Glycobiology Advance Access originally published online on February 16, 2007
Glycobiology 2007 17(7):57R-74R; doi:10.1093/glycob/cwm018
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REVIEW |
The expression and functions of glycoconjugates in neural stem cells
Institute of Molecular Medicine and Genetics and Institute of Neuroscience, Medical College of Georgia, Augusta, GA 30912
1 To whom correspondence should be addressed: Tel: +706 721 0699; Fax: +706 721 8727; E-mail: ryu{at}mcg.edu
Received on January 2, 2007; revised on February 11, 2007; accepted on February 11, 2007
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Abstract |
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Top
Abstract
Neural stem cells and...The mammalian central nervous system is organized by a variety of cells such as neurons and glial cells. These cells are generated from a common progenitor, the neural stem cell (NSC). NSCs are defined as undifferentiated neural cells that are characterized by their high proliferative potential while retaining the capacity for self-renewal and multipotency. Glycoconjugates carrying carbohydrate antigens, including glycoproteins, glycolipids, and proteoglycans, are primarily localized on the plasma-membrane surface of cells and serve as excellent biomarkers at various stages of cellular differentiation. Moreover, they also play important functional roles in determining cell fate such as self-renewal, proliferation, and differentiation. In the present review, we discuss the expression pattern and possible functions of glycoconjugates and carbohydrate antigens in NSCs, with an emphasis on stage-specific embryonic antigen-1, human natural killer antigen-1, polysialic acid-neural cell-adhesion molecule, prominin-1, gp130, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, cystatin C, galectin-1, glycolipids, and Notch.
Key words: development / glycolipids / glycoproteins / niche / proteoglycans
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Neural stem cells and glycoconjugates |
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Neural stem cells (NSCs) are defined as undifferentiated neural cells that are endowed with a high potential for proliferation and the capacity for self-renewal; they also can generate a wide variety of differentiated progeny, such as neurons and glial cells (e.g., astrocytes and oligodendrocytes), and retain their multipotency (Figure 1; Temple 1989
; Reynolds and Weiss 1992; Reynolds et al. 1992; McKay 1997; Temple and Alvarez-Buylla 1999; Gage 2000; Okano 2002). Because of the biological potential of NSCs in neurogenesis and neural repair, there has been tremendous interest lately in their basic biology and clinical use in treating a variety of neurodegenerative disorders and spinal cord injuries.
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During brain development, NSCs appear in the neuroepithelium and contribute to the major cell types of the early neuroectoderm and the neural tube. Radial glia, bipolar cells transiently appearing in the neuroepithelium, are presumed to play important roles as NSCs at this stage (Malatesta et al. 2000
; Hartfuss et al. 2001; Noctor et al. 2001; Götz and Barde 2005). As development proceeds, NSCs are thought to give rise to neurons, astrocytes, and oligodendrocytes (Figure 1). Simultaneously, NSCs become gradually less abundant, and more-restricted neural precursor cells (NPCs) emerge (Note: recently, researchers tend to use the term "NSC" interchangeably with "NPC" without clearly defining the characteristics of the two cell populations. For this reason, in this review we sometimes describe NSCs as "NSCs/NPCs."). In the adult mammalian brain, NSCs are identified as a kind of glial fibrillary acidic protein (GFAP)-positive astrocytes located primarily in two regions: the subventricular zone (SVZ) of the lateral ventricles and the subgranular layer of the dentate gyrus in the hippocampus (Doetsch et al. 1999; Seri et al. 2001). These adult NSCs are thought to be derived from radial glia (Merkle et al. 2004). Although neurogenesis is mostly completed at birth, interest in NSCs in these two regions arises from the fact that NSCs continue to produce new neurons during adulthood. The NSCs in the SVZ migrate through the rostral migratory pathway to yield neurons in the olfactory bulb. NSCs in the subgranular layer migrate a short distance into the granular cell layer and differentiate into hippocampal granule cells. In addition, it has recently been confirmed that the SVZ NSCs give rise to oligodendrocytes in vivo (Menn et al. 2006). Because of these features, NSCs/NPCs have been considered to represent a cellular reservoir for formation of the central nervous system (CNS) and for replacement of cells lost during normal cellular turnover in adulthood. On the other hand, there is an opposing view that there is a lack of neurogenesis in irradiation-induced adult mouse brain, which does not affect learning ability (Meshi et al. 2006).
