the injured striatum after middle cerebral artery occlusion. These neuroblasts in the striatum formed elongated chain-like cell aggregates similar to those in the normal SVZ, and these chains were observed to be closely associated with thin astrocytic processes and blood vessels. Finally, long-term tracing of the green fluorescent-labeled cells with a Cre-loxP system revealed that the SVZ-derived neuroblasts differentiated into mature neurons in the striatum, in which they expressed neuronal-specific nuclear protein and formed synapses with neighboring striatal cells. These results highlight the role of the SVZ in neuronal regeneration after a stroke and its potential as an important therapeutic target for various neurological disorders.

Implication of "Down Syndrome Cell Adhesion Molecule" in the Hippocampal Neurogenesis of Ischemic Monkeys

Hippocampus. 2006  |  Pubmed ID: 16983647

Molecular signals regulating adult neurogenesis in primates are largely unknown. Here the authors used differential display to analyze gene expression changes that occur in dentate gyrus of adult monkeys after transient global cerebral ischemia. Among 14 genes upregulated, the authors focused on Down syndrome cell adhesion molecule (DSCAM) known to play crucial role during neuronal development, and characterized its expression pattern at the protein level. In contrast with approximately threefold upregulation of Dscam gene on days 5 and 7, immunoblotting and immunofluorescence analyses using specific antibodies showed a gradual decrease of DSCAM after ischemia until day 9 followed by recovery on day 15. In the control, immunofluorescence reactivity of DSCAM was detected in dentate gyrus granule cells and CA4 neurons but decreased after ischemia, being compatible with the immunoblotting data. However, in the subgranular zone, cerebral ischemia led to a marked increase of DSCAM-positive cells on days 9 and 15. DSCAM upregulation was seen in two cell types: one is immature neurons positive for polysialylated neural cell adhesion molecule or betaIII-tubulin, while another is astrocytes positive for S100beta. Young astrocytes were in intimate contact with newly generated neurons in the subgranular zone. These data suggest implication of DSCAM in the adult neurogenesis of primate hippocampus upregulated after ischemia.

Role of the Cholinergic System in Regulating Survival of Newborn Neurons in the Adult Mouse Dentate Gyrus and Olfactory Bulb

Genes to Cells : Devoted to Molecular & Cellular Mechanisms. Oct, 2006  |  Pubmed ID: 16999735

Neurogenesis in the subgranular zone of the hippocampal dentate gyrus and olfactory bulbs continues into adulthood and has been implicated in the cognitive function of the adult brain. The basal forebrain cholinergic system has been suggested to play a role in regulating neurogenesis as well as learning and memory in these regions. Herein, we report that highly polysialylated neural cell adhesion molecule (PSA-NCAM)-positive immature cells as well as neuronal nuclei (NeuN)-positive mature neurons in the dentate gyrus and olfactory bulb express multiple acetylcholine receptor subunits and make contact with cholinergic fibers. To examine the function of acetylcholine in neurogenesis, we used donepezil (Aricept), a potent and selective acetylcholinesterase inhibitor that improves cognitive impairment in Alzheimer's disease. Intraperitoneal administrations of donepezil significantly enhanced the survival of newborn neurons, but not proliferation of neural progenitor cells in the subgranular zone or the subventricular zone of normal mice. Moreover, donepezil treatment reversed the chronic stress-induced decrease in neurogenesis. Taken together, these results suggest that activation of the cholinergic system promotes survival of newborn neurons in the adult dentate gyrus and olfactory bulb under both normal and stressed conditions.

Beta-catenin Signaling Promotes Proliferation of Progenitor Cells in the Adult Mouse Subventricular Zone

Stem Cells (Dayton, Ohio). Nov, 2007  |  Pubmed ID: 17673525

The subventricular zone (SVZ) is the largest germinal zone in the mature rodent brain, and it continuously produces young neurons that migrate to the olfactory bulb. Neural stem cells in this region generate migratory neuroblasts via highly proliferative transit-amplifying cells. The Wnt/beta-catenin signaling pathway partially regulates the proliferation and neuronal differentiation of neural progenitor cells in the embryonic brain. Here, we studied the role of beta-catenin signaling in the adult mouse SVZ. beta-Catenin-dependent expression of a destabilized form of green fluorescent protein was detected in progenitor cells in the adult SVZ of Axin2-d2EGFP reporter mice. Retrovirus-mediated expression of a stabilized beta-catenin promoted the proliferation of Mash1+ cells and inhibited their differentiation into neuroblasts. Conversely, the expression of Dkk1, an inhibitor of Wnt signaling, reduced the proliferation of Mash1+ cells. In addition, an inhibitor of GSK3 beta promoted the proliferation of Mash1+ cells and increased the number of new neurons in the olfactory bulb 14 days later. These results suggest that beta-catenin signaling plays a role in the proliferation of progenitor cells in the SVZ of the adult mouse brain.

