Reduced proliferation in the adult mouse subventricular zone increases survival of olfactory bulb interneurons

Neurogenesis in the adult brain is largely restricted to the subependymal zone (SVZ) of the lateral ventricle, olfactory bulb (OB) and the dentate subgranular zone, and survival of adult-born cells in the OB is influenced by factors including sensory experience. We examined, in mice, whether survival of adult-born cells is also regulated by the rate of precursor proliferation in the SVZ. Precursor proliferation was decreased by depleting the SVZ of dopamine after lesioning dopamine neurons in the substantia nigra compacta with 6-hydroxydopamine. Subsequently, we examined the effect of reduced SVZ proliferation on the generation, migration and survival of neuroblasts and mature adult-born cells in the SVZ, rostral migratory stream (RMS) and OB. Proliferating cells in the SVZ, measured by 5-bromo-2-deoxyuridine (BrdU) injected 2 hours prior to death or by immunoreactivity against Ki67, were reduced by 47% or 36%, respectively, 7 days after dopamine depletion, and by 29% or 31% 42 days after dopamine depletion, compared to sham-treated animals. Neuroblast generation in the SVZ and their migration along the RMS were not affected, neither 7 nor 42 days after the 6-hydroxydopamine injection, since the number of doublecortin-immunoreactive neuroblasts in the SVZ and RMS, as well as the number of neuronal nuclei-immunoreactive cells in the OB, were stable compared to control. However, survival analysis 15 days after 6-hydroxydopamine and 6 days after BrdU injections showed that the number of BrdU+ cells in the SVZ was 70% higher. Also, 42 days after 6-hydroxydopamine and 30 days after BrdU injections, we found an 82% increase in co-labeled BrdU+/γ-aminobutyric acid-immunoreactive cell bodies in the granular cell layer, while double-labeled BrdU+/tyrosine hydroxylase-immunoreactive cell bodies in the glomerular layer increased by 148%. We conclude that the number of OB interneurons following reduced SVZ proliferation is maintained through an increased survival of adult-born precursor cells, neuroblasts and interneurons.     

Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal

Neural stem cells (NSCs) in the postnatal mammalian brain self-renew and are a source of neurons and glia. To date, little is known about the molecular and cellular mechanisms regulating the maintenance and differentiation of these multipotent progenitors. We show that Jagged1 is required by mitotic cells in the subventricular zone (SVZ) and stimulates self-renewal of multipotent epidermal growth factor-dependent NSCs. Jagged1-expressing cells line the adult SVZ and are juxtaposed to Notch1-expressing cells, some of which are putative NSCs. In vitro, endogenous Jagged1 acts through Notch1 to promote NSC maintenance and multipotency. In vivo, reducing Jagged1/Notch1 signaling decreases the number of proliferating cells in the SVZ. In addition, soluble Jagged1 promotes self-renewal and neurogenic potential of multipotent neural progenitors in vitro. Our findings suggest a central role for Jagged1 in the NSC niche in the SVZ for maintaining a population of NSCs in the postnatal brain.     

In vitro characterization of subventricular zone isolated neural stem cells,from adult monkey and rat brain

Neural stem cells (NSCs) are multipotent, self-renewable cells who are capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs reside at the subventricular zone (SVZ) of the adult brain permanently to guarantee a lifelong neurogenesis during neural network plasticity or undesirable injuries. Although the specious inaccessibility of adult NSCs niche hampers their in vivo identification, researchers have been seeking ways to optimize adult NSCs isolation, expansion, and differentiation, in vitro. NSCs were isolated from rhesus monkey SVZ, expanded in vitro and then characterized for NSCs-specific markers expression by immunostaining, real-time PCR, flow cytometry, and cell differentiation assessments. Moreover, cell survival as well as self-renewal capacity were evaluated by TUNEL, Live/Dead and colony assays, respectively. In the next step, to validate SVZ-NSCs identity in other species, a similar protocol was applied to isolate NSCs from adult rat’s SVZ as well. Our findings revealed that isolated SVZ-NSCs from both monkey and rat preserve proliferation capacity in at least nine passages as confirmed by Ki67 expression. Additionally, both SVZ-NSCs sources are capable of self-renewal in addition to NESTIN, SOX2, and GFAP expression. The mortality was measured meager with over 95% viability according to TUNEL and Live/Dead assay results. Eventually, the multipotency of SVZ-NSCs appraised authentic after their differentiation into neurons, astrocytes, and oligodendrocytes. In this study, we proposed a reliable method for SVZ-NSCs in vitro maintenance and identification, which, we believe is a promising cell source for therapeutic approach to recover neurological disorders and injuries condition.

