Chemoattractive activity of sonic hedgehog in the adult subventricular zone modulates the number of neural precursors reaching the olfactory bulb

The adult subventricular zone (SVZ) supports neural stem cell self-renewal and differentiation and continually gives rise to new neurons throughout adult life. The mechanisms orienting the migration of neuroblasts from the SVZ to the olfactory bulb (OB) via the rostral migratory stream (RMS) have been extensively studied, but factors controlling neuroblast exit from the SVZ remain poorly explored. The morphogen Sonic Hedgehog (Shh) displays proliferative and survival activities toward neural stem cells and is an axonal chemoattractant implicated in guidance of commissural axons during development. We identify here the presence of Shh protein in SVZ extracts and in the cerebrospinal fluid of adult mice, and we demonstrate that migrating neuroblasts in the SVZ and RMS express the Shh receptor Patched. We show that Shh displays a chemoattractive activity in vitro on SVZ-derived neuronal progenitors, an effect blocked by Cur61414, a Smoothened antagonist. Interestingly, Shh-expressing cells grafted above the RMS of adult mice exert a chemoattractive activity on migrating neuroblasts in vivo, thus inducing their accumulation and deviation from their normal migratory pathway. Furthermore, the adenoviral transfer of Shh into the lateral ventricle or the blocking of Shh present in the SVZ of adult mice using its physiological antagonist Hedgehog interacting protein or neutralizing Shh antibodies provides in vivo evidence that Shh can retain SVZ-derived neuroblasts. The ability to modulate the number of neuroblasts leaving the SVZ and reaching the OB through the chemoattractive activity of Shh suggests a novel degree of plasticity in cell migration of this adult stem cell niche.     

EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells

The subventricular germinal zone (SVZ) retains an active population of stem cells and neural precursor cells throughout adulthood. EphrinB signaling mediates angiogenesis and vasculogenesis in the developing and adult brain. Recent studies indicate that molecules involved in angiogenesis often influence neurogenesis as well. However, little work has been done considering a role for EphB2/EphrinB in adult neural precursor cells. We therefore examined whether the EphB2 receptor tyrosine kinase could directly effect proliferation of SVZ neural precursors and/or direct the cell fate of SVZ cells in vitro. Here, we found that clustered EphB2 increased bromodeoxyuridine (BrdU) incorporation and proliferation of SVZ neurosphere cultures. Immunostaining and RT-PCR analysis for beta-tubulin III (Tuj1) and GFAP indicated 4-day treatment with EphB2 promoted a neuronal phenotype, suggesting that the EphB2 receptor might also direct SVZ cell fate. EphB2 transiently down-regulated SVZ cell mRNA of Notch1 and Zic1, genes that regulate neurogenesis and neuronal differentiation. Notch1 has been implicated in apoptosis of neural precursors, however, a cell viability assay revealed no statistical difference between EphB2-treated and control cultures. When SVZ neurospheres were cultured upon Matrigel, EphB2 attenuated radial migration of SVZ cells in vitro. These results demonstrate that EphB2/EphrinB signaling directly induces SVZ proliferation, decreases migration, and promotes a neuronal fate of SVZ neural precursors independent of cell survival.     

Characterization of novel monoclonal antibodies able to identify neurogenic niches and arrest neurosphere proliferation and differentiation

Two monoclonal antibodies (Nilo1 and Nilo2) were generated after immunization of hamsters with E13.5 olfactory bulb-derived mouse neurospheres. They are highly specific for neural stem and early progenitor cell surface antigens. Nilo positive cells present in the adult mouse subventricular zone (SVZ) were able to initiate primary neural stem cell cultures. Moreover, these antibodies added to neurosphere cultures induced proliferation arrest and interfered with their differentiation. In the lateral ventricles of adult mice, Nilo1 stained a cell subpopulation lining the ventricle and cells located in the SVZ, whereas Nilo2 stained a small population associated with the anterior horn of the SVZ at the beginning of the rostral migratory stream. Co-staining of Nilo1 or Nilo2 and neural markers demonstrated that Nilo1 identifies an early neural precursor subpopulation, whereas Nilo2 detects more differentiated neural progenitors. Thus, these antibodies identify distinct neurogenic populations within the SVZ of the lateral ventricle.     