Regeneration of the Central Nervous System Using Endogenous Repair Mechanisms

Journal of Neurochemistry. Sep, 2007  |  Pubmed ID: 17697047

Recent advances in developmental and stem cell biology have made regeneration-based therapies feasible as therapeutic strategies for patients with damaged central nervous systems (CNSs), including those with spinal cord injuries, Parkinson disease, or stroke. These strategies can be classified into two approaches: (i) the replenishment of lost neural cells and (ii) the induction of axonal regeneration. The first approach includes the activation of endogenous neural stem cells (NSCs) in the adult CNS and cell transplantation therapy. Endogenous NSCs have been shown to give rise to new neurons after insults, including ischemia, have been sustained; this form of neurogenesis followed by the migration and functional maturation of neuronal cells, as well as the responses of glial cells and the vascular system play crucial roles in endogenous repair mechanisms in damaged CNS tissue. In this review, we will summarize the recent advances in regeneration-based therapeutic approaches using endogenous NSCs, including the results of our own collaborative groups.

Cyclin-dependent Kinase 5 is Required for Control of Neuroblast Migration in the Postnatal Subventricular Zone

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2007  |  Pubmed ID: 18032654

At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone.

[Neuronal Migration in the Adult Brain]

Nihon Shinkei Seishin Yakurigaku Zasshi = Japanese Journal of Psychopharmacology. Nov, 2007  |  Pubmed ID: 18154043

Production of new neurons in the subventricular zone (SVZ) and in the dentate gyrus (DG) continues into adulthood. In this paper, we will review our recent studies on migration and survival of new neurons in the adult mouse brain. Neuroblasts generated in the SVZ migrate in chains rostrally toward the olfactory bulb (OB), where they are differentiated into olfactory interneurons. The precise mechanisms controlling neuroblast migration remain unclear. We have recently demonstrated that neuroblast migration parallels cerebrospinal fluid flow caused by integrated beating of ependymal cilia. While SVZ neuroblasts migrate only toward the OB under physiological conditions, we found that they could reach striatum in a mouse model of focal ischemia. The majority of these newly-generated neurons die before they are integrated into the neuronal circuit, even under physiological conditions. We found that long-term administration of donepezil, an acetylcholinesterase inhibitor clinically used for the treatment of Alzheimer's disease, promotes the survival of newly-generated neurons in the OB and the DG. Although there are a lot of subjects to be elucidated, understanding the comprehensive mechanism of adult neurogenesis should be useful for developing successful regenerative therapies for neuropsychological diseases in the future.

Neural Stem Cells: Involvement in Adult Neurogenesis and CNS Repair

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Jun, 2008  |  Pubmed ID: 18339601

Recent advances in stem cell research, including the selective expansion of neural stem cells (NSCs) in vitro, the induction of particular neural cells from embryonic stem cells in vitro, the identification of NSCs or NSC-like cells in the adult brain and the detection of neurogenesis in the adult brain (adult neurogenesis), have laid the groundwork for the development of novel therapies aimed at inducing regeneration in the damaged central nervous system (CNS). There are two major strategies for inducing regeneration in the damaged CNS: (i) activation of the endogenous regenerative capacity and (ii) cell transplantation therapy. In this review, we summarize the recent findings from our group and others on NSCs, with respect to their role in insult-induced neurogenesis (activation of adult NSCs, proliferation of transit-amplifying cells, migration of neuroblasts and survival and maturation of the newborn neurons), and implications for therapeutic interventions, together with tactics for using cell transplantation therapy to treat the damaged CNS.

Cell-cycle-specific Nestin Expression Coordinates with Morphological Changes in Embryonic Cortical Neural Progenitors

Journal of Cell Science. Apr, 2008  |  Pubmed ID: 18349072

During brain development, neural progenitor cells extend across the thickening brain wall and undergo mitosis. To understand how these two completely different cellular events are coordinated, we focused on the transcription pattern of the nestin gene (Nes), which encodes an intermediate filament protein strongly expressed in neural progenitor cells. To visualize nestin expression in vivo, we generated transgenic mice that expressed a destabilized fluorescent protein under the control of Nes second intronic enhancer (E/nestin:dVenus). During the neurogenic stage, when the brain wall thickens markedly, we found that nestin was regulated in a cell-cycle-dependent manner. Time-lapse imaging showed that nestin gene expression was upregulated during G1-S phase, when the neural progenitor cells elongate their fibers. However, nestin expression dramatically declined in G2-M phase, when progenitor cells round up to undergo mitosis. The cell-cycle-dependent phosphorylation of an upstream regulator class III POU transcription factor (Pou3f2 or Brn2) reduced its binding activity to the nestin core enhancer element and was therefore responsible for the decreased Nes transcription in G2-M phase. Collectively, these findings demonstrate precisely orchestrated gene regulation that correlates with the 3D morphological changes in neural progenitor cells in vivo.

