neural stem cells

神经干细胞
  • 文章类型: Journal Article
    在组织工程领域,细胞外基质(ECM)被认为是促进脊髓损伤(SCI)后神经再生的重要元素。牙髓干细胞(DPSC),源自神经c的间充质干细胞,易于在体外收获和培养,表达多种神经营养因子(NTFs)并沉积大量ECM,使它们成为干细胞或基于ECM的SCI治疗的好选择。在本研究中,衍生自DPSC片的脱细胞细胞外基质(dECM)用于治疗SCI。优化实验表明,将DPSC片与1%TritonX-100孵育5分钟是制备DPSCdECM的最佳程序。发现DPSCdECM促进SCI后神经修复和再生,并恢复大鼠后肢运动功能。机械上,DPSCdECM促进神经干细胞的迁移和神经分化,以及小胶质细胞的M2极化,抑制胶质疤痕的形成.这项研究表明,使用DPSCdECM是治疗SCI的潜在策略。
    In the field of tissue engineering, the extracellular matrix (ECM) is considered an important element for promoting neural regeneration after spinal cord injury (SCI). Dental pulp stem cells (DPSCs), mesenchymal stem cells that originate from the neural crest, are easy to harvest and culture in vitro, express a variety of neurotrophic factors (NTFs) and deposit a large amount of ECM, making them a good choice for stem cell- or ECM-based treatment of SCI. In the present study, decellularized extracellular matrix (dECM) derived from DPSC sheets is used for the treatment of SCI. Optimization experiments reveal that incubating DPSC sheets with 1% Triton X-100 for 5 min is the best procedure for preparing DPSC dECM. It is found that DPSC dECM promotes nerve repair and regeneration after SCI and restores hindlimb motor function in rats. Mechanistically, DPSC dECM facilitates the migration and neural differentiation of neural stem cells, as well as M2 polarization of microglia, and inhibits the formation of glial scars. This study suggests that the use of DPSC dECM is a potential strategy for the treatment of SCI.
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  • 文章类型: Journal Article
    神经毒性镉(Cd)暴露对大脑发育的影响尚未得到很好的阐明。为了调查这一点,我们在此使怀孕小鼠在整个妊娠期间接受低剂量Cd。使用单细胞RNA测序(scRNA-seq),我们探索了胚胎大脑对镉暴露的细胞反应,并鉴定出18种不同的细胞亚群,这些亚群对Cd表现出不同的反应。通常,镉暴露阻碍了大脑中细胞的发育和成熟,特别是祖细胞,如神经祖细胞(NPC)和少突胶质细胞祖细胞(OPCs)。它还导致大脑中几乎所有类型的细胞发生显著的细胞亚群变化。此外,Cd暴露降低了后代皮质神经元的树突复杂性。重要的是,这些变化导致皮层中异常的Ca2活性和成熟后代的神经行为变化。这些数据有助于我们了解Cd暴露对大脑发育的影响和机制,并强调了在人群水平上控制环境神经毒物暴露的重要性。
    The effects of neurotoxicant cadmium (Cd) exposure on brain development have not been well elucidated. To investigate this, we have herein subjected pregnant mice to low-dose Cd throughout gestation. Using single-cell RNA sequencing (scRNA-seq), we explored the cellular responses in the embryonic brain to Cd exposure, and identified 18 distinct cell subpopulations that exhibited varied responses to Cd. Typically, Cd exposure impeded the development and maturation of cells in the brain, especially progenitor cells such as neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). It also caused significant cell subpopulation shifts in almost all the types of cells in the brain. Additionally, Cd exposure reduced the dendritic sophistication of cortical neurons in the offspring. Importantly, these changes led to aberrant Ca2+ activity in the cortex and neural behavior changes in mature offspring. These data contribute to our understanding of the effects and mechanisms of Cd exposure on brain development and highlight the importance of controlling environmental neurotoxicant exposure at the population level.