喙端迁移流的发生及其细胞凋亡

目的 研究生后不同日龄小鼠喙端迁移流（RMS）的发育,神经干细胞增殖和凋亡的规律。方法 利用Caspase-8免疫荧光标记法和5’-溴脱氧尿嘧啶核苷（BrdU）法,对小鼠RMS内的神经干细胞增殖和凋亡进行研究（n =92）。结果 生后早期小鼠脑内,尤其是室管下区（SVZ）和RMS,存在大量的增殖细胞。随着小鼠年龄的增加,脑内干细胞逐渐减少,到成年,大脑皮质几乎见不到增殖的神经干细胞,但在SVZ和RMS仍可以看到许多增殖的神经干细胞。在RMS,神经干细胞增殖的同时伴随着细胞凋亡,干细胞的增殖与凋亡存在着正相关关系。结论 RMS的神经干细胞增殖与凋亡有重要的生理意义,通过细胞凋亡,RMS可以调节神经干细胞向嗅球迁移的数量,也可以调节干细胞向颗粒细胞分化。     

Characterization of adult neural stem cells and their relation to brain tumors

The adult mammalian brain contains neural stem cells that are capable of generating new neurons and glia over the course of a lifetime. Neural stem cells reside in 2 germinal niches, the subventricular zone (SVZ) and the dentate gyrus subgranular zone. These primary progenitors have been identified in their niche in vivo; these cells have characteristics of astrocytes. Recent studies have shown that adult SVZ stem cells are derived from radial glia, the stem cells in the developing brain, which in turn are derived from the neuroepithelum, the earliest brain progenitors. Thus, SVZ stem cells are a continuum from neuroepithelium to radial glia to astrocytes, and are contained within what has been considered the lineage for astrocytes. However, it seems that only a small subset of the astrocytes present in the adult brain have stem cell properties. Recent findings have shown that SVZ stem cell astrocytes express a receptor for platelet-derived growth factor (PDGF), suggesting that the ability to respond to specific growth factor stimuli, such as PDGF, epidermal growth factor and others, may be unique to these stem cell astrocytes. Intriguingly, activation of these same signaling pathways is widely implicated in brain tumor formation. Since the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for transformation, the adult neural stem and/or progenitor cells may be likely candidates for the brain tumor cell of origin. Indeed, activation of the PDGF or epidermal growth factor pathways in adult neural stem or progenitor cells confers tumor-like properties on these cells, lending support to this hypothesis.     

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone

The tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. We show here that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuroblastic apoptosis in the subventricular zone is caused by 1-methy-4-phenylpiridinium (MPP+) converted from MPTP through MAO-B

Intraperitoneal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration induces apoptosis of subventricular zone (SVZ) doublecortin (Dcx)-positive neural progenitor cells (migrating neuroblasts, A cells). Actually, a metabolite of MPTP, 1-methy-4-phenylpiridinium (MPP⁺), is responsible for neural progenitor cell toxicity. In the present study, to examine whether the MPTP-induced SVZ cell apoptosis is caused directly by MPP⁺ metabolized through monoamine oxidase B (MAO-B), MPTP or MPP⁺ was intracerebroventricularly (icv) injected into C57BL/6 mice. At Day 1 postinjection, many terminal deoxynucleotidyl transferase-mediated dUTP endlabeling (TUNEL)-positive cells were observed in the SVZ of both low (36 μg) and high (162 μg) dose MPTP- and MPP⁺-injected mice. The number of Dcx-positive A cells showed a significant decrease following high dose of MPTP- or MPP⁺-injection on Days 1 and 3, respectively, whereas that of EGFR-positive C cells showed no change in mice with any treatment. In addition, prior icv injection of a MAO-B inhibitor, R(?)-deprenyl (deprenyl), inhibited MPTP-induced apoptosis, but not MPP⁺-induced apoptosis. MAO-B- and GFAP-double positive cells were detected in the ependyma and SVZ in all mice. It is revealed from these results that icv injection of MPTP induces apoptosis of neural progenitor cells (A cells) in the SVZ via MPP⁺ toxicity. In addition, it is suggested that the conversion from MPTP to MPP⁺ is caused mainly by MAO-B located in ependymal cells and GFAP-positive cells in the SVZ.     

