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First published online September 6, 2007
Stem Cells Vol. 25 No. 12 December 2007, pp. 3016 -3025
doi:10.1634/stemcells.2007-0218; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS

Neural Cell Adhesion Molecule Polysialylation Enhances the Sensitivity of Embryonic Stem Cell-Derived Neural Precursors to Migration Guidance Cues

Tamara Glasera, Claudia Brosea, Isabelle Franceschinib, Katja Hamanna, Alina Smorodchenkoc, Frauke Zippc, Monique Dubois-Dalcqb, Oliver Brüstlea

aInstitute of Reconstructive Neurobiology, Life and Brain Center, University of Bonn and Hertie Foundation, Bonn, Germany;
bUnité de Neurovirologie et Régénération du Système Nerveux, Institut Pasteur, Paris, France;
cInstitute of Neuroimmunology, Charité, Humboldt University of Berlin

Key Words. Embryonic stem cells • Glial precursors • Polysialic acid-neural cell adhesion molecule • Migration • Chemotaxis Transplantation

Correspondence: Oliver Brüstle, M.D., Institute of Reconstructive Neurobiology, University of Bonn Life and Brain Center, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany. Telephone: 49-228-6885-500; Fax: 49-228-6885-501;

Received on March 27, 2007; accepted for publication on August 21, 2007.

First published online in STEM CELLS EXPRESS  September 6, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The development of stem cell-based neural repair strategies requires detailed knowledge on the interaction of migrating donor cells with the host brain environment. Here we report that overexpression of polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), in embryonic stem (ES) cell-derived glial precursors (ESGPs) strikingly modifies their migration behavior in response to guidance cues. ESGPs transduced with a retrovirus encoding the polysialyltransferase STX exhibit enhanced migration in monolayer cultures and an increased penetration of organotypic slice cultures. Chemotaxis assays show that overexpression of PSA results in an enhanced chemotactic migration toward gradients of a variety of chemoattractants, including fibroblast growth factor 2 (FGF2), platelet-derived growth factor, and brain-derived neurotrophic factor (BDNF), and that this effect is mediated via the phosphatidylinositol 3'-kinase (PI3K) pathway. Moreover, PSA-overexpressing ESGPs also exhibit an enhanced chemotactic response to tissue explants derived from different brain regions. The effect of polysialylation on directional migration is preserved in vivo. Upon transplantation into the adult striatum, PSA-overexpressing but not control cells display a targeted migration toward the subventricular zone. On the basis of these data, we propose that PSA plays a crucial role in modulating the ability of migrating precursor cells to respond to regional guidance cues within the brain tissue.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The controlled differentiation of embryonic stem (ES) cells into neural precursors opens fascinating prospects for cell replacement in the central nervous system (CNS). Epigenetic differentiation strategies and genetic lineage selection protocols permit the derivation of defined neural subpopulations from mouse and human ES cells [1]. Although the mere availability of a suitable donor source represents an essential basis for neural cell replacement, the clinical efficacy of grafted cells will critically depend on their ability to migrate and incorporate in adult CNS tissue.

The results of several studies suggest that the polysialylated form of the cell adhesion molecule NCAM (PSA-NCAM) plays a crucial role in mediating precursor cell migration in the adult brain. Furthermore, PSA-NCAM has been shown to modulate axon guidance, synapse formation, and functional plasticity of the nervous system [2–4]. PSA is abundantly expressed in a variety of embryonic tissues, whereas the majority of NCAM in adult tissues lack PSA. In the adult brain, PSA-NCAM expression is restricted to regions that retain the ability to generate neurons or that exhibit a permanent capacity for morphological and/or physiological plasticity such as the subventricular zone (SVZ), the rostral migratory stream (RMS), the olfactory bulb, the dentate gyrus, and the hypothalamo-neurohypophysial system [5–7]. This expression pattern indicates that PSA plays important roles in formation and remodeling of the nervous system through regulation of adhesive properties of NCAM.

Interestingly, re-expression of PSA-NCAM has been demonstrated in some malignant human tumors, including small-cell lung carcinoma, Wilms tumor, neuroblastoma, and rhabdomyosarcoma [8–10]. In these tumors, polysialylation of NCAM appears to increase the metastatic potential and has been correlated with tumor progression and a poor prognosis [9–14].