The fate of NSCs/NPCs, such as proliferation, differentiation, survival, and death, may be regulated by "niche," a specialized microenvironment where NSCs are located in vivo (Doetsch 2003; Alvarez-Buylla and Lim 2004). "Niche" is composed of a group of cells in a specialized tissue location serving extrinsic signals and physical anchors by producing soluble factors, membrane-bound molecules and extracellular matrices (ECMs) to maintain stem cells, such as germ line stem cells, hematopoietic stem cells, epithelial stem cells, intestinal stem cells, and NSCs (Li and Xie 2005). In addition, it is also proposed that "niche" has a role not only to maintain stem cells but also to prevent tumorigenesis by controlling the cellular proliferation (Li and Xie 2005). For adult NSCs, endothelial cells forming blood vessels and the basal lamina have an essential role as components of the niche (Doetsch 2003; Shen et al. 2004). The importance of the signal molecules emanating from the NSC niche is well known. For example, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are important in sustaining multipotency and enhancing the proliferation of NSCs/NPCs and in neurogenesis (Reynolds and Weiss 1992; Reynolds et al. 1992; Kuhn et al. 1997; Vaccarino et al. 1999). ECMs including proteoglycans provide reservoirs for growth factors and matrices for cell attachment and migration. Related to this, one of the receptor molecules of the ECM, ß1 integrin, has been revealed to mediate signaling for cell adhesion and for self-renewal in NSCs (Campos et al. 2004; Leone et al. 2005). Thus, these studies clearly show the essential roles of niche in the maintenance of the properties of NSCs.
Glycoconjugates refer to biopolymers containing one or more carbohydrate units and are roughly classified into three groups based on their structures: proteoglycans, glycoproteins, and glycolipids. These molecules are ubiquitously distributed in the organs, tissues, cells, and body fluids of animals and plants, and are primarily, although not exclusively, localized on the cell surface. Therefore, glycoconjugates are crucial in determining the properties of cells, participating in signal transduction in response to external stimuli, and in mediating cell–cell interaction and adhesion. Because of the importance of these components during development, glycoconjugates in NSCs/NPCs should be systematically studied for the following reasons. First, glycoconjugates are useful neural cell lineage-specific markers. Because glycoconjugates are localized on the cell surface and the expression patterns are frequently and drastically changed during development (Jessell et al. 1990; Cameron and Rakic 1991; Yu 1994; Zhang 2001; Muramatsu and Muramatsu 2004; Yu and Yanagisawa 2006), they are often utilized as specific biomarkers of cells, including NSCs, at different stages of differentiation (Figure 1). Secondly, recent studies clearly implicate the functional relevance of glycoconjugates in NSCs/NPCs in mediating signal transduction and cell–cell recognition and adhesion. There is also a strong possibility that glycoconjugates, especially proteoglycans that are the major components of the ECM, are involved in mediation of the cell fate-regulating signals generated by the NSC niche. In this review, we will discuss the expression pattern and putative functions of glycoconjugates and related proteins in NSCs/NPCs.
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Stage-specific embryonic antigen-1/Lewis x antigen |
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Stage-specific embryonic antigen-1 (SSEA-1, for review, see Muramatsu and Muramatsu 2004
) is the antigen of a monoclonal antibody established by immunization with F9 embryonic carcinoma cells (Solter and Knowles 1978). Studies by Gooi et al. (1981) clearly indicated that the binding reactivity of anti-SSEA-1 antibody to human meconium is inhibited by an oligosaccharide containing the Lewis x structure [Lex; Galß1-4(Fuc
1-3)GlcNAcß-]. Thus, the antigenic epitope of SSEA-1 has been considered to be Lex (Note: strictly, "SSEA-1" is not equal to "Lex". However, in this review we will describe both as SSEA-1 because SSEA-1 and Lex have not been clearly distinguished in the literature discussed in this section. SSEA-1 is also known as CD15 according to the cluster of differentiation (CD) nomenclature.
The expression of SSEA-1 was originally reported as being confined to undifferentiated cells, but not in the differentiated cells (Solter and Knowles 1978, 1979; Knowles et al. 1980). These early reports underline the importance of SSEA-1 as a marker for undifferentiated cells. In fact, it is well known that SSEA-1 is utilized as a specific marker of mouse embryonic stem (ES) cells (Muramatsu and Muramatsu 2004). More recent studies, however, have indicated that SSEA-1 is also expressed in more differentiated tissues and cells. In the CNS, SSEA-1 is abundantly expressed in the developing mouse brain (Fox et al. 1981; Yamamoto et al. 1985; Dasgupta et al. 1996; Ashwell and Mai 1997). Furthermore, SSEA-1 expression was found in human embryonic NSCs (Klassen et al. 2001), in mouse postnatal, adult, and embryonic NSCs/NPCs (Klassen et al. 2001; Capela and Temple 2002; Kim and Morshead 2003; Corti et al. 2005; Yanagisawa, Taga, et al. 2005), and in mouse embryonic retinal progenitor cells (Koso et al. 2006). Because these cells are not fully differentiated, the expression may represent the immature status of the cells as previously suggested. On the other hand, in the developing quail peripheral nervous system (PNS), SSEA-1 is expressed in more mature neural cells, the primary sensory neurons differentiated from neural crest cells (Sieber-Blum 1989).
