Notch信号通路对神经干细胞增殖分化的作用

神经干细胞作为一种具有自我更新能力和多向分化潜能的细胞,它的增殖和分化受到多种源于自身或外在、邻近或远程细胞信号通路的调控,各种细胞因子及胞间通讯在神经干细胞的增殖和分化中发挥着重要的作用。近年来的多种研究表明,Notch信号通路正是这样一种可以通过相邻细胞的配体与受体相互作用,从而传递信号,进一步发挥其生物学功能的重要信号通路。该通路参与了神经干细胞维持自我形态及向多种具有不同功能的神经细胞分化的过程．对于研究神经干细胞的增殖和分化具有巨大的意义。该文将就当前Notch信号通路对神经干细胞增殖分化影响的相关研究进行简要综述。     

Wnt信号通路与神经干细胞

神经干细胞增殖、分化机制的研究为神经系统疾病治疗提供了新的途径,具有巨大的潜在应用价值和理论研究意义。业已发现,Wnt信号通路对神经干细胞的增殖发挥着决定性作用,但新近的研究却表明Wnt信号能够明显促进神经干细胞向神经元分化,这种不同的表现可能与神经干细胞的内在特点、周围环境及靶基因的不同有关。本文试从Wnt信号通路及其在调控神经干细胞的增殖、分化中的作用加以综述。     

Notch信号通路在脑生发区神经干细胞维持与神经再生中的作用

成年神经再生在各种脑损伤、神经系统变性疾病的修复中发挥了重要的作用。在啮齿类和灵长类动物脑中,其主要发生在侧脑室(lateral ventricle,LV)的脑室下区(subventricular zone,SVZ)和海马齿状回(dentate gyrus,DG)颗粒下区(subgranular zone,SGZ)。神经干细胞(neural stem cells,NSCs)的增殖与分化之间的平衡调控是成年神经再生的重要机制。成年神经再生过程包含几个阶段,每个阶段均受到多种内源性和外源性因素的调节。Notch信号通路在成年NSCs的维持中发挥了重要的作用。该文将对Notch信号通路在脑生发区NSCs的维持与神经再生中的作用机制及其研究进展进行综述。     

川芎嗪对体外培养的胎鼠神经干细胞增殖和分化的影响

目的：探讨川芎嗪（TMP）在体外神经干细胞（NSCs）增殖与分化中的作用。方法：原代提取孕14 d雌性大鼠的胎鼠大脑皮层分离培养,并作免疫荧光染色鉴定,取传代培养第3代的NSCs进行实验。实验分为对照组、β-巯基乙醇阳性对照组、TMP诱导组和TMP+EGTA组（n=4）。采用BrdU法和MTT法观察川芎嗪对NSCs增殖数量的影响,采用蛋白免疫印迹法检测NSCs的分化表达情况。结果：实验成功分离纯化原代NSCs,培养3~5 d可见部分神经球形成,具备典型的NSCs形态并表达NSCs特异抗原巢蛋白;BrdU法和MTT法结果均显示,与对照组和β-巯基乙醇阳性对照组相比,TMP组NSCs增殖数量明显增多（P<0.05）;蛋白免疫印迹结果显示,TMP组和TMP+EGTA组NSCs的神经元分化率明显增高,TMP+EGTA组分化率增高更明显（P<0.05）。结论：TMP能显著增强NSCs的增殖和神经元分化率。减少细胞外Ca²⁺可促进TMP诱导NSCs向神经元分化,Ca²⁺信号在TMP诱导NSCs向神经元分化过程中起重要作用。     