The polysialylation of NCAM is executed by two closely related enzymes, the Golgi-associated polysialytransferases PST (i.e., ST8SiaIV/PST) and STX (i.e., ST8SiaIV/STX), whose expression and activity are spatially and temporally regulated [15, 16]. Deletion of both polysialyltransferase genes results in a severe phenotype with specific brain wiring defects, progressive hydrocephalus, postnatal growth retardation, and precocious death [17].

Several lines of evidence suggest that PSA-NCAM can regulate cell motility. Impairment of the migration of olfactory precursor cells in the rostral migratory stream was observed in NCAM-deficient mice [18, 19] and when the polysialic acid moiety was removed [19, 20]. A number of studies have shown that PSA-NCAM-positive neural precursors can be mobilized and recruited after demyelination [21–23]. Mouse neural precursors overexpressing PSA-NCAM and grafted into the developing chick neural crest have been found to switch their migration from a dorsolateral to a ventral route [24]. The results of a very recent study indicate that virus-mediated transfer of PST into astrocytes enhances the recruitment of SVZ progenitors to a cortical lesion site [4]. In vitro studies also showed that PSA-NCAM is required for the directed migration of oligodendrocyte progenitors toward a gradient of platelet-derived growth factor (PDGF) [25] and that overexpression of PSA-NCAM in Schwann cells strongly enhances their motility [26]. Although these observations have been attributed to an enhanced motility of PSA-NCAM-positive neural cells, the precise nature of this effect has remained largely enigmatic.

In the present study, we explored overexpression of the polysialylated form of NCAM as a tool to enhance the migratory properties of ES cell-derived neural cells. Specifically, overexpression of the polysialyltransferase STX was used to generate ESGPs with high-level PSA expression. Migration studies performed with these cells in vitro and in vivo not only demonstrate that PSA induces migration but point to an important novel function of PSA in enhancing the cells' responsiveness to defined chemotactic factors and putative guidance cues of various brain regions. We conclude that PSA-NCAM modulates the cellular response of ESGPs to soluble guidance cues and enhances their directed migration in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Culture and Differentiation of ES Cells
Culture and differentiation of mouse ES cells (line CJ7, kindly provided by T. Gridley [27]) was performed as described [28] (Fig. 1A). Briefly, ES cells proliferating on mitotically inactivated murine embryonic fibroblasts (MEFs) were passaged and propagated on gelatin-coated dishes (0.1%; Sigma-Aldrich, Steinheim, Germany, http://www.sigmaaldrich.com) for 2 days to eliminate MEFs. The cells were then transferred to bacterial culture dishes to allow for EB formation and initiation of differentiation. Four-day-old EBs were plated in defined ITSFn medium containing 5 µg/ml insulin (Intergene, Purchase, NY, http://www.intergene.com), 50 µg/ml human apo-transferrin (Intergene), 30 nM sodium selenite (Sigma-Aldrich), and 2.5 µg/ml fibronectin (Life Technologies, Karlsruhe, Germany, http://www.lifetech.com) to promote the survival of neuroepithelial cells [29]. After 5 days, cells were further propagated on polyornithine-coated dishes (15 µg/ml; Sigma-Aldrich) in the presence of 10 ng/ml fibroblast growth factor 2 (FGF2), which permits the proliferation of multipotential ES cell-derived neural precursors (ESNPFGF2·PROL). To obtain ESGPs, cells were cultured in the presence of 10 ng/ml FGF2 and 20 ng/ml epidermal growth factor (EGF) (ESGPFGF2·EGF·PROL). For some experiments, these cells were further proliferated in medium containing 10 ng/ml FGF2 and 10 ng/ml PDGF (ESGPFGF2·PDGF·PROL). Differentiation of the individual cell populations was induced by withdrawing the growth factors for 4 days (ESNPFGF2·DIFF, ESGPFGF2·EGF·DIFF, ESGPFGF2·PDGF·DIFF). All growth factors were purchased from R&D Systems Inc. (Wiesbaden, Germany, http://www.rndsystems.com).