Taking advantage of the expression pattern and the cell-surface localization of SSEA-1, strategies employing the anti-SSEA-1 antibody have been developed to sort NSCs from brain tissues by flow cytometry. These strategies led to sorting of specific stem-cell populations from the SVZs of adult mouse brain (Capela and Temple 2002; Corti et al. 2005), the germinal zones of embryonic mouse forebrain (Kim and Morshead 2003; Corti et al. 2005), and embryonic mouse retina (Koso et al. 2006). These findings reinforced the concept that SSEA-1 is a specific marker for NSCs/NPCs. The possibility of using SSEA-1 as a marker for selecting human NSCs that express this antigen (Klassen et al. 2001) should also be considered. SSEA-1 expression, however, is not limited to NSCs/NPCs. For this reason, it would be advisable to use anti-SSEA-1 antibody with probes to confirmatory markers or dyes for efficient sorting of NSCs/NPCs, as performed by Kim and Morshead (2003) and Corti et al. (2005).
One of the molecular mechanisms underlying the characteristic SSEA-1 expression pattern in the developing brain has been elucidated. Shimoda et al. (2002) found that SSEA-1 expression was significantly down-regulated in the embryonic brain of a rat small eye strain. In the small eye strain, the activity of fucosyltransferase 9, a key enzyme for SSEA-1 synthesis (Kudo et al. 1998; Nishihara et al. 1999), was also reduced. Because the small eye strain has mutations in the Pax6 transcription factor gene, this study suggests that the expression of SSEA-1 in the developing rat brain is positively regulated by Pax6 via induction of fucosyltransferase 9. Because mouse radial glia and mouse embryonic cortical NPCs express Pax6 (Heins et al. 2002; Abematsu et al. 2006), the expression of SSEA-1 in NSCs/NPCs may also be regulated by Pax6.
As compared with its expression pattern, the functional roles of SSEA-1 in NSCs/NPCs are not well understood. SSEA-1 has been generally considered to be involved in cell adhesion and compaction of the mouse embryo at the morula stage and also in embryonic carcinoma cells (Fenderson et al. 1984; Eggens et al. 1989). In NSCs/NPCs, anti-SSEA-1 antibody does not seem to affect the formation of mouse embryonic neurospheres (floating clonal aggregates formed by NSCs in vitro), but it inhibits cell migration from the neurospheres in experimental conditions (von Holst et al. 2006; Yanagisawa, Taga, et al. 2005). Analysis of mice deficient in fucosyltransferase 9 and SSEA-1 revealed increased anxiety-like behavior of the mice, but no discernible morphological phenotype in brain development (Kudo et al. 2004, 2006). This finding suggests that the function of SSEA-1 may be compensated by other molecules or is not essential for brain development in the mouse embryo.
To determine the functional roles of SSEA-1 in NSCs/NPCs, it is also essential to identify the carrier molecules. SSEA-1 is a common carbohydrate epitope carried by glycoproteins, proteoglycans, and glycolipids. At present, the SSEA-1 epitope has been suggested to be associated with chondroitin sulfate proteoglycans (CSPGs) in mouse embryonic NPCs (Kabos et al. 2004), ß1 integrin in mouse embryonic NPCs (Yanagisawa, Taga, et al. 2005), Wnt-1 in mouse embryonic brain (Capela and Temple 2006), and a glycolipid [Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glcß1-1'Cer] in mouse embryonic NPCs (Yanagisawa, Taga, et al. 2005).
As we discussed, SSEA-1 is expressed in mouse embryonic stem cells (ES cells), but not in human ES cells. Instead of SSEA-1, SSEA-3 [-3GalNAcß1-3Gal1-4Galß1-; Kannagi et al. 1983] and SSEA-4 [SSEA-4; NeuAc2-3Galß1-3GalNAcß1-; Kannagi et al. 1983] are expressed in human ES cells and are commonly used as the cell surface markers (for review, see Muramatsu and Muramatsu 2004). Recently, it has been reported that SSEA-4 is expressed in human embryonic NSCs. Piao et al. (2006) found that a small number of cells in the neurospheres, but not in differentiated cells, prepared from human embryonic forebrains (4.5–12 weeks of gestation) and spinal cords (4.5–9.5 weeks of gestation) express SSEA-4 with other ES cell marker proteins, Tra-1-60 and Tra-1-81. Similarly, Barraud et al. (2007) found that SSEA-4-expressing cells are enriched in the NSCs prepared from human embryo forebrains (7–9 weeks of gestation) and the expression is associated with prominin-1 (see Prominin-1). They suggest that a combination of SSEA-4 with prominin-1 can be used for immunological sorting of NSCs from developing human forebrains, as performed for mouse NSCs by Kim and Morshead (2003) and Corti et al. (2005). Further studies to elucidate the molecular properties and functions of SSEA-4 in NSCs remain to be done.
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Human natural killer-1 antigen |
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Neural crest cells of the vertebrate embryo are transitory cells that detach from the neural fold of the neural tube and migrate during development to diverse locations (Anderson 1997
; Sieber-Blum 2000; Christiansen et al. 2000). Neural crest cells exhibit multipotency (Bronner-Fraser and Fraser 1988) and contain a stem-cell population of neural crest stem cells (Stemple and Anderson 1992; Morrison et al. 1999), which are capable of self-renewal and have the multipotency to differentiate into autonomic neurons by stimulation with bone morphogenetic protein (BMP) 2 (Shah et al. 1996), into Schwann cells by stimulation with glial growth factor (Shah et al. 1994), and into smooth muscles by stimulation with transforming growth factor-ß (Shah et al. 1996) (Figure 2).