Glutamate enhances survival and proliferation of neural progenitors derived from the subventricular zone

Extracellular glutamate levels increase as a consequence of perinatal hypoxia/ischemia, causing the death of neurons and oligodendrocytes. Precursors in the subventricular zone (SVZ) also die following perinatal hypoxia/ischemia; therefore we hypothesized that glutamate would stimulate the death of neural precursors. Here we demonstrate using calcium imaging that SVZ derived neural stem/progenitor cells respond to both ionotropic and metabotropic excitatory amino acids. Therefore, we tested the effects of high levels of glutamate receptor agonists on the proliferation, survival, and differentiation of SVZ derived neural stem/progenitor cells in vitro. We show that high levels of glutamate, up to 1 mM, are not toxic to neural precursor cultures. In fact, stimulation of either the kainate receptor or group 2 metabotropic glutamate receptors (group 2 mGluR) reduces basal levels of apoptosis and increases neural precursor proliferation. Furthermore, group 2 mGluR activation expands the number of multipotent progenitor cells present in these cultures while maintaining equivalent mature cell production. We conclude that the glutamate released following perinatal hypoxia/ischemia may act to acutely promote the proliferation of multipotent precursors in the subventricular zone.     

Beta-catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone

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.     

Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells

Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. Here, we show that neural stem cells are generated independent of RBP-Jkappa, a key molecule in Notch signaling, by using RBP-Jkappa(-/-) embryonic stem cells in an embryonic stem cell-derived neurosphere assay. However, Notch pathway molecules are essential for the maintenance of neural stem cells; they are depleted in the early embryonic brains of RBP-Jkappa(-/-) or Notch1(-/-) mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1(+/-) adult brains. Both neuronal and glial differentiation in vitro were enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch.     

The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke

Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a γ secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a γ secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.     

Induction of neurogenesis in nonconventional neurogenic regions of the adult central nervous system by niche astrocyte-produced signals

The central nervous system (CNS) of adult mammals regenerates poorly; in vivo, neurogenesis occurs only in two restricted areas, the hippocampal subgranular zone (SGZ) and the subventricular zone (SVZ). Neurogenic potential depends on both the intrinsic properties of neural progenitors and the environment, or niche, in which progenitor cells reside. Isolation of multipotent progenitor cells from broad CNS regions suggests that the neurogenic potential of the adult CNS is dictated by local environmental cues. Here, we report that astrocytes in the neurogenic brain regions, the SGZ and SVZ, of adult mice release molecular signals, such as sonic hedgehog (Shh), that stimulate adult neural progenitors to reenter the cell cycle and generate new neurons in vitro and in vivo. Transplantation of SGZ astrocytes or application of Shh caused de novo neurogenesis from the non-neurogenic neocortex of adult mice. These findings identify a molecular target that can activate the dormant neurogenic potential from nonconventional neurogenic regions of the adult CNS and suggest a novel mechanism of neural replacement therapy for treating neurodegenerative disease and injury without transplanting exogenous cells.     

Adult neural stem cells from the mouse subventricular zone are limited in migratory ability compared to progenitor cells of similar origin

The subventricular zone (SVZ) in the forebrain is the largest source of neural stem cells and progenitor cells in the adult CNS. To assess the ability of adult neural stem cells to survive, differentiate and migrate, we have compared the behavior of dissociated, neurosphere-derived stem cells with that of progenitor cells in transplantation experiments. This ability was first tested in vivo, offering the stem cells the possibility to migrate along the rostral migratory stream (RMS), their specific pathway. In addition, the differential behaviors of the two classes of cells were also compared in vitro by grafting them into organotypic slice cultures containing either tangential (embryonic cerebral cortex) or radial (early postnatal cerebellar cortex) migratory routes. Most of the grafted adult neurosphere-derived stem cells survived and integrated in vivo, and a proportion of them differentiate into neurons, oligodendrocytes or astrocytes. However, they were unable to migrate along the RMS and remained in the vicinity of the injection site. In contrast, SVZ progenitor cells were able to migrate toward the olfactory bulb and, once there, to acquire the phenotype of granule cells, as previously reported. In vitro, neural stem cells exhibited a better migratory ability, although they only migrated for short distances, particularly, in forebrain slices. Nevertheless, the average distance covered by progenitor cells was a two-fold longer than that covered by neural stem cells, corroborating that this class of more specified cells has higher migratory ability. These results suggest that the in vitro conditions of expanding SVZ-derived stem cells, required to maintain them in an immature stage might modify their intrinsic properties, preventing their differentiation into neuroblasts and their subsequent migration.     