未成熟钠通道调控大鼠胚胎神经干细胞分化

钠通道在各类神经元上高表达,参与细胞多种生理功能的调节,是神经元实现功能活动的基本单位．未成熟神经元上钠/钙通道所诱发和自发的电位活动对后期的发育成熟至关重要．然而,发育中的钠通道是否参与神经干细胞(neural stem cells, NSCs)分化的调控尚不清楚．本研究证明,未成熟的钠通道参与NSCs分化调控．Western印迹结果显示,在分化第1,3,5,7 d的NSCs上钠通道和胞外信号调节激酶（ERK）的蛋白表达与分化时间正相关.免疫组化结果发现,与对照组比较,加入电压门控钠通道阻断剂TTX可明显下调NeuN、GFAP和Gal-c在NSCs中的表达（P＜0.05）,提示钠通道参与NSCs分化的调控.当采用veratridine激动钠通道后,激光共聚焦检测到细胞内Ca2+浓度明显升高,免疫组化和Western印迹结果显示细胞内Ca2+浓度明显升高,p-ERK表达量明显上调;相反,TTX可明显阻断Veratridine所引起的细胞内Ca2+浓度上调,并使p-ERK峰值明显降低和延后（P＜0.05）．研究结果表明,未成熟钠通道可通过激活ERK信号途径促进NSCs的分化．钠通道的这种作用可能是由钙离子介导的,其详尽机制有待进一步研究．     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

Wnt信号转导通路与神经发育研究进展

Wnt蛋白是一类分泌型糖蛋白家族,Wnt信号蛋白与细胞表面的多种受体相互作用,参与诸多生命过程。对神经系统发育的研究表明,Wnt信号通路在神经发生,神经祖细胞增值、分化,神经干细胞的自我更新,轴突导向等过程中起重要调控作用。多项研究已经证实,Wnt通路失调与诸多神经系统疾病有密切关系。Wnt信号通路的突变或异常,将会引起神经系统发育缺陷。然而,对Wnt非经典信号通路的研究,尤其是新受体Ryk的调控作用的认识迄今仍不全面。根据国内外相关研究,阐述了经典Wnt信号通路Wnt/β-catenin途径的同时也对Wnt/Ryk非经典信号途径这一研究新领域做了讨论。在非经典信号通路中,Ryk-ICD的剪接对于前体细胞的神经分化起重要作用。本文分析了Wnt/β-catenin和Wnt/Ryk信号通路在神经发育中的作用,有助于深入理解神经发育过程中Wnt信号通路的作用机制。然而,Ryk-ICD引导因子、分子机制等问题仍待进一步研究,而这将有利于理解神经干细胞分化机理。     

神经干细胞巢成分及调节机制的研究进展

神经干细胞（NSCs）是一类具有自我更新和多向分化潜能的细胞。在特定的条件下能够分化成神经元、星形胶质细胞和少突胶质细胞,从而参与神经发生和损伤修复。调节NSCs的特定微环境,通常称为神经干细胞巢,包括多个细胞群,其贡献目前正在积极探索。了解NSCs及其微环境成分之间的相互作用,对于开发治疗神经退行性疾病及脊髓损伤的疗法至关重要。本篇综述描述并讨论了最新的研究,确定了新的成分在神经干细胞巢中的作用。这些发现给这个领域带来了新的概念。本综述评估这些最新进展,提高对NSCs微环境及其对NSCs功能的影响的认识。     

BDNF及其下游通路与GABA能神经元发育相关性的研究进展

脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)是一种具有神经营养作用的蛋白质,广泛分布于中枢神经系统内。BDNF及其下游信号通路在γ-氨基丁酸(γ-aminobutyric acid, GABA)能神经元存活、生长、分化、发育等方面均发挥重要的作用。GABA能神经元可以通过释放抑制性神经递质GABA调节神经元活性,进而维持神经环路的正常功能。多种疾病的发生发展都与GABA能神经元发育的异常密切相关。该文将就BDNF及其下游通路与GABA能神经元发育的相关性进行综述,希望为疾病的治疗提供新的方向。     