     

Development of neural stem cell in the adult brain

New neurons are continuously generated in the dentate gyrus of the mammalian hippocampus and in the subventricular zone of the lateral ventricles throughout life. The origin of these new neurons is believed to be from multipotent adult neural stem cells. Aided by new methodologies, significant progress has been made in the characterization of neural stem cells and their development in the adult brain. Recent studies have also begun to reveal essential extrinsic and intrinsic molecular mechanisms that govern sequential steps of adult neurogenesis in the hippocampus and subventricular zone/olfactory bulb, from proliferation and fate specification of neural progenitors to maturation, navigation, and synaptic integration of the neuronal progeny. Future identification of molecular mechanisms and physiological functions of adult neurogenesis will provide further insight into the plasticity and regenerative capacity of the mature central nervous system.     

Gene profiles within the adult subventricular zone niche: proliferation, differentiation and migration of neural progenitor cells in the ischemic brain

Focal cerebral ischemia induces neurogenesis in the subventricular zone (SVZ) of the adult human brain. Neurogenesis is controlled by proliferation, differentiation, and migration of neural progenitor cells. This article reviews emerging data that changes of cell cycle kinetics of neural progenitor cells induced by stroke contribute to increased neural progenitor cell proliferation and that gene profiles control proliferation, differentiation, and migration of neural progenitor cells within the SVZ niche. A better understanding of gene profiles that control the biological function of adult SVZ neural progenitor cells could lead to more selective and effective treatments to enhance neurogenesis during stroke recovery.     

Cdk2 is critical for proliferation and self-renewal of neural progenitor cells in the adult subventricular zone

We investigated the function of cyclin-dependent kinase 2 (Cdk2) in neural progenitor cells during postnatal development. Chondroitin sulfate proteoglycan (NG2)–expressing progenitor cells of the subventricular zone (SVZ) show no significant difference in density and proliferation between Cdk2^−/− and wild-type mice at perinatal ages and are reduced only in adult Cdk2^−/− mice. Adult Cdk2^−/− SVZ cells in culture display decreased self-renewal capacity and enhanced differentiation. Compensatory mechanisms in perinatal Cdk2^−/− SVZ cells, which persist until postnatal day 15, involve increased Cdk4 expression that results in retinoblastoma protein inactivation. A subsequent decline in Cdk4 activity to wild-type levels in postnatal day 28 Cdk2^−/− cells coincides with lower NG2⁺ proliferation and self-renewal capacity similar to adult levels. Cdk4 silencing in perinatal Cdk2^−/− SVZ cells abolishes Cdk4 up-regulation and reduces cell proliferation and self- renewal to adult levels. Conversely, Cdk4 overexpression in adult SVZ cells restores proliferative capacity to wild-type levels. Thus, although Cdk2 is functionally redundant in perinatal SVZ, it is important for adult progenitor cell proliferation and self-renewal through age-dependent regulation of Cdk4.     

Prospective isolation of adult neural stem cells from the mouse subependymal zone

Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

Ezrin is a member of the ezrin–radixin–moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell–matrix recognition, cell–cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.     