[Adult Neurogenesis in Physiological and Pathological Conditions]

Brain and Nerve = Shinkei Kenkyū No Shinpo. Apr, 2008  |  Pubmed ID: 18421973

Generation of new neurons persists in the two restricted regions of adult brain, the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Newly generated neurons are functionally integrated into the neuronal circuits, which is involved in regulation of brain plasticity. Endogenous neuronal production in the DG and SVZ is expected to provide a continuous source of new neurons that replace degenerated neurons in the injured brain. Recent studies indicate that adult neurogenesis is modified by various brain insults including stroke, epilepsy and neurodegenerative disorders. While up-regulation of neurogenesis in these situations may partially contributes to restoration and regeneration of damaged neural tissues, inadequate cell differentiation and/or excessive supply of new neurons should disturb existing neural circuits. For development of successful regenerative medicine for injured brain, we need to understand more precise and comprehensive mechanisms regulating adult neurogenesis.

[Neuronal Migration in the Adult Brain]

Nihon Shinkei Seishin Yakurigaku Zasshi = Japanese Journal of Psychopharmacology. Apr, 2008  |  Pubmed ID: 18516984

Production of new neurons in the subventricular zone (SVZ) continues into adulthood. Neuroblasts generated in the SVZ migrate in chains rostrally toward the olfactory bulb (OB), where they are differentiated into olfactory interneurons. In this paper, we will review our recent studies on production, migration and survival of newly-generated neurons in the adult mouse brain. Although the precise mechanisms controlling the migration of neuroblasts remain unclear, some molecules related to cell adhesion, cytoskeletal regulation or attractive/repulsive cues have been shown to be involved in this process. We have recently demonstrated that neuroblast migration parallels cerebrospinal fluid flow caused by integrated beating of ependymal cilia. While SVZ neuroblasts migrate only toward the OB under physiological conditions, we found that they could reach striatum in a mouse model of focal ischemia using blood vessels as their scaffold. The majority of the newly-generated neurons are known to die before they are integrated into neuronal circuits. However, we found that their survival could be promoted by the long-term administration of donepezil, an acetylcholinesterase inhibitor that is widely used for the treatment of Alzheimer's disease. Understanding more precise and comprehensive mechanisms of adult neurogenesis should lead to future development of regenerative therapies for neuropsychiatric diseases.

[Neurogenesis in the Adult Subventricular Zone]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Jun, 2008  |  Pubmed ID: 18536349

Epigenetic Regulation of Neural Cell Differentiation Plasticity in the Adult Mammalian Brain

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2008  |  Pubmed ID: 19004774

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

Adult Neurogenesis and Its Alteration Under Pathological Conditions

Neuroscience Research. Mar, 2009  |  Pubmed ID: 19118585

Even in the adult brain, neural stem cells in the dentate gyrus and subventricular zone continue to produce neuronal precursors, which migrate and differentiate into functional mature neurons. This physiological neurogenesis is thought to be involved in neuronal plasticity. Moreover, recent studies indicate that adult neurogenesis can change in response to various brain insults, including psychiatric diseases, stroke, and neurodegenerative disorders. Although increased neurogenesis in these pathological conditions could contribute to the restoration and regeneration of the damaged brain, an inadequate and/or excessive supply of new neurons, or suppressed neurogenesis, could contribute to their pathophysiology. To develop successful regenerative treatments for the injured brain, we need to understand more precisely and comprehensively the mechanisms regulating adult neurogenesis under both physiological and pathological conditions.

Efhc1 Deficiency Causes Spontaneous Myoclonus and Increased Seizure Susceptibility

Human Molecular Genetics. Mar, 2009  |  Pubmed ID: 19147686

Mutations in EFHC1 gene have been previously reported in patients with epilepsies, including those with juvenile myoclonic epilepsy. Myoclonin1, also known as mRib72-1, is encoded by the mouse Efhc1 gene. Myoclonin1 is dominantly expressed in embryonic choroid plexus, post-natal ependymal cilia, tracheal cilia and sperm flagella. In this study, we generated viable Efhc1-deficient mice. Most of the mice were normal in outward appearance, and both sexes were found to be fertile. However, the ventricles of the brains were significantly enlarged in the null mutants, but not in the heterozygotes. Although the ciliary structure was found intact, the ciliary beating frequency was significantly reduced in null mutants. In adult stages, both the heterozygous and null mutants developed frequent spontaneous myoclonus. Furthermore, the threshold of seizures induced by pentylenetetrazol was significantly reduced in both heterozygous and null mutants. These observations seem to further suggest that decrease or loss of function of myoclonin1 may be the molecular basis for epilepsies caused by EFHC1 mutations.