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  • 文章类型: Journal Article
    ADP核糖基化因子样GTP酶2(Arl2)对于控制各种生物体中的线粒体融合和微管组装至关重要。Arl2通过微管生长调节果蝇中神经干细胞的不对称分裂。然而,哺乳动物Arl2在皮质发育过程中的功能尚不清楚。这里,我们证明小鼠Arl2通过调节微管生长在皮质发生中起新的作用,但不是线粒体的功能。Arl2敲低(KD)导致神经祖细胞(NPC)增殖受损和神经元迁移。小鼠NPC中的Arl2KD显着降低了中心体微管的生长和中心体蛋白Cdk5rap2和γ-微管蛋白的离域。此外,Arl2通过使用AlphaFold多聚体的模拟预测与Cdk5rap2物理关联,通过共免疫沉淀和邻近连接测定进行了验证。值得注意的是,Cdk5rap2过表达显着挽救了Arl2KD引起的神经发生缺陷。因此,Arl2通过微管生长通过中心体蛋白Cdk5rap2在小鼠皮质发育中起重要作用。
    ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.
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  • 文章类型: Journal Article
    影响年轻人的最普遍的罕见遗传疾病是脊髓性肌萎缩症(SMA),这是由端粒基因存活运动神经元(SMN)1的功能丧失突变引起的。SMA病理生理学的高度异质性取决于SMN2的拷贝数,SMN2是一种可以转录相同蛋白质的独立着丝粒基因,尽管它以较慢的速度表示。SMA影响运动神经元。然而,根据病情的严重程度,各种不同的组织和器官也可能受到影响。新的药物治疗,比如Spinraza,Onasemnogeneabeparvovec-xioi,和Evrysdi,被认为是疾病修饰剂,因为它们的使用可以改变患者的表型。由于已经报道了受SMA影响的细胞中的氧化应激,我们研究了抗氧化疗法对具有分化为运动神经元潜能的神经干细胞(NSC)的影响.抗氧化剂可以通过各种途径发挥作用;例如,其中一些通过核因子(红系衍生的2)-样2(NRF2)发挥功能。我们发现姜黄素能够通过激活NRF2的核易位在健康和受SMA影响的NSC中诱导积极作用,这可能使用与通过抗氧化剂反应元件和抗氧化剂分子的产生的经典氧化还原调节不同的机制。
    The most prevalent rare genetic disease affecting young individuals is spinal muscular atrophy (SMA), which is caused by a loss-of-function mutation in the telomeric gene survival motor neuron (SMN) 1. The high heterogeneity of the SMA pathophysiology is determined by the number of copies of SMN2, a separate centromeric gene that can transcribe for the same protein, although it is expressed at a slower rate. SMA affects motor neurons. However, a variety of different tissues and organs may also be affected depending on the severity of the condition. Novel pharmacological treatments, such as Spinraza, Onasemnogene abeparvovec-xioi, and Evrysdi, are considered to be disease modifiers because their use can change the phenotypes of the patients. Since oxidative stress has been reported in SMA-affected cells, we studied the impact of antioxidant therapy on neural stem cells (NSCs) that have the potential to differentiate into motor neurons. Antioxidants can act through various pathways; for example, some of them exert their function through nuclear factor (erythroid-derived 2)-like 2 (NRF2). We found that curcumin is able to induce positive effects in healthy and SMA-affected NSCs by activating the nuclear translocation of NRF2, which may use a different mechanism than canonical redox regulation through the antioxidant-response elements and the production of antioxidant molecules.