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The human natural killer-1 (HNK-1) antigen (CD57) is a carbohydrate antigen whose structure has been established as HSO3-3GlcAß1-3Galß1-4GlcNAc- (Chou et al. 1986
; Ariga et al. 1987). By studying model compounds, Tokuda et al. (1998) established the minimal carbohydrate epitope structure as HSO3-3GlcAß1-3Galß1-. HNK-1 is a well-known cell surface marker that is one of the common antigens in the nervous system (Tucker et al. 1984; Vincent and Thiery 1984; Bronner-Fraser 1986). In the avian and rodent nervous systems, HNK-1 is distributed on the surface of neural crest cells and is required for their proper migration during development (Bronner-Fraser 1987; Holley and Yu 1987; Nagase et al. 2003). Therefore, regulation of neural crest cell migration by HNK-1 may be a general mechanism in all vertebrates. However, Tucker et al. (1988) reported that mouse neural crest cells are negative for HNK-1, indicating that the mouse neural crest cells differ from avian and rat neural crest cells in the expression pattern of the HNK-1 epitope. It remains to be determined whether the requirement of the HNK-1 epitope for mouse neural crest cell migration is compensated for by the presence of other molecules.
Interest in the HNK-1 epitope arises from its association with a number of cell-adhesion molecules (Kruse et al. 1984; Jungalwala 1994). Of the carrier molecules of HNK-1 antigen, glycoproteins [e.g. L1, P0, and neural cell-adhesion molecule (NCAM); Kruse et al. 1984], glycolipids (e.g. SGPG and SGLPG; Chou et al. 1986; Ariga et al. 1987), and proteoglycans (e.g. CSPGs; Domowicz et al. 1995; Pettway et al. 1996) have been characterized. Because of the wide distribution on various glycoconjugates, HNK-1 is expected to play important functional roles in neural development. So far, mice deficient in glucuronyltransferase (GlcAT-P) or HNK-1 sulfotransferase, the key enzymes of HNK-1 antigen synthesis, have been established (Yamamoto et al. 2002; Senn et al. 2002). In these mice, brain development is generally normal; however, adult mice deficient for GlcAT-P or HNK-1 sulfotransferase exhibit reduced long-term potentiation and defective spatial memory formation. These results suggest a functional role of the HNK-1 antigen in synaptic plasticity of the hippocampus, but not in brain development.
More recently, HNK-1 expression in mouse embryonic NPCs was confirmed to be exclusively associated with glycoproteins and/or proteoglycans (Yanagisawa, Taga, et al. 2005). On the other hand, ventrally emigrating neural-tube cells, multipotent cells that also appear from neural tubes but differ from neural crest cells, are reported to lack expression of HNK-1 (Dickinson et al. 2004). At present, the detailed expression pattern, the nature of the carrier glycoproteins/proteoglycans, and the functional roles of HNK-1 in the NPCs are not yet completely elucidated.
Other glycoconjugate markers reported to be present in neural crest and neural crest-derived cells include GD3 ganglioside in mouse neural crest cells (Stainier et al. 1991), SSEA-1 in quail committed sensory neurons (Sieber-Blum 1989), B30 gangliosides (two unidentified gangliosides recognized by the B30 antibody—one migrates slightly above GM1, and the other migrates between GD1a and GD3 on thin-layer chromatography) in mouse sensory neurons (Stainier et al. 1991), polysialic acid-neural cell adhesion molecule (PSA-NCAM) in mouse and rat sensory and/or autonomic neurons (Boisseau et al. 1991; Stemple and Anderson 1992), and O4 antigen (sulfatide) in rat Schwann cells (Stemple and Anderson 1992), and mouse Schwann cells and their precursors (Dong et al. 1999) (Figure 2).
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Polysialic acid–neural cell adhesion molecule |
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Unlike SSEA-1 and HNK-1, the polysialic acid (PSA) carbohydrate structure (Finne et al. 1983
) exclusively carried by the neural cell adhesion molecule (NCAM) is expressed in NSCs/NPCs. PSA is a linear homopolymer containing 8–100 2–8-linked sialic acid residues (SA2-8SA2-; Rutishauser and Landmesser 1996), whose synthesis is catalyzed by polysialyltransferases, ST8SiaII (also known as STX) and ST8SiaIV (also known as PST) (Angata and Fukuda 2003). As expected from its characteristic carbohydrate structure, PSA presents interesting features, such as highly negative charges and a high level of hydration, and an excellent ability to bind cations. PSA-NCAM is abundantly expressed during neural development. Although expression is drastically decreased in adult brain, certain regions (e.g., the SVZ, olfactory bulb, and hippocampus) have continuously expressed PSA-NCAM (Angata and Fukuda 2003; Seki and Arai 1993). Taking advantage of the expression pattern, Pennartz et al. (2004) successfully established a method to purify PSA-NCAM-positive NSCs/NPCs from adult mouse brain. It should be noted, however, that cells expressing PSA-NCAM in the postnatal rat brain behave like glial precursor cells in vitro and are mostly committed to a glial fate (Ben-Hur et al. 1998). More detailed studies about the lineage of the PSA-NCAM-positive cells are still needed.