Roles of Disrupted-in-schizophrenia 1-interacting Protein Girdin in Postnatal Development of the Dentate Gyrus

Neuron. Sep, 2009  |  Pubmed ID: 19778507

Disrupted-In-Schizophrenia 1 (DISC1), a susceptibility gene for major psychiatric disorders, regulates neuronal migration and differentiation during mammalian brain development. Although roles for DISC1 in postnatal neurogenesis in the dentate gyrus (DG) have recently emerged, it is not known how DISC1 and its interacting proteins govern the migration, positioning, and differentiation of dentate granule cells (DGCs). Here, we report that DISC1 interacts with the actin-binding protein girdin to regulate axonal development. DGCs in girdin-deficient neonatal mice exhibit deficits in axonal sprouting in the cornu ammonis 3 region of the hippocampus. Girdin deficiency, RNA interference-mediated knockdown, and inhibition of the DISC1/girdin interaction lead to overextended migration and mispositioning of the DGCs resulting in profound cytoarchitectural disorganization of the DG. These findings identify girdin as an intrinsic factor in postnatal development of the DG and provide insights into the critical role of the DISC1/girdin interaction in postnatal neurogenesis in the DG.

Identification of Tumor-initiating Cells in a Highly Aggressive Brain Tumor Using Promoter Activity of Nucleostemin

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2009  |  Pubmed ID: 19805150

Controversy remains over whether the cancer stem cell (CSC) theory applies to all tumors. To determine whether cells within a highly aggressive solid tumor are stochastically or hierarchically organized, we combined a reporter system where the nucleostemin (NS) promoter drives GFP expression (termed NS-GFP) with a mouse brain tumor model induced by retroviral Ras expression on a p16(Ink4a)/p19(Arf)-deficient background. The NS-GFP system allowed us to monitor the differentiation process of normal neural stem/precursor cells by analyzing GFP fluorescence intensity. In tumor-bearing mice, despite the very high frequency of tumorigenic cells, we successfully identified the NS-GFP(+) cells as tumor-initiating cells (T-ICs). The clonal studies conclusively established that phenotypical heterogeneity can exist among the cells comprising a genetically homogeneous tumor, suggesting that this aggressive brain tumor follows the CSC model. Detailed analyses of the NS-GFP(+) brain tumor cells revealed that T-ICs showed activation of the receptor tyrosine kinase c-Met, which functions in tumor invasiveness. Thus, the NS-GFP system provides a powerful tool to elucidate stem cell biology in normal and malignant tissues.

Various Facets of Vertebrate Cilia: Motility, Signaling, and Role in Adult Neurogenesis

Proceedings of the Japan Academy. Series B, Physical and Biological Sciences. 2009  |  Pubmed ID: 19838012

Cilia are microtubule-based cellular organelles that are widely distributed in vertebrate tissues. They were first observed hundreds of years ago. Recent studies indicate that this small organelle plays important roles in numerous physiological phenomena, including tissue morphogenesis, signal transduction, determination of left-right asymmetry during development, and adult neurogenesis. Ciliopathies, syndromes resulting from a genetic disorder of cilial components, frequently have complex effects involving many organ systems, owing to the broad distribution of cilia in the body.

[Epigenetic Mechanisms Regulating Neural Cell Fate Determination]

No to Hattatsu. Brain and Development. Nov, 2009  |  Pubmed ID: 19928537

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in specifiying the fate of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work, we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which were expressed predominantly in neurons in the central nervous system, inhibited astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vive (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

[Endogenous Repair Mechanisms in the Brain]

Rinshō Shinkeigaku = Clinical Neurology. Nov, 2009  |  Pubmed ID: 20030223

Most of neurons are generated by neural stem cells in the developing brain at the embryonic or neonatal stages. However, recent studies indicate that adult brain also contains neural stem cells that continuously generate new neurons. Neurogenesis can be observed in the adult subventricular zone (SVZ) at the lateral wall of the lateral ventricles of various animal species including primates. Young neurons generated in the SVZ migrate over long distances and mature after they reach their final destinations where they function. In this talk, I will present our recent studies using animal models on the mechanisms of neuronal production, migration and maturation in the adult brain under physiological and pathological conditions, and discuss the possibility of their application into regeneration therapies for ischemic brain diseases.

Musashi1 As a Marker of Reactive Astrocytes After Transient Focal Brain Ischemia

Neuroscience Research. Apr, 2010  |  Pubmed ID: 20036290

The synthesis of glial-fibrillary acidic protein (GFAP) or the re-expression of progenitor markers such as Nestin increases in reactive astrocytes after brain ischemia. We investigated the dynamics of reactive astrocytes after transient focal brain ischemia by examining the expression of Musashi1 (Msi1), an RNA-binding protein and another marker of neural stem/progenitor cells. In ischemic striatum induced by middle cerebral artery occlusion (MCAO), an increase in Msi1-immunoreactivity was observed from 2 days after MCAO, persisting until 14 days. The proliferation of Msi1-positive cells was observed from 4 days after MCAO and reached a peak at 7 days. These Msi1-positive cells were regarded as reactive astrocytes based on their co-expression with GFAP or Nestin and their morphology. Msi1-positive cells were located in the peri-infarct area in a region similar, but not identical, to that of Nestin-positive cells. The Msi1(+)Nestin(+) cells were located much closer to the ischemic core than the Msi1(+)Nestin(-) cells. The present study revealed that Msi1-expression, similar to Nestin, is induced after brain ischemia and may be involved in the reactivation of astrocytes, including their proliferation. However, the difference in the distributions of Msi1 and Nestin suggests that some of their features may differ in reactive astrocytes.

