Quantitative proteomic analysis of membrane proteins involved in astroglial differentiation of neural stem cells by SILAC labeling coupled with LC-MS/MS

Membrane proteins play a critical role in the process of neural stem cell self-renewal and differentiation. Here, we apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the self-renewing and the astroglial differentiating cells. High-resolution analysis on a linear ion trap-Orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 700 distinct membrane proteins during the astroglial differentiation. Of the 735 quantified proteins, seven cell surface proteins display significantly higher expression levels in the undifferentiated state membrane compared to astroglial differentiating membrane. One cell surface protein transferrin receptor protein 1 may serve as a new candidate for NSCs surface markers. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that most of overexpressed membrane proteins in the astroglial differentiation neural stem cells are involved in cellular growth, nervous system development, and energy metabolic pathway. Taken together, this study increases our understanding of the underlying mechanisms that modulate complex biological processes of neural stem cell proliferation and differentiation.     

Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and β-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and β-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, β-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas β-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the β-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and β-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and β-III-tubulin during the neural differentiation of H9 cells.     

磷脂酶D1信号对神经干细胞向神经分化的重要影响

磷脂酶D1(PLD1)在细胞生长、存活、分化、膜转运和细胞骨架组织等多种功能的调控中发挥重要作用。近年来研究发现,PLD1在神经干细胞（NSCs）向神经元的分化中也起关键作用。PLD1参与多种信号通路如Rho家族GTP酶和Ca²⁺信号通路的调节,影响轴突生长、突触发育及其可塑性。因此,PLD1作为神经系统中一种重要的信号分子引起了广泛的关注。本文综述了PLD1的结构、功能、作用机制及其在NSCs向神经分化中的调控作用,对深入研究NSCs的分化和神经元的再生有重要的指导意义。     

HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development.     

Lewis X-carrying N-glycans regulate the proliferation of mouse embryonic neural stem cells via the Notch signaling pathway

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells. Several signaling pathways have been shown to be involved in the fate determination process of NSCs, but the molecular mechanisms underlying the maintenance of neural cell stemness remain largely unknown. Our previous study showed that human natural killer carbohydrate epitopes expressed specifically by mouse NSCs modulate the Ras-MAPK pathway, raising the possibility of regulatory roles of glycoprotein glycans in the specific signaling pathways involved in NSC fate determination. To address this issue, we performed comparative N-glycosylation profiling of NSCs before and after differentiation in a comprehensive and quantitative manner. We found that Lewis X-carrying N-glycans were specifically displayed on undifferentiated cells, whereas pauci-mannose-type N-glycans were predominantly expressed on differentiated cells. Furthermore, by knocking down a fucosyltransferase 9 with short interfering RNA, we demonstrated that the Lewis X-carrying N-glycans were actively involved in the proliferation of NSCs via modulation of the expression level of Musashi-1, which is an activator of the Notch signaling pathway. Our findings suggest that Lewis X carbohydrates, which have so far been characterized as undifferentiation markers, actually operate as activators of the Notch signaling pathway for the maintenance of NSC stemness during brain development.     

The RNA helicase DDX6 regulates cell-fate specification in neural stem cells via miRNAs

In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein–protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Epigenetic mechanisms regulating fate specification of neural stem cells

Neural stem cells (NSCs) possess the ability to self-renew and to differentiate along neuronal and glial lineages. These processes are defined by the dynamic interplay between extracellular cues including cytokine signalling and intracellular programmes such as epigenetic modification. There is increasing evidence that epigenetic mechanisms involving, for example, changes in DNA methylation, histone modification and non-coding RNA expression are closely associated with fate specification of NSCs. These epigenetic alterations could provide coordinated systems for regulating gene expression at each step of neural cell differentiation. Here we review the roles of epigenetics in neural fate specification in the mammalian central nervous system.     

Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A1 in mouse hepatitis virus RNA synthesis

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has previously been shown to bind mouse hepatitis virus (MHV) RNA at the 3' end of both plus and minus strands and modulate MHV RNA synthesis. However, a mouse erythroleukemia cell line, CB3, does not express hnRNP A1 but still supports MHV replication, suggesting that alternative proteins can replace hnRNP A1 in cellular functions and viral infection. In this study, we set out to identify these proteins. UV cross-linking experiments revealed that several CB3 cellular proteins similar in size to hnRNP A1 interacted with the MHV RNA. These proteins were purified by RNA affinity column with biotinylated negative-strand MHV leader RNA and identified by mass spectrometry to be hnRNP A2/B1, hnRNP A/B, and hnRNP A3, all of which belong to the type A/B hnRNPs. All of these proteins contain amino acid sequences with strong similarity to the RNA-binding domains of hnRNP A1. Some of these hnRNPs have previously been shown to replace hnRNP A1 in regulating RNA splicing. These proteins displayed MHV RNA-binding affinity and specificity similar to those of hnRNP A1. hnRNP A2/B1, which is predominantly localized to the nucleus and shuttles between the nucleus and the cytoplasm, was shown to relocalize to the cytoplasm in MHV-infected CB3 cells. Furthermore, overexpression of hnRNP A/B in cells enhanced MHV RNA synthesis. Our findings demonstrate that the functions of hnRNP A1 in MHV RNA synthesis can be replaced by other closely related hnRNPs, further supporting the roles of cellular proteins in MHV RNA synthesis.     

Cytoskeleton‐associated proteins are enriched in human embryonic‐stem cell‐derived neuroectodermal spheres

The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2‐DE using protein extracts from hESC‐derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis‐related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton‐associated proteins (CSAP) such as Fascin‐1, Cofilin‐1, and Stathmin‐1. Western‐blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin‐1 and Stathmin‐1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin‐1 in (sub‐)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton.     

Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation

In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.     

Quantitative analysis of gene expression in living adult neural stem cells by gene trapping

The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral-vector gene-trap approach that uses beta-lactamase-induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of 'tagged' adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed.     

Comprehensive Quantitative Comparison of the Membrane Proteome,Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells

Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post-translational modifications reported for hESCs and NSCs. This study revealed potential markers to distinguish NSCs from hESCs and will contribute to improve our understanding on the differentiation process.Pluripotent embryonic stem cell (ESC)¹-derived neural stem cells (NSCs) can differentiate into neurons and glia cells of the central nervous system (1), including specialized neuron types like dopaminergic, representing a potential source for treatment of neurological diseases, such as Parkinson′s disease. Therefore, a better understanding of the cellular processes behind the changes of hESCs into NSCs, including solid markers for each cell type, is fundamental to move forward with a successful regenerative cell therapy and to investigate the early human neurogenesis processes.Many markers have been reported for the two types of stem cells (2, 3), however several of these markers are also identified in other stem or progenitor cells such as CD133 (Prominin-1) (4). Discovery of cell surface specific markers for differentiated stem cells is highly relevant for future clinical applications. In particular being able to distinguish the developmental stages of the differentiation from parental stem cells to fully mature cells would allow a correct manipulation and isolation of the cell type of interest. Moreover, such study would increase our understanding on the whole process of differentiation from embryonic cells to neural cells. Because plasma membrane-associated proteins are the key interface between cell and the surrounding environment, and they frequently present large extracellular domains suitable for antibody detection, they represent a great marker candidate potential. In addition, these proteins are very important in the cellular signaling process and cell–cell interaction and communication, processes very important for cellular differentiation. Furthermore, most membrane bound proteins involved in the abovementioned process are frequently regulated or otherwise manipulated to alter interaction partners and function by post-translational modifications (PTMs) such as phosphorylation and glycosylation.Protein phosphorylation and glycosylation are the most common PTMs in nature and they play an important role in many protein regulatory functions and cellular and biological processes. Protein phosphorylation is a dynamic PTM involved in many different cell signaling events like cell cycle, protein synthesis, protein degradation, differentiation, as well cellular proliferation and apoptosis (5). On the other hand, protein glycosylation has several roles in cell–cell interaction, cell-matrix interaction, communication and cellular signaling (6–8). A nine carbon sugar unit termed sialic acid (SA) can be bound to the nonreducing ends of glycans attached to certain membrane proteins, secreted proteins and lipids (9). SA is involved in many physiological processes such as cell–cell interaction and molecular recognition, and is important in different pathophysiological process, including brain development and cancer metastasis (10–14).The mass spectrometry-based proteomic approach is a powerful tool for characterization of proteins and their PTMs. Because of the low abundance of protein PTMs in comparison to their nonmodified counterpart in the cell or tissue, their detection and characterization are almost only possible by using advanced enrichment strategies. Although many studies have reported the proteome and phosphoproteome of hESCs and early differentiated stages (15–20), there are a limited number of studies available comparing the individual stages between hESCs and NSC (21, 22) especially at the PTM level. For example, Chaerkady and collaborators have reported the quantitative temporal proteomic analysis of hESC differentiation into neural cell types, including motor neurons and astrocytes (21). The authors identified a total of 1251 proteins including proteins differentially regulated during neural differentiation such as the solute carrier protein 3 member 2 (SLC3A2), a cell surface protein highly expressed only in the hESCs stage. However, the focus of this study was not on membrane bound proteins and the authors did not analyze any PTM profile during differentiation, which may also elicit important and more selective information regarding the molecular events underlying the different stages.To obtain more information about membrane proteins involved in the changes of hESCs to NSCs and also to investigate potential markers for the two distinct cellular stages, we performed a comprehensive quantitative mass-spectrometry-based proteome and PTM-ome study of membrane fractions isolated from hESCs and NSCs. We focused the PTM study on phosphorylation and SA N-linked glycosylation. This study allowed us to identify several significantly regulated proteins in hESCs and NSCs, including proteins involved in the early embryonic development as well as in the neural development. In the latter group of proteins we could identify Crumbs homolog 2 (CR2) as a potential novel NSC marker. By using selective reaction monitoring (SRM) we were able to verify a number of potential markers including CRB2 at protein and PTM level as well as CMP-N-acetylneuraminate-poly-alpha-2,8-sialyltransferase (SIA8D) at the PTM level across different cell lines beside the one used in this study. In addition, calmodulin-dependent protein kinase-2 (CaMKII) could be an important kinase for the NSC stage, because we identified the sequence recognition motif (R-X-X-pS/T) highly up-regulated in these cells. This is the consensus site for several kinases including CaMKII. Moreover, the analysis of the regulated dataset revealed an over-representation of the extracellular matrix (ECM)-receptor pathways, which are involved in diverse processes such as differentiation and proliferation (23). To our knowledge this comparative proteome study of the membrane-associated proteins that include quantitative analysis of protein phosphorylation and SA N-linked glycosylation is the most diverse set of PTMs reported for hESCs and NSCs.     

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells (NSCs/NPCs). It involves the proliferation and neuronal fate specification/differentiation of NSCs, as well as migration, maturation and functional integration of the neuronal progeny into neuronal network. NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Contrary to previous dogma that neurogenesis happens only during development, it is generally accepted now that neurogenesis can take place throughout life in mammalian brains. This raises a new therapeutic potential of applying stem cell therapy for stroke, neurodegenerative diseases and other diseases. However, the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks. Uncovering the underlying mechanisms that drive the differentiation, migration and maturation of specific neuronal lineages for use in regenerative medicine is, therefore, crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis. Here, we focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.