Dopaminergic modulation of neurogenesis in the subventricular zone of the adult brain

Dopaminergic receptors are expressed on neural precursor cells (NPCs) in the subventricular zone (SVZ) and are known to regulate NPC proliferation and differentiation fate in this region. We now report that this optimally requires the simultaneous activation of both D1-like and D2-like dopaminergic receptors with the agonists Bromocriptine, SKF-38393 and 7-OH-pipat maleate (BSP) in vitro. This is consistent with our previous findings that dopamine stimulates NPC proliferation through an EGF paracrine mechanism within the SVZ. Furthermore this combined dopamine agonist therapy rescues NPC proliferation in the SVZ in the 6-OHDA animal model of PD and importantly significantly increases neuronal differentiation in the olfactory bulb to a greater extent than we showed previously with L-dopa. This result has implications for the use of dopaminergic therapies in PD and in the development of such therapies focusing on upregulating SVZ neurogenesis.     

Architecture and cell types of the adult subventricular zone: In search of the stem cells

Neural stem cells are maintained in the subventricular zone (SVZ) of the adult mammalian brain. Here, we review the cellular organization of this germinal layer and propose lineage relationships of the three main cell types found in this area. The majority of cells in the adult SVZ are migrating neuroblasts (type A cells) that continue to proliferate. These cells form an extensive network of tangentially oriented pathways throughout the lateral wall of the lateral ventricle. Type A cells move long distances through this network at high speeds by means of chain migration. Cells in the SVZ network enter the rostral migratory stream (RMS) and migrate anteriorly into the olfactory bulb, where they differentiate into interneurons. The chains of type A cells are ensheathed by slowly proliferating astrocytes (type B cells), the second most common cell type in this germinal layer. The most actively proliferating cells in the SVZ, type C, form small clusters dispersed throughout the network. These foci of proliferating type C cells are in close proximity to chains of type A cells. We discuss possible lineage relationships among these cells and hypothesize which are the neural stem cells in the adult SVZ. In addition, we suggest that interactions between type A, B, and C cells may regulate proliferation and initial differentiation within this germinal layer. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 234–248, 1998     

A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis

According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular–subventricular zone (V‐SVZ). Both processes will ultimately lead to the generation of adult‐born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult‐born OB interneurons largely derive from an additional NSC‐type resident in the basal V‐SVZ. Despite being both capable of self‐renewal and long‐term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch‐related genes also differs between the two NSC groups, and Notch activation is greatest in apical NSCs. Apical downregulation of Notch‐effector Hes1 decreases Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation‐dependent increase in neurogenesis is paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis, whereas apical NSCs impart Notch‐mediated lateral inhibition across the V‐SVZ.     

Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.     

Role and mechanism of neural stem cells of the subventricular zone in glioblastoma

Glioblastoma multiforme (GBM), the most frequently occurring malignant brain tumor in adults, remains mostly untreatable. Because of the heterogeneity of invasive gliomas and drug resistance associated with the tumor microenvironment, the prognosis is poor, and the survival rate of patients is low. Communication between GBMs and non-glioma cells in the tumor microenvironment plays a vital role in tumor growth and recurrence. Emerging data have suggested that neural stem cells (NSCs) in the subventricular zone (SVZ) are the cells-of-origin of gliomas, and SVZ NSC involvement is associated with the progression and recurrence of GBM. This review highlights the interaction between SVZ NSCs and gliomas, summarizes current findings on the crosstalk between gliomas and other non-glioma cells, and describes the links between SVZ NSCs and gliomas. We also discuss the role and mechanism of SVZ NSCs in glioblastoma, as well as the interventions targeting the SVZ and their therapeutic implications in glioblastoma. Taken together, understanding the biological mechanism of glioma-NSC interactions can lead to new therapeutic strategies for GBM.     