Subventricular Zone-derived Neural Progenitor Cells Migrate Along a Blood Vessel Scaffold Toward the Post-stroke Striatum

Stem Cells (Dayton, Ohio). Mar, 2010  |  Pubmed ID: 20073084

The subventricular zone (SVZ) of the adult brain contains neural stem cells that have the capacity to regenerate new neurons after various insults. Brain ischemia causes damage to brain tissue and induces neural regeneration together with angiogenesis. We previously reported that, after ischemic injury in mice, SVZ-derived neural progenitor cells (NPCs) migrate into the striatum, and these NPCs are frequently associated with blood vessels in the regenerating brain tissue. Here we studied the role of blood vessels during the neural regeneration in more detail. BrdU administration experiments revealed that newly generated NPCs were associated with both newly formed and pre-existing blood vessels in the ischemic striatum, suggesting that the angiogenic environment is not essential for the neuron-blood vessel interaction. To observe migrating NPCs and blood vessels simultaneously in damaged brain tissue, we performed live imaging of cultured brain slices after ischemic injury. In this system, we virally labeled SVZ-derived NPCs in Flk1-EGFP knock-in mice in which the blood vessels are labeled with EGFP. Our results provide direct evidence that SVZ-derived NPCs migrate along blood vessels from the SVZ toward the ischemic region of the striatum. The leading process of the migrating NPCs was closely associated with blood vessels, suggesting that this interaction provides directional guidance to the NPCs. These findings suggest that blood vessels play an important role as a scaffold for NPCs migration toward the damaged brain region.

Coupling Between Hydrodynamic Forces and Planar Cell Polarity Orients Mammalian Motile Cilia

Nature Cell Biology. Apr, 2010  |  Pubmed ID: 20305650

In mammals, motile cilia cover many organs, such as fallopian tubes, respiratory tracts and brain ventricles. The development and function of these organs critically depend on efficient directional fluid flow ensured by the alignment of ciliary beating. To identify the mechanisms involved in this process, we analysed motile cilia of mouse brain ventricles, using biophysical and molecular approaches. Our results highlight an original orientation mechanism for ependymal cilia whereby basal bodies first dock apically with random orientations, and then reorient in a common direction through a coupling between hydrodynamic forces and the planar cell polarity (PCP) protein Vangl2, within a limited time-frame. This identifies a direct link between external hydrodynamic cues and intracellular PCP signalling. Our findings extend known PCP mechanisms by integrating hydrodynamic forces as long-range polarity signals, argue for a possible sensory role of ependymal cilia, and will be of interest for the study of fluid flow-mediated morphogenesis.

Regulation of Adult Neural Progenitor Cells by Galectin-1/beta1 Integrin Interaction

Journal of Neurochemistry. Jun, 2010  |  Pubmed ID: 20367753

Neural stem cells (NSCs) proliferate and generate new neurons in the adult brain. A carbohydrate-binding protein (lectin), Galectin-1, is expressed in the NSCs in the subependymal zone (SEZ) of the adult mouse brain. The infusion and knockout of Galectin-1 in the SEZ results in an increase and decrease, respectively, of NSCs and subsequently born progenitor cells. The molecular mechanism of this effect, however, has been unknown. Previous studies outside the brain suggest that Galectin-1 binds to a carbohydrate structure of beta1 Integrin and modulates cell adhesion. Here, we studied the functional interaction between Galectin-1 and beta1 Integrin in the adult mouse SEZ. Beta1 Integrin was purified from adult SEZ tissue by binding to a Galectin-1 affinity column, and this binding depended on Galectin-1's carbohydrate-binding activity. In adult brain sections, Galectin-1-binding activity was detected on beta1 Integrin-expressing cells in the SEZ. Furthermore, in the adult SEZ, the simultaneous infusion of a beta1 Integrin-neutralizing antibody with Galectin-1 protein reversed the increasing effect of Galectin-1 on the number of adult neural progenitor cells (NPCs). Finally, intact beta1 Integrin was required for Galectin-1's function in NPC adhesion in vitro. These results suggest that the interaction between beta1 Integrin and Galectin-1 plays an important role in regulating the number of adult NPCs through mechanisms including cell adhesion.