The remarkable chemical structure and expression pattern of PSA-NCAM suggest its importance in brain development. In fact, it has been shown that PSA-NCAM regulates myelination, axon guidance, synapse formation, and functional plasticity of the nervous system. Mice deficient in polysialyltransferase present developmental and behavioral defects, including reduction of long-term potentiation and long-term depression (in ST8SiaIV/PST-deficient mice; Eckhardt et al. 2000) and misguidance of mossy fibers and ectopic synapse formation in the hippocampus (in ST8SiaII/STX-deficient mice; Angata et al. 2004). Surprisingly, no defect in NSCs/NPCs has been reported by these loss-of-function experiments in mice. A gain-of-function experiment by inducing stable over-expression of PSA in mouse NSCs, however, suggests that PSA-NCAM plays an important role during NSC/NPC differentiation and migration; in mouse NSCs/NPCs over-expressing PSA, migration is enhanced and oligodendrocytogenesis is suppressed, although the cells' multipotency and survival are not affected (Franceschini et al. 2004). Furthermore, it has been reported that enzymatic digestion of PSA represses migration and induces premature neuronal differentiation of adult mouse NSCs (Petridis et al. 2004). Thus, it is considered that PSA-NCAM may not be essential for NSCs/NPCs, but that it serves to modify the cell fate possibly by its characteristic chemical structure.
PSA-NCAM is expressed not only in the CNS but also in the PNS. Boisseau et al. (1991) found that expression of high PSA-NCAM was restricted to an early neuronal lineage cells derived from neural crest cells. Recently, it has been reported that BMP enhances migration, neurite fasciculation, and clustering of neuronal cells via promotion of polysialylation of NCAM in the enteric nervous system formed from neural crest cells (Fu et al. 2006; Faure et al. 2007). These studies suggest the importance of regulation of glycoconjugate expression by cytokine signaling in the developing nervous system.
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Prominin-1 |
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Prominin-1, also known as CD133 or AC133 (human homolog), is a pentaspan membrane glycoprotein (Shmelkov et al. 2005
) originally identified as an antigen expressed on the apical surface of neuroepithelial stem cells and in several other epithelia, including kidney proximal tubules (Weigmann et al. 1997). This antigen, however, has been shown to be expressed not only in embryonic NSCs but also in adult NSCs (Sawamoto et al. 2001), hematopoietic stem cells (Yin et al. 1997) and brain cancer stem cells (Hemmati et al. 2003; Singh et al. 2003), indicating its specificity as a somatic stem-cell marker.
Researchers have taken advantage of the expression pattern on the neuroepithelial cell surface to sort NSCs using flow cytometry with anti-pominin-1 antibody. In 2000, Uchida et al. established a method to isolate NSCs directly from fresh human fetal brains. In their study, cells positive for prominin-1 and negative for CD34 (sialomucin) and CD45 (T200 glycoprotein) were NSCs capable of forming neurospheres and differentiating into both neurons and glial cells. NSCs positive for prominin-1 have also been sorted from mouse postnatal cerebellum (Lee et al. 2005).
Investigators using electromicroscopic analysis have found that prominin-1 is specifically associated with plasma membrane protrusions that have a microvilli-like structure on the apical surface of neuroepithelial cells (Weigmann et al. 1997). Interestingly, during the early stages of neurogenesis, the apical plasma membrane protrusions containing prominin-1 are released to the lumen of the neural tube as a novel class of extracellular membrane particles with diameters of approximately 600 and 50–80 nm (Marzesco et al. 2005). The release of the prominin-1-containing particles is not limited to the developing brain, and the occurrence of the particles is confirmed in various adult human body fluids, including seminal fluid, saliva, urine, and lacrimal fluid. Although the physiological significance of prominin-1 is still unclear, its characteristic localization pattern on the apical plasma membrane and its presence in the novel class of particles suggest that prominin-1 may play important roles in the maintenance of membrane protuberances and cell polarity, and in interaction with the niche. In fact, analysis of a family with autosomal recessive retinal degeneration has revealed that a single nucleotide deletion of a protamin-1 gene causing the frame-shift mutation disrupts transportation of prominin-1 to the cell surface and leads to retinal degeneration due to impaired photoreceptor disk morphogenesis (Maw et al. 2000).