Figure 1
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  		Figure 1. Baseline PSA expression of embryonic stem (ES) cell-derived neural precursors and retroviral-mediated gene transfer of STX. (A): Schematic representation of the in vitro development of ES cells into ESNPs and ESGPs (green) and their differentiation into neurons, oligodendrocytes, and astrocytes (red). (B, C): Quantitative PSA immunofluorescence (B) and Western blot (C) analyses were used to determine levels of PSA in proliferating and differentiating precursors. The percentage of PSA-positive cells was much higher in differentiating versus proliferating cells. Western blot analysis at various stages detected the PSA chain as a wide band with a molecular mass of 180–220 kDa and confirmed the weak PSA expression in proliferating ESGPs. (D–F): ESGPFGF2·EGF·PROL were transduced with STX- or EGFP-expressing retrovirus. (D): Immunofluorescence of ESGPFGF2·EGF·PROL revealed an enhanced polysialylation of NCAM in STX-transduced cells compared with EGFP-transduced control cells. Quantitative immunofluorescence (E) and Western blot analysis (F) of proliferating ESGPs (ESGPFGF2·EGF·PROL and ESGPFGF2·PDGF·PROL) and their differentiated progeny (ESGPFGF2·EGF·DIFF and ESGPFGF2·PDGF·DIFF) demonstrated a strong and stable increase of PSA expression in STX-transduced cells. Quantitative results shown in (B) and (E) are based on at least three independent experiments and are expressed as mean ± SEM. Abbreviations: EGFP, enhanced green fluorescent protein; ESGP, embryonic stem cell-derived glial precursor; ESNP, embryonic stem cell-derived neural precursor; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; PSA, polysialic acid.

 
All migration experiments were done with the glial precursors growing in the presence of FGF2 and EGF (ESGPFGF2·EGF·PROL; stage 2a; Fig. 1A). In experiments focusing on in vitro differentiation, further propagation in the presence of FGF2 and PDGF for 4 days (ESGPFGF2·PDGF·PROL; stage 3a; Fig. 1A) was used to promote the generation of oligodendrocyte progenitors.

Construction of Retroviral Vectors and Viral Transduction
The retroviral vectors were derived from the bicistronic Moloney murine leukemia virus pREV-HW3 expressing placental alkaline phosphatase (plap) and neomycin phosphotransferase (neoR) [30]. Details of virus construction and transduction are given elsewhere ([24, 31, 32]; supplemental online Methods).

Proliferation Assay, Immunocytochemical Analysis, and Western Blot Analysis
Detailed protocols are given in the supplemental online Methods.

In Vitro Migration Assay
Cell migration in vitro was measured using a scratch wound healing assay. STX/PSA-overexpressing and enhanced green fluorescent protein (EGFP)-expressing control ESGPs were plated on 3.5-cm uncoated cell culture dishes (8 x 105 cells per dish). After 24 hours, a small area was disrupted by scratching a line through the subconfluent cell monolayer using a sterile pipette tip, resulting in a gap approximately 1 mm wide. Immediately after scratching, the cells were washed with medium. Repopulation of the scratch was monitored after 24 hours. Cells were fixed and stained for PSA and 4,6-diamidino-2-phenylindole (DAPI), and cell migration was evaluated by counting the cells that had migrated into the scratched area (migrating cells were counted using a x10 Zeiss objective (Carl Zeiss, Jena, Germany, http://www.zeiss.com) that allowed cells to be quantified between the wound edges). For control experiments STX-transduced cells were pretreated with the enzyme endoneuraminidase (EndoN; kindly provided by G. Rougon) for 5 hours to remove PSA from NCAM before the cells were exposed to the scratch assay. Data were expressed as mean ± SEM of different experiments, with at least three scratches per experiment.

Slice Cultures
Preparation of and transplantation into entorhinal-hippocampal and cerebellar myelin-deficient rat slice cultures were performed as described [33, 34] and are presented in the supplemental online Methods and supplemental online Figure 1.