New Neurons Clear the Path of Astrocytic Processes for Their Rapid Migration in the Adult Brain

Neuron. Jul, 2010  |  Pubmed ID: 20670830

In the long-range neuronal migration of adult mammals, young neurons travel from the subventricular zone to the olfactory bulb, a long journey (millimeters to centimeters, depending on the species). How can these neurons migrate through the dense meshwork of neuronal and glial processes of the adult brain parenchyma? Previous studies indicate that young neurons achieve this by migrating in chains through astrocytic tunnels. Here, we report that young migrating neurons actively control the formation and maintenance of their own migration route. New neurons secrete the diffusible protein Slit1, whose receptor, Robo, is expressed on astrocytes. We show that the Slit-Robo pathway is required for morphologic and organizational changes in astrocytes that result in the formation and maintenance of the astrocytic tunnels. Through this neuron-glia interaction, the new neurons regulate the formation of the astrocytic meshwork that is needed to enable their rapid and directional migration in adult brain.

Planar Polarity of Multiciliated Ependymal Cells Involves the Anterior Migration of Basal Bodies Regulated by Non-muscle Myosin II

Development (Cambridge, England). Sep, 2010  |  Pubmed ID: 20685736

Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.

Expression and Proliferation-promoting Role of Diversin in the Neuronally Committed Precursor Cells Migrating in the Adult Mouse Brain

Stem Cells (Dayton, Ohio). Nov, 2010  |  Pubmed ID: 20827749

The subventricular zone (SVZ) is the largest neurogenic region in the adult rodent brain. In the adult SVZ, unlike in the embryonic brain, neuronally committed precursor cells (neuroblasts) maintain their proliferative activity while migrating toward the olfactory bulb (OB), suggesting that they are inhibited from exiting the cell cycle. Little is known about the mechanisms underlying the unique ability of adult neuroblasts to proliferate during migration. Here, we studied the expression and function of Diversin, a component of the Wnt signaling pathways. In the neonatal and adult mouse brain, Diversin expression was observed in neuroblasts and mature neurons in the SVZ and hippocampus. Retrovirus-mediated overexpression of Diversin promoted the proliferation of neuroblasts and increased the number of neuroblasts that reached the OB. Conversely, the knockdown of Diversin decreased the proliferation of neuroblasts. Our results indicate that Diversin plays an important role in the proliferation of neuroblasts in the SVZ of the adult brain.

Protein Phosphatase 1γ is Responsible for Dephosphorylation of Histone H3 at Thr 11 After DNA Damage

EMBO Reports. Nov, 2010  |  Pubmed ID: 20948546

The DNA-damage-induced transcriptional suppression of cell cycle regulatory genes correlates with a reduction in histone H3-Thr 11 phosphorylation (H3-pThr 11) on their promoters that is partly mediated by the dissociation of Chk1 from chromatin. In this study, we identify protein phosphatase 1γ (PP1γ) as a phosphatase responsible for DNA-damage-induced H3-pThr 11 dephosphorylation. PP1γ is activated after DNA damage, which is mainly mediated by a reduction in Cdk-dependent phosphorylation of PP1γ at Thr 311. The depletion of PP1γ sensitizes HCT116 cells to DNA damage. Our results suggest that the ataxia telangiectasia, mutated and Rad3-related-Chk1 axis regulates H3-pThr 11 dephosphorylation on DNA damage, at least in part by the activation of PP1γ through Chk1-dependent inhibition of Cdks.

Migration of New Neurons Towards the Injured Brain Tissue

Rinshō Shinkeigaku = Clinical Neurology. Nov, 2010  |  Pubmed ID: 21921491

Transplantation of Human Neural Stem/progenitor Cells Overexpressing Galectin-1 Improves Functional Recovery from Focal Brain Ischemia in the Mongolian Gerbil

Molecular Brain. 2011  |  Pubmed ID: 21951913

Transplantation of human neural stem/progenitor cells (hNSPCs) is a promising method to regenerate tissue from damage and recover function in various neurological diseases including brain ischemia. Galectin-1(Gal1) is a lectin that is expressed in damaged brain areas after ischemia. Here, we characterized the detailed Gal1 expression pattern in an animal model of brain ischemia. After brain ischemia, Gal1 was expressed in reactive astrocytes within and around the infarcted region, and its expression diminished over time. Previously, we showed that infusion of human Gal1 protein (hGal1) resulted in functional recovery after brain ischemia but failed to reduce the volume of the ischemic region. This prompted us to examine whether the combination of hNSPCs-transplantation and stable delivery of hGal1 around the ischemic region could reduce the ischemic volume and promote better functional recovery after brain ischemia. In this study, we transplanted hNSPCs that stably overexpressed hGal1 (hGal1-hNSPCs) in a model of unilateral focal brain ischemia using Mongolian gerbils. Indeed, we found that transplantation of hGal1-hNSPCs both reduced the ischemic volume and improved deficits in motor function after brain ischemia to a greater extent than the transplantation of hNSPCs alone. This study provides evidence for a potential application of hGal1 with hNSPCs-transplantation in the treatment of brain ischemia.