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gp130 |
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gp130 (CD130) is a cell surface glycoprotein originally found as a receptor component and signal transducer of interleukin (IL)-6 (Taga et al. 1989
; Taga and Kishimoto 1997; Fukuda and Taga 2005). Further studies revealed that this molecule mediates signaling activated by all eight IL-6 family of cytokines, such as IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M, cardiotrophin-1, cardiotrophin-like cytokines/novel neurotrophin-1/B-cell stimulating factor 3, and neuropoietin. Of the signaling pathways activated by the IL-6 family cytokines via gp130, the Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) pathway, the Ras–mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3 kinase–Akt pathway are known. Although the expression of gp130 is ubiquitous and exhibits no significant pattern in NSCs, the functional aspect is too important to ignore.
Thus far, gp130 has been shown to play critical roles in NSCs/NPCs. First, it is involved in the induction of astrocyte differentiation. It has been reported that the IL-6 family of cytokines induce astrocyte differentiation of rat and mouse embryonic NPCs via activation of a gp130 homo-/hetero-dimer and the JAK–STAT pathway (Bonni et al. 1997, Nakashima, Yanagisawa, et al. 1999; Fukuda and Taga 2005). Among the IL-6 family of cytokines having this activity (Fukuda and Taga 2005; Ohno et al. 2006), cardiotrophin-1 is proposed to be a bona fide inducer for astrocyte differentiation in the developing brain (Barnabe-Heider et al. 2005). The involvement of gp130 in astrocyte differentiation has been confirmed in physiological conditions; gp130-knockout mice exhibit reduction of astrocyte numbers in the developing brain (Nakashima, Wiese et al. 1999). Astrocytic differentiation, however, is not regulated only by the IL-6 family of cytokines, gp130, and the downstream JAK–STAT pathway. For instance, the positive and negative cross-talk of gp130 signaling with BMP signaling (Nakashima, Yanagisawa, et al. 1999), neurogenin-2 (a basic helix-loop-helix [bHLH] transcription factor; Sun et al. 2001), or Notch–hairy-enhancer of split (HES) signaling (Kamakura et al. 2004) has been clarified. In addition, the epigenetic status of astrocytic genes in NPCs is also important for the gp130-mediated astrocyte differentiation (Takizawa et al. 2001).
Secondly, gp130 is involved in maintenance of the self-renewal property of NSCs. Shimazaki et al. (2001) reported that CNTF maintains embryonic and adult NSCs in an undifferentiated state by blocking the differentiation via gp130 signaling. They also found that activation of gp130 leads to an increase in Notch-signaling functioning in NSC maintenance and proliferation (Chojnacki et al. 2003). On the other hand, CNTF is not considered to be a secreted cytokine because of its lack of a secretory signal sequence in the gene and the cytosolic localization of the protein. It should be noted that other IL-6 families of cytokines such as neuropoietin are suggested to share the biological function of CNTF (Derouet et al. 2004). Therefore, the self-renewal property of NSCs may be maintained by multiple IL-6 families of cytokines.
In addition to the above, gp130 signaling has been reported to support NPC survival via activation of the phosphatidylinositol 3 kinase-Akt pathway (Chang et al. 2004). As compared with these diverse biological activities, however, the structure and function of the carbohydrate chain(s) carried by gp130 are totally unknown. The carbohydrate chain of gp130 may be involved in the receptor-ligand interaction, such as that by glycosaminoglycans of proteoglycans.
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Chondroitin sulfate proteoglycans |
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Proteoglycans, the major components of the ECM, are a class of glycosylated proteins having covalently linked glycosaminoglycans, sulfated carbohydrate chains made of repeating disaccharides. Based on the components of disaccharides, proteoglycans are divided into certain subclasses. For instance, proteoglycans having chondroitin sulfate glycosaminoglycans are classified as CSPGs, whereas proteoglycans having heparan sulfate glycosaminoglycans are classified as heparan sulfate proteoglycans (HSPGs). Both subclasses of proteoglycans are known to be expressed in NSCs/NPCs.
As CSPGs expressed in NSCs/NPCs, tenascin C (Garcion et al. 2004; Kabos et al. 2004), aggrecan (Kabos et al. 2004), neurocan (Ida et al. 2006), phosphacan (Kabos et al. 2004; Ida et al. 2006; von Holst et al. 2006), and neuroglycan C (Ida et al. 2006) have been reported. Among these components, the importance of tenascin C and phosphacan has been well studied. Tenascin C is a CSPG robustly expressed in the SVZ and considered to contribute to generation of the stem cell niche. By analyzing tenascin C-knockout mice, Garcion et al. (2004) clearly demonstrated that this CSPG regulates the NSC differentiation via modulation of cytokine signaling in developing mouse brains. Similarly, phosphacan, a CSPG having the glycosaminoglycan epitope recognized by 473HD antibody and representing a neurite outgrowth activity (Faissner et al. 1994), is also localized in the postnatal and adult NSC niche. In considering the characteristic expression pattern of phosphacan, von Holst et al. (2006) isolated cells positive for 473HD from mouse embryonic brain by immunopanning or sorting with magnetic beads, and demonstrated that these 473HD-positive cells exhibit NSC features. Interestingly, modification of 473HD epitope by chondroitinase ABC or 473HD antibody prevented 473HD-positive NSCs from neurosphere formation, suggesting the physiological significance of phosphacan and glycosaminoglycan in NSC self-renewal.