Chemotaxis Assay
Chemotaxis was assayed using 6.5-mm polycarbonate transwell filter inserts with an 8-µm pore size (Millipore, Billerica, MA, http://www.millipore.com). Cells (1.5 x 105 per insert) were plated onto the polycarbonate filter in culture medium supplemented with FGF2 and EGF until the migration assay to prevent premature cell differentiation. To analyze chemotactic signals derived from the adult rat brain, the brains were dissected in ice-cold phosphate-buffered saline and sectioned sagittally (500 µm) using a tissue chopper or vibratome. Tissue explants from SVZ, olfactory bulb, hippocampus, and cerebellum were isolated with a biopsy punch (2-mm diameter; Stiefel Laboratorium, Offenbach, Germany, http://www.stiefel.com). To distinguish between directed migration (chemotaxis) and random movement (chemokinesis), tissue slices of equal sizes were placed either in both the top and bottom wells (gradient-independent) or in the bottom well only (gradient-dependent). Similarly, PDGF, FGF2, and brain-derived neurotrophic factor (BDNF) (50 ng/ml) were added to the bottom well only or to both wells to evaluate potential chemotactic responses to these molecules. In experiments using EndoN, cells were pretreated with the enzyme (1:600) for 4–5 hours, and the same concentration of the enzyme was also added to the top and the bottom well during the migration assay to prevent the recovery of PSA. In experiments using the pharmacological pathway inhibitors, cells were untreated (vehicle control) or treated with the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin (1 µM) and LY294002 (50 µM) and the mitogen-activated protein kinase (MAPK) inhibitor PD98059 (50 µM) (Calbiochem, San Diego, http://www.emdbiosciences.com) 30 minutes before and during the incubation with PDGF, FGF2, or BDNF. Twelve hours later, cells on the top of the filter were scraped, and the remaining cells on the bottom side of the filter were fixed, stained with DAPI, and counted under the fluorescence microscope. Twenty microscopic fields were counted at x40 magnification to determine mean cell numbers and standard deviation. The ratio of transmigrated cells after single-well versus two-well exposure to the brain explants/factors is a measure of directed chemotaxis ("chemotactic index"; supplemental online Fig. 2). Data were based on at least three independent experiments, which were analyzed for statistical significance using Student's t test.

Cell Transplantation, Tissue Processing, and Analysis
Details for cell transplantation, tissue processing, and analysis have been described previously [35] and are presented in the supplemental online Methods.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Generation of PSA-Overexpressing ESGPs Using Retroviral Gene Transfer of the Polysialyltransferase STX
We have previously shown that glial precursors with the potential to differentiate into astrocytes and oligodendrocytes can be reliably obtained from ES cells. To that end, pan-neural precursors derived from plated embryoid bodies are expanded in FGF2 (ESNPFGF2·PROL) [29] and then propagated in FGF2/EGF to promote the generation of ESGPs (ESGPFGF2·EGF·PROL; Fig. 1A; details given in Materials and Methods). Further proliferation in FGF2/PDGF can be used to enrich for myelinating oligodendrocytes (ESGPFGF2·PDGF·PROL) [28]. We first examined PSA expression of ESNPs and ESGPs before and after differentiation following a 4-day growth factor withdrawal. PSA immunofluorescence (Fig. 1B) and Western blot (Fig. 1C) analyses both showed that proliferating ES cell-derived precursors express comparatively low levels of PSA. Indeed, only 16% ± 3% of ESGPFGF2·EGF·PROL (Fig. 1A, stage 2a) and 10% ± 1% of ESGPFGF2·PDGF·PROL (Fig. 1A, stage 3a) were found to exhibit detectable PSA immunoreactivity, whereas PSA expression after 4 days of differentiation (Fig. 1A, stages 2b and 3b) was 2–3-fold higher (Fig. 1B).

To increase the level of precursor cell polysialylation, ESGPs growing in the presence of FGF2 and EGF were infected with a retrovirus expressing the PSA-synthesizing enzyme STX. Control populations were generated by transduction with EGFP-expressing or enhanced cyan fluorescent protein (ECFP)-expressing virus, and transduced cells were selected by neomycin resistance. Immunofluorescence analysis demonstrated that 68% ± 10% of STX-transduced ESGPFGF2·EGF·PROL were expressing PSA, compared with 12% ± 3% of control ESGPs (Fig. 1D,1E). STX transduction not only increased the number of PSA-positive cells but also amplified the immunocytochemical signal in positive cells, suggesting that overexpression of the enzyme leads to an overall increase in polysialylation of NCAM. Quantitative immunofluorescence (Fig. 1E) and Western blot analysis (Fig. 1F) showed that PSA overexpression of STX-transduced populations was maintained during proliferation of the ESGPs (ESGPFGF2·EGF·PROL and ESGPFGF2·PDGF·PROL; Fig. 1A, stages 2a, 3a) and following differentiation (ESGPFGF2·EGF·DIFF and ESGPFGF2·PDGF·DIFF; Fig. 1A, stages 2b, 3b). Overall, the number of PSA-positive cells in the STX-transduced population ranged between 68% and 73%. In contrast, EGFP-transduced control cells exhibited much lower levels of PSA, with an increase upon differentiation.