Neuronal Regeneration in a Zebrafish Model of Adult Brain Injury

Disease Models & Mechanisms. Oct, 2011  |  Pubmed ID: 22028327

Neural stem cells in the subventricular zone (SVZ) of the adult mammalian forebrain are a potential source of neurons for neural tissue repair after brain insults such as ischemic stroke and traumatic brain injury (TBI). Recent studies show that neurogenesis in the ventricular zone (VZ) of the adult zebrafish telencephalon has features in common with neurogenesis in the adult mammalian SVZ. Here, we established a zebrafish model to study injury-induced neurogenesis in the adult brain. We show that the adult zebrafish brain possesses a remarkable capacity for neuronal regeneration. Telencephalon injury prompted the proliferation of neuronal precursor cells (NPCs) in the VZ of the injured hemisphere, compared with in the contralateral hemisphere. The distribution of NPCs, viewed by BrdU labeling and ngn1-promoter-driven GFP, suggested that they migrated laterally and reached the injury site via the subpallium and pallium. The number of NPCs reaching the injury site significantly decreased when the fish were treated with an inhibitor of γ-secretase, a component of the Notch signaling pathway, suggesting that injury-induced neurogenesis mechanisms are at least partly conserved between fish and mammals. The injury-induced NPCs differentiated into mature neurons in the regions surrounding the injury site within a week after the injury. Most of these cells expressed T-box brain protein (Tbr1), suggesting they had adopted the normal neuronal fate in this region. These results suggest that the telencephalic VZ contributes to neural tissue recovery following telencephalic injury in the adult zebrafish, and that the adult zebrafish is a useful model for regenerative medicine.

Cellular Composition and Organization of the Subventricular Zone and Rostral Migratory Stream in the Adult and Neonatal Common Marmoset Brain

The Journal of Comparative Neurology. Mar, 2011  |  Pubmed ID: 21246550

The adult subventricular zone (SVZ) of the lateral ventricle contains neural stem cells. In rodents, these cells generate neuroblasts that migrate as chains toward the olfactory bulb along the rostral migratory stream (RMS). The neural-stem-cell niche at the ventricular wall is conserved in various animal species, including primates. However, it is unclear how the SVZ and RMS organization in nonhuman primates relates to that of rodents and humans. Here we studied the SVZ and RMS of the adult and neonatal common marmoset (Callithrix jacchus), a New World primate used widely in neuroscience, by electron microscopy, and immunohistochemical detection of cell-type-specific markers. The marmoset SVZ contained cells similar to type B, C, and A cells of the rodent SVZ in their marker expression and morphology. The adult marmoset SVZ had a three-layer organization, as in the human brain, with ependymal, hypocellular, and astrocyte-ribbon layers. However, the hypocellular layer was very thin or absent in the adult-anterior and neonatal SVZ. Anti-PSA-NCAM staining of the anterior SVZ in whole-mount ventricular wall preparations of adult marmosets revealed an extensive network of elongated cell aggregates similar to the neuroblast chains in rodents. Time-lapse recordings of marmoset SVZ explants cultured in Matrigel showed the neuroblasts migrating in chains, like rodent type A cells. These results suggest that some features of neurogenesis and neuronal migration in the SVZ are common to marmosets, humans, and rodents. This basic description of the adult and neonatal marmoset SVZ will be useful for future studies on adult neurogenesis in primates.

Galectin-1 is Expressed in Early-type Neural Progenitor Cells and Down-regulates Neurogenesis in the Adult Hippocampus

Molecular Brain. 2011  |  Pubmed ID: 21269521

In the adult mammalian brain, neural stem cells (NSCs) proliferate in the dentate gyrus (DG) of the hippocampus and generate new neurons throughout life. A multimodal protein, Galectin-1, is expressed in neural progenitor cells (NPCs) and implicated in the proliferation of the NPCs in the DG. However, little is known about its detailed expression profile in the NPCs and functions in adult neurogenesis in the DG.

Girdin is an Intrinsic Regulator of Neuroblast Chain Migration in the Rostral Migratory Stream of the Postnatal Brain

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2011  |  Pubmed ID: 21632933

In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed "chain migration." The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin's C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell-cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS.

Strategies for Regenerating Striatal Neurons in the Adult Brain by Using Endogenous Neural Stem Cells

Neurology Research International. 2011  |  Pubmed ID: 21766028

Currently, there is no effective treatment for the marked neuronal loss caused by neurodegenerative diseases, such as Huntington's disease (HD) or ischemic stroke. However, recent studies have shown that new neurons are continuously generated by endogenous neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain, including the human brain. Because some of these new neurons migrate to the injured striatum and differentiate into mature neurons, such new neurons may be able to replace degenerated neurons and improve or repair neurological deficits. To establish a neuroregenerative therapy using this endogenous system, endogenous regulatory mechanisms that can be co-opted for efficient regenerative interventions must be understood, along with any potential drawbacks. Here, we review current knowledge on the generation of new neurons in the adult brain and discuss their potential for use in replacing striatal neurons lost to neurodegenerative diseases, including HD, and to ischemic stroke.