Exendin-4对成年小鼠脑室下区神经干细胞分化作用的体外研究*

目的:研究艾塞那肽(Ex-4)对成年小鼠脑室下区(SVZ)神经干细胞(NSCs)分化的影响及机制。方法:提取5周龄C57BL/6J小鼠SVZ的NSCs,100 nmol/L Ex-4处理分化14 d观察细胞形态,用免疫荧光检测巢蛋白(nestin)和胰高糖素样肽-1受体(GLP-1R)的表达。用shRNA敲低GLP-1R,将研究分为四组:对照组,Ex-4组,GLP-1R敲低组,GLP-1R敲低+Ex-4组。100 nmol/L Ex-4处理14 d后免疫荧光标记β-微管蛋白3(β-tublin III)和胶质纤维酸性蛋白(GFAP)并统计β-tublin III阳性细胞比例,Western blot检测环磷腺苷效应元件结合蛋白(CREB)的活化。为进一步研究Ex-4对MAPK和PI3K通路的影响,分别以丝裂原活化蛋白激酶(MAPK)抑制剂U0126 0.07 μmol/L预处理细胞30 min、磷脂酰肌醇-3激酶(PI3K)抑制剂LY294002 50 μmol/预处理细胞2 h,将研究分为: 对照组,Ex-4组,U0126组,U0126+Ex-4组,LY294002组,LY294002+Ex-4组,Western blot检测各组CREB的活化,各组实验独立重复三次。结果:成功从C57BL/6J小鼠SVZ提取NSCs,免疫荧光提示NSCs中nestin以及GLP-1R阳性。相对于对照组,Ex-4组分化为神经元的比例更高。GLP-1R敲低+Ex-4组中神经元比例与对照组基本一致(P＜0.01),β-tublin III阳性的细胞显示出GLP-1R以及CREB活化阳性。Western blot显示Ex-4组中CREB显著活化,GLP-1R敲低+Ex-4组的CERB活化与对照组基本一致(P＜0.01)。U0126+Ex-4组与Ex-4组CERB活化水平一致,LY294002+Ex-4组与对照组CERB活化水平一致(P＜0.01)。 结论:Ex-4通过GLP-1R受体促进成年小鼠SVZ中NSCs分化为神经元,这一作用可能通过PI3K/ CREB通路来实现。     

Effects of the monomeric, oligomeric, and fibrillar Abeta42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone

The incidence of amyloid plaques, composed mainly of beta-amyloid peptides (Abeta), does not correlate well with the severity of neurodegeneration in patients with Alzheimer's disease (AD). The effects of Abeta(42) on neurons or neural stem cells (NSCs) in terms of the aggregated form remain controversial. We prepared three forms of oligomeric, fibrillar, and monomeric Abeta(42) peptides and investigated their effects on the proliferation and neural differentiation of adult NSCs, according to the degree of aggregation or concentration. A low micromolar concentration (1 micromol/L) of oligomeric Abeta(42) increased the proliferation of adult NSCs remarkably in a neurosphere assay. It also enhanced the neuronal differentiation of adult NSCs and their ability to migrate. These results provide us with valuable information regarding the effects of Abeta(42) on NSCs in the brains of patients with AD.     

Expression of Dbn1 during mouse brain development and neural stem cell differentiation

Dbn1 is a newly discovered gene in the drebrin gene family of mice. Previous studies have reported that Dbn1 is specifically expressed in the mouse brain suggesting its potential role in brain development. However, a detailed analysis of Dbn1 expression during mouse brain development has not been demonstrated. Here, we describe the expression pattern of Dbn1 and the coexpression of Dbn1 and actin during the development of the mouse brain from embryonic day 14 (E14) to adulthood and during the differentiation of neural stem cells (NSCs), as determined using immunohistochemistry, double-labeling immunofluorescence, and quantitative real-time polymerase chain reaction. During mouse brain development, Dbn1 expression level was high at E14, attenuated postnatally, reached its highest point at postnatal day 7 (P7), and showed a very low level at adulthood. Imaging data showed that Dbn1 was mainly expressed in the hippocampus, ventricular zone, and cortex, where NSCs are densely distributed, and that the intracellular distribution of Dbn1 was predominantly located in the cytoplasm edges and neurites. Moreover, the signal for colocalization of Dbn1 with actin was intense at E14, P0, and P7, but it was weak at adulthood. During NSC differentiation, Dbn1 mRNA expression increased after the onset of differentiation and reached its highest point at 3 days, followed by a decrease in expression. The imaging data showed that Dbn1 was increasingly expressed in the extending neurites in accordance with the cell morphological changes that occur during differentiation. Furthermore, obvious colocalization signals of Dbn1 with actin were found in the neurites and dendritic spines. Collectively, these results suggest that Dbn1 may play a key role in mouse brain development and may regulate NSC differentiation by filamentous actin.     