Another important CSPG is nerve/glial antigen 2 (NG2), which was originally identified in rat. The mouse homolog is also known as AG2. NG2 is a CSPG highly expressed in adult and embryonic brains (Stallcup 2002). Cells positive for NG2 have been considered to be committed oligodendrocyte progenitors because they exclusively express oligodendrocyte markers and give rise to oligodendrocyte lineage cells (Dawson et al. 2000; Stallcup 2002). For instance, O-2A progenitor cells, glial precursor cells capable of differentiating into oligodendrocytes and type-2 astrocytes (Raff et al. 1983), are positive for NG2 (Stallcup and Beasley 1987; Levine and Stallcup 1987). It has also been asserted that NG2-glia, an immature glial-cell population capable of giving rise to oligodendrocytes, express this proteoglycan (Nishiyama et al. 2005). More recently, however, it has been reported that NG2-positive cells in postnatal mouse brain exhibit characteristics of NSCs, such as multipotency to differentiate into oligodendrocytes and astrocytes as well as neurons (Belachew et al. 2003; Aguirre and Gallo 2004; Aguirre et al. 2004). In addition, it has been confirmed that neurosphere-forming NSCs prepared from rat telencephalons express NG2 (Ida et al. 2006). In light of these different findings, it is tempting to suggest that there are heterogeneous populations of NG2-positive cells giving rise to different cell types. The bona fide lineages and physiological functions of NG2-positive cells remain to be determined. With respect to its functional roles, NG2 has been shown to have a high affinity for bFGF and platelet-derived growth factor-AA, critical mitogens to NSCs/NPCs and oligodendrocyte progenitor cells (Goretzki et al. 1999). The high affinity for growth factors is reminiscent of the critical roles of HSPGs as a bFGF signal cofactor during brain development (see the Heparan sulfate proteoglycans section). Interestingly, NG2-knockout mice exhibit no defect in hippocampal neurogenesis (Thallmair et al. 2006), suggesting that, unlike other proteoglycans such as HSPGs, NG2 may be nonessential for NSCs/NPCs proliferation.
Recently, it has been reported that mice deficient in an Olig2 bHLH transcription factor exhibit severe defects in NG2-positive cells in developing brains and spinal cords (Ligon et al. 2006). This study indicates that development of NG2-positive cells requires Olig transcription factors, especially Olig2.
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Heparan sulfate proteoglycans |
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NSCs/NPCs express CSPGs as well as HSPGs, the other major component of ECM. Expression of a truncated form of perlecan (Joseph et al. 1996
), glypican-4 (K-glypican) (Hagihara et al. 2000; Ford-Perriss et al. 2003), and syndecan (Ford-Perriss et al. 2003) has been demonstrated in the neuroepithelium. Among these, glypican-4 (Hagihara et al. 2000) and syndecan (-1 and -3; Zou et al. 2006) have been shown to be expressed in neurospheres and mouse embryonic NPCs, respectively. Notably, the expression of glypican-4, a glycosylphosphatidylinositol-anchored HSPG, in NPCs disappears immediately after bFGF depletion to induce multilineage differentiation (Hagihara et al. 2000), suggesting the possibility of glypican-4 as a specific and sensitive marker of undifferentiated NSCs. Furthermore, treatment of NPCs with a recombinant glypican-4 partially inhibits bFGF-induced proliferation of NPCs (Hagihara et al. 2000), indicating the involvement of glypican-4 in bFGF signaling in NSCs/NPCs, one of the most important functions of HSPGs in the cells.
At present, HSPGs are known to bind to many factors such as morphogens and mitogens including bFGF, a critical growth factor that sustains multipotency and induces proliferation of NSCs/NPCs. This association suggests the role of HSPGs in mediating signal transduction. For example, bFGF signaling is mediated by receptors, such as FGFR1IIIc and FGFR3IIIc, but an additional cofactor is required for formation of the receptor complex. The cofactor forming a complex with bFGF and FGFR is HSPG (Nurcombe et al. 1993). In NSCs/NPCs, active HSPGs secreted from the cells exhibit high affinity for bFGF and selectivity to different FGF receptors via the heparan sulfate chains (Brickman et al. 1995). The importance of HSPGs for FGF signaling in NPCs/NPCs is further demonstrated in physiological conditions. Brains of conditional knockout mice lacking EXT1, a heparan sulfate (HS)-polymerizing enzyme essential for HS synthesis, exhibit various severe defects, such as thinning of the cortex (Inatani et al. 2003). In NPCs prepared from the knockout mice, bFGF- or fibroblast growth factor 8-induced proliferation is reduced. These reports suggest that HSPGs play a critical role in bFGF-induced proliferation of NSCs/NPCs mediated by highly regulated signaling mechanisms. As a cofactor of HSPG for the bFGF signaling during neurogenesis, a truncated form of perlecan secreted from neuroepithelial cells has been reported (Aviezer et al. 1994; Joseph et al. 1996). In NSCs/NPCs, glypican-4 has also been proposed to act as a potent cofactor for bFGF signaling (Hagihara et al. 2000).