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  • 文章类型: Journal Article
    原理:脑室下区(SVZ)的成人神经发生对于维持神经稳态至关重要,它的失调会导致神经系统疾病的失语症和组织愈合延迟,如帕金森病(PD)。尽管在SVZ神经发生中发现了复杂的调节网络,动态维持神经干/祖细胞(NSPCs)响应生理和病理刺激的分子机制仍未完全阐明。方法:我们建立了一个RNA结合基序蛋白24(Rbm24)敲除模型,以研究其对SVZ中成人神经发生的影响。采用免疫荧光,免疫印迹,电生理学,RNA测序,和体外实验。进一步的研究利用PD小鼠模型,连同遗传和药理操作,阐明Rbm24参与PD病理。结果:Rbm24,细胞稳态的多方面转录后调节因子,从发育到衰老在SVZ中表现出广泛的表达。Rbm24的缺失显着损害成年SVZ的NSPC增殖,最终导致嗅球神经发生塌陷。值得注意的是,Rbm24在维持成年NSPCs中Notch1mRNA的稳定性中起着特定的作用。Rbm24/Notch1信号轴在PD小鼠的SVZ中显著下调。值得注意的是,Rbm24的过表达拯救了PD小鼠成年神经发生和嗅觉功能障碍的破坏,这些影响受到DAPT的阻碍,Notch1的有效抑制剂。结论:我们的发现强调了Rbm24/Notch1信号轴在生理和病理情况下调节成人SVZ神经发生的关键作用。这为NSPC稳态的动态调节提供了有价值的见解,并为PD和相关神经系统疾病提供了潜在的针对性干预措施。
    Rationale: Adult neurogenesis in the subventricular zone (SVZ) is essential for maintaining neural homeostasis, and its dysregulation contributes to anosmia and delayed tissue healing in neurological disorders, such as Parkinson\'s disease (PD). Despite intricate regulatory networks identified in SVZ neurogenesis, the molecular mechanisms dynamically maintaining neural stem/progenitor cells (NSPCs) in response to physiological and pathological stimuli remain incompletely elucidated. Methods: We generated an RNA binding motif protein 24 (Rbm24) knockout model to investigate its impact on adult neurogenesis in the SVZ, employing immunofluorescence, immunoblot, electrophysiology, RNA-sequencing, and in vitro experiments. Further investigations utilized a PD mouse model, along with genetic and pharmacological manipulations, to elucidate Rbm24 involvement in PD pathology. Results: Rbm24, a multifaceted post-transcriptional regulator of cellular homeostasis, exhibited broad expression in the SVZ from development to aging. Deletion of Rbm24 significantly impaired NSPC proliferation in the adult SVZ, ultimately resulting in collapsed neurogenesis in the olfactory bulb. Notably, Rbm24 played a specific role in maintaining Notch1 mRNA stability in adult NSPCs. The Rbm24/Notch1 signaling axis was significantly downregulated in the SVZ of PD mice. Remarkably, overexpression of Rbm24 rescued disruption of adult neurogenesis and olfactory dysfunction in PD mice, and these effects were hindered by DAPT, a potent inhibitor of Notch1. Conclusions: Our findings highlight the critical role of the Rbm24/Notch1 signaling axis in regulating adult SVZ neurogenesis under physiological and pathological circumstances. This provides valuable insights into the dynamic regulation of NSPC homeostasis and offers a potential targeted intervention for PD and related neurological disorders.
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  • 文章类型: Journal Article
    胶质母细胞瘤(GBM)是最普遍和侵袭性的恶性原发性脑肿瘤。侧脑室(LVs)近端GBM更具侵袭性,可能是因为脑室下区域接触。尽管如此,GBM和神经干/祖细胞(NSC/NPCs)之间的串扰还不是很清楚。使用细胞特异性蛋白质组学,我们显示LV近端GBM通过诱导衰老阻止神经干细胞的神经元成熟。此外,GBM脑肿瘤起始细胞(BTIC)在与NPC相互作用时增加组织蛋白酶B(CTSB)的表达。慢病毒敲低和重组蛋白实验表明,细胞固有和可溶性CTSB均可促进BTIC中与恶性肿瘤相关的表型。可溶性CTSB阻止NPCs中的神经元成熟,同时促进衰老,提供LV-肿瘤接近和神经发生破坏之间的联系。最后,我们显示患者左心室近端CTSB上调,显示了这种串扰在人类GBM生物学中的相关性。这些结果证明了蛋白质组学分析在肿瘤微环境研究中的价值,并为GBM的新治疗策略提供了方向。
    Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially because of subventricular zone contact. Despite this, cross-talk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. In addition, GBM brain tumor-initiating cells (BTICs) increase expression of cathepsin B (CTSB) upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal that both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Last, we show LV-proximal CTSB up-regulation in patients, showing the relevance of this cross-talk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.