Migration of Neuronal Precursors from the Telencephalic Ventricular Zone into the Olfactory Bulb in Adult Zebrafish

The Journal of Comparative Neurology. Dec, 2011  |  Pubmed ID: 21800305

In the brain of adult mammals, neuronal precursors are generated in the subventricular zone in the lateral wall of the lateral ventricles and migrate into the olfactory bulbs (OBs) through a well-studied route called the rostral migratory stream (RMS). Recent studies have revealed that a comparable neural stem cell niche is widely conserved at the ventricular wall of adult vertebrates. However, little is known about the migration route of neuronal precursors in nonmammalian adult brains. Here, we show that, in the adult zebrafish, a cluster of neuronal precursors generated in the telencephalic ventricular zone migrates into the OB via a route equivalent to the mammalian RMS. Unlike the mammalian RMS, these neuronal precursors are not surrounded by glial tubes, although radial glial cells with a single cilium lined the telencephalic ventricular wall, much as in embryonic and neonatal mammals. To observe the migrating neuronal precursors in living brain tissue, we established a brain hemisphere culture using a zebrafish line carrying a GFP transgene driven by the neurogenin1 (ngn1) promoter. In these fish, GFP was observed in the neuronal precursors migrating in the RMS, some of which were aligned with blood vessels. Numerous ngn1:gfp-positive cells were observed migrating tangentially in the RMS-like route medial to the OB. Taken together, our results suggest that the RMS in the adult zebrafish telencephalon is a functional migratory pathway. This is the first evidence for the tangential migration of neuronal precursors in a nonmammalian adult telencephalon.

Sensory Input Regulates Spatial and Subtype-specific Patterns of Neuronal Turnover in the Adult Olfactory Bulb

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2011  |  Pubmed ID: 21832189

Throughout life, new neurons are added and old ones eliminated in the adult mouse olfactory bulb. Previous studies suggested that olfactory experience controls the process by which new neurons are integrated into mature circuits. Here we report novel olfactory-experience-dependent mechanisms of neuronal turnover. Using two-photon laser-scanning microscopy and sensory manipulations in adult live mice, we found that the neuronal turnover was dynamically controlled by olfactory input in a neuronal subtype-specific manner. Olfactory input enhanced this turnover, which was characterized by the reiterated use of the same positions in the glomeruli by new neurons. Our results suggest that olfactory-experience-dependent modification of neuronal turnover confers structural plasticity and stability on the olfactory bulb.

Endogenous Erythropoietin from Astrocyte Protects the Oligodendrocyte Precursor Cell Against Hypoxic and Reoxygenation Injury

Journal of Neuroscience Research. Oct, 2011  |  Pubmed ID: 21833990

The hypoxia-responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT-PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT-PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury.

Planar Polarity of Ependymal Cilia

Differentiation; Research in Biological Diversity. Feb, 2012  |  Pubmed ID: 22101065

Ependymal cells, epithelial cells that line the cerebral ventricles of the adult brain in various animals, extend multiple motile cilia from their apical surface into the ventricles. These cilia move rapidly, beating in a direction determined by the ependymal planar cell polarity (PCP). Ciliary dysfunction interferes with cerebrospinal fluid circulation and alters neuronal migration. In this review, we summarize recent studies on the cellular and molecular mechanisms underlying two distinct types of ependymal PCP. Ciliary beating in the direction of fluid flow is established by a combination of hydrodynamic forces and intracellular planar polarity signaling. The ciliary basal bodies' anterior position on the apical surface of the cell is determined in the embryonic radial glial cells, inherited by ependymal cells, and established by non-muscle myosin II in early postnatal development.

A Role for MDia, a Rho-regulated Actin Nucleator, in Tangential Migration of Interneuron Precursors

Nature Neuroscience. Jan, 2012  |  Pubmed ID: 22246438

In brain development, distinct types of migration, radial migration and tangential migration, are shown by excitatory and inhibitory neurons, respectively. Whether these two types of migration operate by similar cellular mechanisms remains unclear. We examined neuronal migration in mice deficient in mDia1 (also known as Diap1) and mDia3 (also known as Diap2), which encode the Rho-regulated actin nucleators mammalian diaphanous homolog 1 (mDia1) and mDia3. mDia deficiency impaired tangential migration of cortical and olfactory inhibitory interneurons, whereas radial migration and consequent layer formation of cortical excitatory neurons were unaffected. mDia-deficient neuroblasts exhibited reduced separation of the centrosome from the nucleus and retarded nuclear translocation. Concomitantly, anterograde F-actin movement and F-actin condensation at the rear, which occur during centrosomal and nuclear movement of wild-type cells, respectively, were impaired in mDia-deficient neuroblasts. Blockade of Rho-associated protein kinase (ROCK), which regulates myosin II, also impaired nuclear translocation. These results suggest that Rho signaling via mDia and ROCK critically regulates nuclear translocation through F-actin dynamics in tangential migration, whereas this mechanism is dispensable in radial migration.