Overexpression of PSA Does Not Interfere with ESGP Differentiation into Astrocytes and Oligodendrocytes
The results of several studies suggest that polysialylation influences neural precursor cell differentiation both in vitro and in vivo [36–39]. When CNS neural precursors are engineered to overexpress the polysialyltransferase PST, differentiation into mature oligodendrocytes is delayed in vitro and in vivo, most likely because of the time required for downregulation of the PST transgene [24]. In contrast, no effect on differentiation was observed with STX-overexpressing Schwann cells [26]. To determine whether PSA overexpression influences the potential of ESGPs to differentiate into oligodendrocytes and astrocytes, we analyzed expression of the astrocytic intermediate filament glial fibrillary acidic protein (GFAP) and the oligodendroglial marker 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) by both immunofluorescence and Western blot analysis. Four days after growth factor withdrawal, STX-transduced ESGPFGF2·PDGF·DIFF (Fig. 1A, stage 3b) had generated 62% ± 2% GFAP-positive and 28% ± 1% CNP-immunoreactive cells. These values equaled those observed in the EGFP-transduced control cells (60% ± 4% and 25% ± 1%, respectively; Fig. 2A). Similarly, STX-and EGFP-transduced ESGPFGF2·EGF·DIFF and ESGPFGF2·PDGF·PROL (Fig. 1A, stages 2b, 3a) showed no significant differences in the number of GFAP- and CNP-positive cells (Fig. 2A). In general, the percentage of astrocytes generated was very similar in ESGPFGF2·EGF·DIFF and ESGPFGF2·PDGF·DIFF cultures, whereas both STX-transfected and control cells generated more oligodendrocytes after propagation in PDGF/FGF2 (Fig. 2A, compare stages 2b and 3b), a growth factor combination known to enhance oligodendrocyte progenitor proliferation. Western blot analysis confirmed the comparable astrocyte and oligodendrocyte formation by STX- and EGFP-transduced cells at the individual in vitro differentiation stages (Fig. 2B). These data indicate that PSA overexpression has no overt effect on the astrocytic and oligodendroglial differentiation potential of ESGPs.


Figure 2
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  		Figure 2. Overexpression of polysialic acid does not affect lineage-specific differentiation of ESGPs into astrocytes and oligodendrocytes. STX- and EGFP-transduced ESGPFGF2·EGF·PROL were induced to differentiate into astrocytes and oligodendrocytes by growth factor withdrawal (ESGPFGF2·EGF·DIFF) or further proliferated in the presence of FGF2 and PDGF (ESGPFGF2·PDGF·PROL) followed by growth factor withdrawal-induced differentiation (ESGPFGF2·PDGF·DIFF). Lineage-specific differentiation was determined by quantitative immunofluorescence (A) and Western blot analysis (B) with antibodies to GFAP and CNP. Quantitative results are based on at least three independent experiments and expressed as mean ± SEM. Abbreviations: CNP, 2'3'-cyclic nucleotide 3'-phosphodiesterase; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; ESGP, embryonic stem cell-derived glial precursor; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; PDGF, platelet-derived growth factor.

 
As the expression of PSA on oligodendrocytes might interfere with myelination [24], we examined whether PSA overexpression disrupts myelination by STX-transduced ESGPFGF2·PDGF·PROL in organotypic slice cultures derived from the cerebellum of P9 myelin-deficient (md) rats. Fourteen days after deposition on the slices, the donor cells were identified with an antibody to proteolipid protein (PLP), a myelin protein deficient in the md rat. The STX-transduced donor cells readily contacted host axons and enwrapped them with PLP-positive sheaths (supplemental online Fig. 3). Although additional studies are required to fully characterize the differentiation potential in vivo, these data indicate that PSA-overexpressing ESGPs can in principle generate myelinating oligodendrocytes.