In addition to bFGF, midkine, a heparan-binding growth factor, has also been reported to induce proliferation and survival of mouse embryonic NPCs (Zou et al. 2006). The midkine signaling is mediated by multimolecules including HSPGs. In the NPCs, the importance of HSPGs, such as syndecan-1 and -3, as components of the midkine receptors is proposed (Zou et al. 2006). On the other hand, NPCs prepared from mouse embryo deficient in syndecan-3 are reported to exhibit no defect in proliferation and differentiation (Hienola et al. 2006).
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N-glycosylated cystatin C |
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In addition to HSPGs, another glycoconjugate has been reported as a cofactor of bFGF signaling. Taupin et al. (2000) found that a soluble factor is required for bFGF-induced proliferation of adult rat hippocampus-derived NSCs in conditioned media of the NSC cultures. Purification and characterization of the autocrine/paracrine factor revealed that it is the glycosylated form of cystatin C, a cysteine protease inhibitor belonging to family 2 of the cystatin superfamily. In addition, it also has been reported that cystatin C secreted by mouse embryonic neurospheres enhances differentiation of ES cells to NSCs, probably in cooperation with bFGF (Kato et al. 2006
). These reports suggest that cystatin C is an important glycoconjugate in supporting NSC development and maintenance, although the exact physiological role of cystatin C in NSCs remains unclear.
In contrast to these reports, cystatin C has been proposed not be a factor involved in self-renewal of NSCs, but rather it induces astrocyte differentiation of an immature astrocyte cell line (Kumada et al. 2004). At this time, the origin of this functional discrepancy is not yet clear. One possibility could be related to differences in the methodologies used by the two researchers: Kumada et al. (2004) introduced a cDNA library of immature mouse brains to an immature astrocyte cell line to seek astrocyte inducers; on the other hand, Taupin et al. (2000) used conditioned media of an adult rat hippocampus-derived NSC culture to identify an unknown FGF cofactor. Therefore, there is a possibility that cystatin C analyzed in these studies had different glycosylation patterns, which led to the different biological activities of cystatin C. In fact, the N-glycan of cystatin C is found to be important and essential for the mitogenic activity of cystatin C in NSCs (Taupin et al. 2000). Evaluation of the physiological functions of cystatin C remains to be done, but studies regarding cystatin C and HSPG (Nurcombe et al. 1993; Brickman et al. 1995) may reinforce the importance of the carbohydrate chains for the functions of cofactors in bFGF signaling.
In addition to cystatin C, another molecule belonging to the cystatin superfamily, cystatin B, is expressed in mouse embryonic and adult NSCs and differentiated neurons and glial cells from the NSCs (Brannvall et al. 2003).
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Galectin-1 |
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Galectin-1 is an animal lectin with Gal-binding specificity (Barondes et al. 1994
). Although galectin-1 is not a glycoconjugate, we introduce it as a glycoconjugate-related molecule regulating the fate of NSCs. It has recently been shown that a conditioned medium of OP9 stromal cells contains a factor that enhances neurosphere formation and proliferation of adult mouse NSCs (Sakaguchi et al. 2006). This unknown factor was purified and identified as galectin-1 by tandem mass spectrometry. Further experiments revealed that mouse adult NSCs as well as OP9 cells express galectin-1. Importantly, galectin-1-deficient mice exhibit a decrease of NSCs in the SVZ (Sakaguchi et al. 2006), clearly indicating that galectin-1 plays a role in maintenance of adult NSCs under physiological conditions. The molecular mechanism by which galectin-1 enhances proliferation of the NSCs is unknown. It is tempting, however, to suggest that galectin-1 binds to the carbohydrate chains of signaling molecules and modulates the signal transduction as do HSPGs or and/or cystatin C. |
b-series gangliosides |
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Glycolipids are lipid molecules linked with one or more carbohydrate units. The molecules are classified into glycosphingolipids (GSLs) and glycoglycerolipids based on their core lipid moiety. In their core lipid moiety, GSLs and glycoglycerolipids contain ceramide (Cer) and glycerol, respectively. GSLs containing one or more sialic acid residue(s) are referred to as gangliosides. GSLs are found in virtually all vertebrate cells and body fluids and are particularly abundant in the nervous system. In cells, they are localized primarily, but not exclusively, on the plasma membrane. Although not fully clarified, information regarding the biological functions of GSLs is emerging rapidly. For example, GSLs may serve as mediators in cell–cell recognition and adhesion, as receptors for bioactive molecules, and as modulators of signal transduction (Hakomori 1990
; Ledeen and Wu 2002; Hakomori 2003; Yu et al. 2004). In the developing brain, gangliosides are presumed to modulate Cer-induced apoptosis and to maintain cellular survival and differentiation (Bieberich et al. 2001). Furthermore, it is well known that serious neurological deficits develop in animals or humans with aberrant ganglioside metabolism