The autophagy regulators Ambra1 and Beclin 1 are required for adult neurogenesis in the brain subventricular zone

Autophagy is a conserved proteolytic mechanism required for maintaining cellular homeostasis. The role of this process in vertebrate neural development is related to metabolic needs and stress responses, even though the importance of its progression has been observed in a number of circumstances, both in embryonic and in postnatal differentiating tissues. Here we show that the proautophagic proteins Ambra1 and Beclin 1, involved in the initial steps of autophagosome formation, are highly expressed in the adult subventricular zone (SVZ), whereas their downregulation in adult neural stem cells in vitro leads to a decrease in cell proliferation, an increase in basal apoptosis and an augmented sensitivity to DNA-damage-induced death. Further, Beclin 1 heterozygosis in vivo results in a significant reduction of proliferating cells and immature neurons in the SVZ, accompanied by a marked increase in apoptotic cell death. In sum, we propose that Ambra1- and Beclin 1-mediated autophagy plays a crucial role in adult neurogenesis, by controlling the survival of neural precursor cells.In the adult mammalian brain, neural stem cells are localized in two regions: in the subventricular zone (SVZ), a layer extending along the wall of the lateral ventricle, and in the subgranular zone of the dentate gyrus in the hippocampus.¹ SVZ stem cells are strictly controlled under physiological conditions and are believed to replenish dying cells. In addition to their effect in maintaining brain homeostasis, they are also involved in neuronal replacement in response to injury.² Although several factors are known to affect neurogenesis, understanding of the mechanisms that regulate adult neurogenic niches and their metabolism is still incomplete. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved cellular turnover process in which bulk cytoplasmic materials, long-lived proteins or damaged organelles are sequestered and delivered to lysosomes for degradation.³ A complex crosstalk takes place between apoptosis and autophagy that determines the death or life of cells.⁴ Beclin 1 has a key role in autophagy initiation;⁵ it regulates the autophagy-promoting activity of the Class III PI 3-kinase Vps34,⁶ and is involved in the recruitment of membranes to form the key autophagy vesicles, named autophagosomes. Beclin 1 also interacts with Bcl-2,⁷ and plays an important function in the regulation of cell survival.⁸ Ambra1 (activating molecule in Beclin 1-regulated autophagy) is another modulator of autophagy, which is phosphorylated by the upstream autophagy kinase Ulk1 and acts on Ulk1 stability and function.^{9, 10} Ambra1 also interacts with Beclin 1 upon autophagic stimuli, thereby promoting the binding between Beclin 1 and its target kinase, Vps34. The binding between Ambra1 and mitochondrial Bcl-2 is also important for cell survival.¹¹ Moreover, Ambra1 is crucial for nervous system development and is expressed from early neurulation onwards, with a high specificity for the neural plate.¹²In contrast with studies on the pro-survival impact of autophagy in post-mitotic cells and in disease models, the role of autophagy in the maintenance and function of adult neural stem cells (ANSCs) is poorly understood. Here we have found that expression of upstream autophagy-regulating genes in the adult neurogenic region of SVZ, in physiological conditions, plays a crucial role in the regulation of adult neurogenesis.