Selective upregulation of 3-phosphoglycerate dehydrogenase (Phgdh) expression in adult subventricular zone neurogenic niche

In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-phosphoglycerate dehydrogenase (Phgdh), an enzyme involved in de novo synthesis of l-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that l-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. In the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of l-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain.     

Analysis of extracellular signal-regulated kinase 2 function in neural stem/progenitor cells via nervous system-specific gene disruption

Extracellular signal-regulated kinase 2 (ERK2) is involved in a variety of cell fate decisions during development, but its exact role in this process remains to be determined. To specifically focus on the role of ERK2 in the brain, and to avoid early lethalities, we used a conditional gene-targeting approach to preferentially inactivate Erk2 in the embryonic mouse brain. The resulting mutant mice were viable and were relatively normal in overall appearance. However, the loss of Erk2 resulted in a diminished proliferation of neural stem cells in the embryonic ventricular zone (VZ), although the survival and differentiation of these cells was unaffected. The multipotent neural progenitor cells (NPCs) isolated from ERK2-deficient brains also showed impaired proliferation, reduced self-renewal ability, and increased apoptosis. By neurosphere differentiation analysis we further observed that lineage-restricted glial progenitors were increased in ERK2-deficient mice. The decline in the self-renewal ability and multipotency of NPCs resulting from the loss of ERK2 was found to be caused at least in part by upregulation of the JAK-STAT signaling pathway and reduced G1/S cell cycle progression. Furthermore, by global expression analysis we found that neural stem cell markers, including Tenascin C NR2E1 (Tlx), and Lgals1 (Galectin-1), were significantly downregulated, whereas several glial lineage markers were upregulated in neurospheres derived from ERK2-deficient mice. Our results thus suggest that ERK2 is required both for the proliferation of neural stem cells in the VZ during embryonic development and in the maintenance of NPC multipotency by suppressing the commitment of these cells to a glial lineage.     

An endogenous vitamin K-dependent mechanism regulates cell proliferation in the brain subventricular stem cell niche

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K-dependent proteins (VKDPs) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin ((S(-)-3-acetonylbenzyl)-4-hydroxycoumarin)), a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDPs production, in vitro, by exposure of SVZ cell cultures to warfarin or, in vivo, by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anticoagulant factors, protein S and its structural homolog Gas6, as the two only VKDPs produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, and Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDPs in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem-like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDPs, and anticoagulants in NSC biology in health and disease.     

Ephrin-A2 reverse signaling negatively regulates neural progenitor proliferation and neurogenesis

The number of cells in an organ is regulated by mitogens and trophic factors that impinge on intrinsic determinants of proliferation and apoptosis. We here report the identification of an additional mechanism to control cell number in the brain: EphA7 induces ephrin-A2 reverse signaling, which negatively regulates neural progenitor cell proliferation. Cells in the neural stem cell niche in the adult brain proliferate more and have a shorter cell cycle in mice lacking ephrin-A2. The increased progenitor proliferation is accompanied by a higher number of cells in the olfactory bulb. Disrupting the interaction between ephrin-A2 and EphA7 in the adult brain of wild-type mice disinhibits proliferation and results in increased neurogenesis. The identification of ephrin-A2 and EphA7 as negative regulators of progenitor cell proliferation reveals a novel mechanism to control cell numbers in the brain.     

A profusion of neural stem cells in the brain of the spiny mouse,Acomys cahirinus

The vast majority of neural stem cell studies have been conducted on the brains of mice and rats, the classical model rodent. Non-model organisms may, however, give us some important insights into how to increase neural stem cell numbers for regenerative purposes and with this in mind we have characterized these cells in the brain of the spiny mouse, Acomys cahirinus. This unique mammal is highly regenerative and damaged tissue does not scar or fibrose. We find that there are more than three times as many stem cells in the SVZ and more than 3 times as many proliferating cells compared to the CD-1 outbred strain of lab mouse. These additional cells create thick stem cell regions in the wall of the SVZ and very obvious columns of cells moving into the rostral migratory stream. In the dentate gyrus, there are more than 10 times as many cells proliferating in the sub-granular layer and twice the number of doublecortin expressing neuroblasts. A preliminary analysis of some stem cell niche genes has identified Sox2, Notch1, Shh, and Noggin as up-regulated in the SVZ of Acomys and Bmp2 as being down-regulated. The highly increased neural stem cell numbers in Acomys may endow this animal with increased regenerative properties in the brain or improved physiological performance important for its survival.