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  • 文章类型: Journal Article
    再生,恢复受损或缺失细胞的复杂过程,组织,和器官,物种之间差异很大。斑马鱼因其令人印象深刻的再生能力而成为一种非凡的模型生物,特别是在心脏等器官中,fin,视网膜,脊髓,和大脑。与哺乳动物不同,斑马鱼可以再生有限或没有疤痕,这种现象与干细胞和免疫细胞的激活密切相关。这篇综述探讨了免疫反应和炎症在斑马鱼和小鼠再生过程中的独特作用。强调其不同再生能力背后的细胞和分子机制。通过关注斑马鱼的端脑再生并将其与啮齿动物的端脑再生进行比较,这次审查强调了控制良好的重要性,急性,斑马鱼的非持续性免疫反应,这促进了有利于再生的环境。从了解斑马鱼再生机制中获得的知识为人类神经退行性疾病和脑损伤(中风和创伤性脑损伤)的治疗带来了巨大的希望,以及再生医学方法的进步。
    Regeneration, the complex process of restoring damaged or absent cells, tissues, and organs, varies considerably between species. The zebrafish is a remarkable model organism for its impressive regenerative abilities, particularly in organs such as the heart, fin, retina, spinal cord, and brain. Unlike mammals, zebrafish can regenerate with limited or absent scarring, a phenomenon closely linked to the activation of stem cells and immune cells. This review examines the unique roles played by the immune response and inflammation in zebrafish and mouse during regeneration, highlighting the cellular and molecular mechanisms behind their divergent regenerative capacities. By focusing on zebrafish telencephalic regeneration and comparing it to that of the rodents, this review highlights the importance of a well-controlled, acute, and non-persistent immune response in zebrafish, which promotes an environment conducive to regeneration. The knowledge gained from understanding the mechanisms of zebrafish regeneration holds great promises for the treatment of human neurodegenerative diseases and brain damage (stroke and traumatic brain injuries), as well as for the advancement of regenerative medicine approaches.
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  • 文章类型: Journal Article
    背景:神经再生不足和抑制性局部微环境是脊髓损伤(SCI)修复的主要障碍。内源性神经干细胞(NSC)的活化和分化命运调节是最有前途的修复方法之一。二甲双胍的抗氧化作用已被广泛研究,抗炎,抗衰老,和自噬调节特性在中枢神经系统疾病。然而,二甲双胍对内源性神经干细胞的影响尚待阐明。
    方法:用CCK-8法评价神经干细胞的增殖和分化能力,EdU/Ki67染色和免疫荧光染色。采用WesternBlot和免疫荧光染色检测NSC中铁凋亡相关关键蛋白表达的变化。活性氧的水平,使用相应的测定试剂盒测量谷胱甘肽和组织铁。用透射电镜和JC-1荧光探针观察线粒体形态和膜电位的变化。通过BBB评分评估大鼠SCI后的运动功能恢复,LSS得分,CatWalk步态分析,和电生理测试。使用Western印迹检查AMPK途径的表达。
    结果:二甲双胍在体外和体内均能促进神经干细胞的增殖和神经元分化。此外,在体外建立了使用erastin处理的神经干细胞的铁凋亡模型,二甲双胍治疗可以逆转关键的铁凋亡相关蛋白表达的变化,增加谷胱甘肽合成,减少活性氧的产生,改善线粒体膜电位和形态。此外,服用二甲双胍可改善SCI后大鼠的运动功能恢复和组织学结局。值得注意的是,加入化合物C后,二甲双胍的所有上述有益作用均完全消除,AMP激活蛋白激酶(AMPK)的特异性抑制剂。
    结论:二甲双胍,由典型的AMPK依赖性调节驱动,促进内源性神经干细胞的增殖和神经元分化,同时抑制铁凋亡,从而促进SCI后运动功能的恢复。我们的研究进一步阐明了二甲双胍在SCI中的保护机制,为其作为SCI治疗剂的候选资格提供了新的机制见解。
    BACKGROUND: Inadequate nerve regeneration and an inhibitory local microenvironment are major obstacles to the repair of spinal cord injury (SCI). The activation and differentiation fate regulation of endogenous neural stem cells (NSCs) represent one of the most promising repair approaches. Metformin has been extensively studied for its antioxidative, anti-inflammatory, anti-aging, and autophagy-regulating properties in central nervous system diseases. However, the effects of metformin on endogenous NSCs remains to be elucidated.
    METHODS: The proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot.
    RESULTS: Metformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK).
    CONCLUSIONS: Metformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.