溶血磷脂酸促进离体大鼠胚胎神经干细胞的增殖及其向胆碱能神经元分化

溶血磷脂酸（1ysophosphatidic acid,LPA）是一种细胞外磷脂信号。本研究用[^3H]-胸腺嘧啶掺入法、免疫细胞化学和Western blot等技术,观察了LPA对体外培养的大鼠胚胎神经干细胞（neural stem cells,NSCs）的增殖以及向MAF2标记的一般神经元和ChAT标记的胆碱能神经元的分化的影响。结果显示：（1）在特殊的无血清培养基中加入低浓度的LPA（0．01-1．0μmol/L）后,NSCs对【^3H】-胸腺嘧啶的摄入呈剂量依赖性增加,表明LPA对NSCs有显著的促增殖作用;（2）在培养基中加入胎牛血清以诱导NSCs的分化,发现低浓度的LPA增加MAF2阳性和ChAT阳性神经元的比例,0．1μmol／L LPA引起的增加达到峰值;（3）Western blot分析显示LPA促进了MAP2和ChAT的表达;（4）在诱导NSCs出现分化早期,用倒置显微镜观察到低浓度的LPA明显促进细胞突起的生长和细胞的迁移。以上结果表明,低浓度LPA在一定范围内可以促进NSCs的增殖、并分化为一般的MAP2阳性神经元和特殊的胆碱能神经元,而且LPA可以促进在分化早期出现的神经元或神经胶质细胞前体细胞的迁移和突起生长。     

Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.     

Stem cell factor and granulocyte colony-stimulating factor promote neuronal lineage commitment of neural stem cells

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally discovered as growth factors for hematopoietic stem cells (HSCs). It has been well defined that SCF and G-CSF contribute to regulation of lineage commitment for HSCs. However, little is known about whether SCF and G-CSF play roles in the determination and differentiation of neural stem cells (NSCs). Here we demonstrate the novel function of SCF and G-CSF in controlling cell cycle and cell fate determination of NSCs. We also observe that SCF and G-CSF promote neuronal differentiation and inhibit astroglial differentiation at the early stage of differentiation. In addition, our research data reveal that SCF in combination with G-CSF has a dual function in promoting cell cycle exit and directing neuronal fate commitment at the stage of NSC dividing. This coordination effect of SCF+G-CSF on cell cycle arrest and neuronal differentiation is through enhancing neurogenin 1 (Ngn1) activity. These findings extend current knowledge regarding the role of SCF and G-CSF in the regulation of neurogenesis and provide insights into the contribution of hematopoietic growth factors to brain development and remodeling.     

TAM Receptors Support Neural Stem Cell Survival,Proliferation and Neuronal Differentiation

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III⁺) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.     

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

While a mother’s excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.     

Role of phospholipase D1 in neurite outgrowth of neural stem cells

Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.     

Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells

Secreted proteoglycan molecule Tsukushi (TSK) regulates various developmental processes, such as early body patterning and neural plate formation by interacting with major signaling pathways like Wnt, BMP, Notch etc. In central nervous system, TSK inhibits Wnt signaling to control chick retinal development. It also plays important roles for axon guidance and anterior commissure formation in mouse brain. In the present study, we investigated the role of TSK for the development and proper functioning of mouse hippocampus. We found that TSK expression is prominent at hippocampal regions of early postnatal mouse until postnatal day 15 and gradually declines at later stages. Hippocampal dimensions are affected in TSK knockout mice (TSK-KO) as shown by reduced size of hippocampus and dentate gyrus (DG). Interestingly, neural stem cell (NSC) density at the neural niche of DG was higher in TSK-KO compared with wild-type. The ratio of proliferating NSCs as well as the rate of overall cell proliferation was also higher in TSK-KO hippocampus. Our in vitro study also suggests an increased number of neural stem/progenitor cells residing in TSK-KO hippocampus. Finally, we found that the terminal differentiation of NSCs in TSK-KO was disturbed as the differentiation to neuronal cell lineage was increased while the percentages of astrocytes and oligodendrocytes were decreased. Overall, our study establishes the involvement of TSK in hippocampal development, NSC maintenance and terminal differentiation at perinatal stages.     

WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition, WNT pathway mutations are associated with medulloblastoma, the most common malignant brain tumor in children. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover, mice that express activated β-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region, whereas expression of the same protein in GNPs impairs proliferation. Although β-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation.     

Copine1 regulates neural stem cell functions during brain development

Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development.