Enhanced Migration of PSA-Overexpressing ESGPs in Monolayer and Slice Cultures
To study the migratory properties of the PSA-overexpressing glial precursors (ESGPFGF2·EGF·PROL; Fig. 1A, stage 2a), we first performed a scratch assay. After a 24-hour incubation period, the cells were fixed, and the number of cells that had invaded the gap created by the scratch was counted. STX-transduced cells frequently populated the entire scratch area, whereas only a few EGFP-transduced cells had bridged the gap (Fig. 3A,3B). When STX-transduced cells were pretreated with EndoN, an enzyme that removes PSA from NCAM, cell motility was reduced to the level of EGFP-transduced cells (Fig. 3C). Overall, we observed a fivefold increase in the migration capacity of STX/PSA-overexpressing ESGPs (Fig. 3D). As differences in repopulation of the gap could, in principle, also be due to differences in cell proliferation, we labeled with 5-bromo-2'-deoxyuridine (BrdU) for 8, 16, and 24 hours and found closely similar levels of BrdU-positive cells in wild-type, EGFP-transduced, and STX-transduced populations (Fig. 3E). Thus, PSA overexpression does not affect ESGP proliferation but enhances the migration of ESGPs in monolayer culture.


Figure 3
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  		Figure 3. Enhanced migration of STX-transduced ESGPs in monolayer cultures (scratch assay). (A–C): Scratches (1 mm) were generated in subconfluent monolayers of STX- and EGFP-transduced ESGPFGF2·EGF·PROL. After 24 h, fixed cells were immunostained with anti-polysialic acid antibody and 4,6-diamidino-2-phenylindole (DAPI). Images show the scratch area of EGFP-transduced (A) and STX-transduced (B) ESGPs. (C): STX-transduced ESGPs were pretreated with EndoN for 5 h. Note that only STX-transduced cells had completely bridged the gap created by the scratch. Shown are representative data taken from at least three independent experiments. (D): Migration was quantified by counting the DAPI-positive cells that had migrated into the damaged area. *, p < .05 compared with EGFP control cells (Student's t test). (E): No differences in proliferation were noted among wt, STX-transduced, and EGFP-transduced ESGPs as determined by BrdU incorporation. Cells were exposed to BrdU for the indicated time periods. Quantitative results in (D) and (E) are based on at least three independent experiments and expressed as mean ± SEM. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; DAPI, 4',6'-diamidino-2'-phenylindole; EGFP, enhanced green fluorescence protein; EndoN, endoneuraminidase; ESGP, ES cell-derived glial precursor; h, hour(s); wt, wild-type.

 
Although this study focuses on ESGPs, we became, at this point, interested in whether ESNPs would respond similarly to a PSA overexpression. Indeed, PSA-overexpressing ESNPs exhibited an enhanced migratory potential in the scratch assay without a change in cell proliferation (supplemental online Fig. 4A–4C). PSA-overexpressing ESNPs also showed a slightly accelerated neuronal differentiation, which was not, however, associated with a general change in lineage commitment (supplemental online Fig. 4D–4G).

To determine whether the migration of PSA-overexpressing ESGPs is also enhanced in CNS tissue, ESGPFGF2·EGF·PROL; Figure 1A, stage 2a) were double-infected with the STX and ECFP virus or the ECFP virus alone before being deposited on the surface of acute hippocampal slices made from P10 SV129 mice. Twenty-four and 48 hours after transplantation, 20-µm cryosections were cut through the slices to detect the location of ECFP fluorescent cells in both groups. After 24 hours, the majority of ECFP-infected cells had invaded up to 40 µm of the slices, and only occasional cells were found at 60 and 80 µm depths. In contrast, STX/ECFP-infected cells had penetrated the slice up to a depth of 120 µm (supplemental online Fig. 1). This difference was maintained after a 48-hour incubation, where STX-transduced cells had migrated up to 180 µm in the slice compared with 120 µm observed with control cells. These in vitro transplantation data demonstrate that STX-transduced ESGPs migrate deeper and faster into acute hippocampal slices.