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  • 文章类型: Journal Article
    组蛋白甲基化是调节染色质结构和基因表达以用于适当的细胞和生理过程的关键表观遗传修饰。异常的组蛋白甲基化模式与许多疾病有关。因此,监测活细胞和物种中的组蛋白甲基化动态对于阐明其调控机制和确定潜在的治疗靶标至关重要.然而,目前检测组蛋白甲基化的方法受限于它们的低敏感性和特异性。为了克服这一挑战,我们开发了一种基因编码的生物传感器,名为Phaser-Trim(基于相分离的基因编码的H3K9三甲基化报告基因),通过相分离液滴的产生和消失来检测活细胞和物种中H3K9me3的动态变化。Phaser-Trim显示出明显的表型特征的优势,操作方便,定量精度,生物相容性,高特异性,和优越的成像性能与高信号背景比(SBR)的体内动物成像。使用Phaser-Trim,我们已经成功地检测了果蝇神经干细胞分化过程中H3K9me3水平的动力学。此外,Phaser-Trim还有望在高通量筛选系统中应用,以促进新型抗癌药物的发现。
    Histone methylation is a key epigenetic modification that regulates the chromatin structure and gene expression for proper cellular and physiological processes. Aberrant histone methylation patterns are implicated in many diseases. Therefore, monitoring histone methylation dynamics in living cells and species is essential for elucidating its regulatory mechanisms and identifying potential therapeutic targets. However, current methods for detecting histone methylation are limited by their low sensitivity and specificity. To overcome this challenge, we have developed a genetically encoded biosensor named Phaser-Trim (Phase separation based genetically encoded reporter for H3K9 Trimethylation) to detect the dynamic changes of H3K9me3 in living cells and species through the generation and disappearance of phase-separated droplets. Phaser-Trim demonstrates advantages of clear phenotypic characteristics, convenient operation, quantitative accuracy, biocompatibility, high specificity, and superior imaging performance with high signal-to-background ratio (SBR) for in vivo animal imaging. Using Phaser-Trim, we have successfully detected the dynamics of the H3K9me3 level during the differentiation of neural stem cells in Drosophila. Furthermore, Phaser-Trim also holds promise for application in high-throughput screening systems to facilitate the discovery of novel anticancer drugs.
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  • 文章类型: Journal Article
    虽然细胞外基质(ECM)应力松弛越来越受到人们的重视,以调节干细胞的命运承诺和其他行为,与传统的2D细胞培养相比,细胞如何处理组织样三维(3D)几何形状中的应力松弛线索仍然未知。这里,我们开发了一种寡核苷酸交联的基于透明质酸的ECM平台,具有可调的应力松弛特性,可用于2D或3D。引人注目的是,应力松弛有利于3D中的神经干细胞(NSC)神经发生,但在2D中抑制它。RNA测序和功能研究暗示膜相关蛋白血影蛋白是应力松弛线索的关键3D特异性换能器。限制压力会促使血影蛋白募集到F-肌动蛋白细胞骨架,在那里它机械地加强皮质并增强机械传导信号。血影蛋白表达的增加也伴随着转录因子EGR1的表达增加,我们先前在3D中显示了介导NSC刚度依赖性谱系的承诺。我们的工作强调光谱是3D应力松弛线索的重要分子传感器和换能器。
    While extracellular matrix (ECM) stress relaxation is increasingly appreciated to regulate stem cell fate commitment and other behaviors, much remains unknown about how cells process stress-relaxation cues in tissue-like three-dimensional (3D) geometries versus traditional 2D cell culture. Here, we develop an oligonucleotide-crosslinked hyaluronic acid-based ECM platform with tunable stress relaxation properties capable of use in either 2D or 3D. Strikingly, stress relaxation favors neural stem cell (NSC) neurogenesis in 3D but suppresses it in 2D. RNA sequencing and functional studies implicate the membrane-associated protein spectrin as a key 3D-specific transducer of stress-relaxation cues. Confining stress drives spectrin\'s recruitment to the F-actin cytoskeleton, where it mechanically reinforces the cortex and potentiates mechanotransductive signaling. Increased spectrin expression is also accompanied by increased expression of the transcription factor EGR1, which we previously showed mediates NSC stiffness-dependent lineage commitment in 3D. Our work highlights spectrin as an important molecular sensor and transducer of 3D stress-relaxation cues.
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