PSA-NCAM Modulates the Response of ESGPs to Defined Chemoattractants
To determine whether overexpression of PSA induces merely random cell motility or affects directional migration of ESGPs, we performed an in vitro transfilter migration assay. STX- or ECFP-infected ESGPs were exposed to gradients of three factors known to provide directional cues for neural cell migration, that is, PDGF, FGF2, and BDNF [25, 40–45]. Directed migration or chemotaxis was measured by placing these factors in the bottom well only and distinguished from chemokinesis by adding the factors to both the upper and lower wells of the chamber, thereby eliminating a chemical gradient (supplemental online Methods; supplemental online Fig. 2). Addition of PDGF, FGF2, and BDNF to the lower compartment clearly increased directed migration of PSA-overexpressing cells compared with ECFP-transduced control cells (Fig. 4A). In contrast, migration was not significantly affected when the factors were placed in both the top and bottom wells, indicating a specific chemotactic response of PSA-overexpressing cells. To determine whether the chemotactic effect is PSA-specific, the cells were treated with EndoN, which significantly reduced the ability of STX-transduced cells to migrate along a gradient of PDGF, FGF2, and BDNF. Notably, no significant differences in chemotaxis were observed between STX- and ECFP-transduced cells upon EndoN treatment (Fig. 4A).


Figure 4
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  		Figure 4. PSA-NCAM enhances the response of ESGPs to chemotactic cues in vitro. (A): A transfilter chemotaxis assay was used to evaluate the effect of PSA overexpression on migration in response to concentration gradients of putative chemoattractive factors. STX-transduced precursors exhibited increased chemotaxis toward PDGF, FGF2, and BDNF compared with controls. The effect was abolished by EndoN, indicating that the enhanced migration is PSA-specific. (B): The phosphatidylinositol 3'-kinase inhibitors LY294002 (50 µM) and wortmannin (1 µM) both suppressed the PSA-mediated chemotactic responses, whereas inhibition of the mitogen-activated protein kinase signaling by PD98059 (50 µM) had no effect on transfilter migration. (C): Using transwell chambers, STX- and ECFP-transduced ESGPs were cocultured with tissue explants from the SVZ, OB, HC, and CB. STX-transduced cells showed a clearly enhanced chemotactic response to all four brain regions. Data are expressed as net chemotactic index already corrected for chemokinetic movements (details given in Materials and Methods) and represent mean ± SEM representative of one of five independent experiments. *, p < .05; **, p < .01 (Student's t test). Abbreviations: BDNF, brain-derived neurotrophic factor; CB, cerebellum; ECFP, enhanced cyan fluorescent protein; Endo N, endoneuraminidase; ESGP, ES cell-derived glial precursor; FGF, fibroblast growth factor; HC, hippocampus; OB, olfactory bulb; PDGF, platelet-derived growth factor; PSA-NCAM, polysialic acid neural cell adhesion molecule; SVZ, subventricular zone.

 
The enhanced chemotactic responses of PSA-expressing cells might be mediated by specific signal transduction pathways [46–48], and growing evidence indicates that the preferential activation of phosphatidylinositol 3'-kinase (PI3K) is important for directional movement of a wide range of motile cell types [49–52]. PDGF, FGF2, and BDNF activate specific transmembrane receptor tyrosine kinases (i.e., PDGF and FGF receptors and TrkB) to initiate distinct signal transduction pathways via mitogen-activated protein kinases (MAPKs) and PI3K [53]. PI3K can also be induced by focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, which in turn can be phosphorylated upon binding to the activated NCAM-Fyn complex [54, 55]. To investigate the potential roles of MAPK and PI3K pathways in PSA-mediated chemotactic responses, we tested the effect of the pharmacological PI3K inhibitors LY294002 and wortmannin and the MAPK inhibitor PD9805 in the transwell migration assay. Although inhibition of the MAPK pathway had little effect on transfilter migration, both PI3K inhibitors suppressed the directional movement of ESGPs in response to PDGF, FGF2, and BDNF in a dose-dependent manner (Fig. 4B; supplemental online Fig. 5), supporting an involvement of PI3K but not MAPK activity in this process. Together, these data indicate that PSA not only increases cell motility of ESGPs but promotes directional migration in response to chemoattractants and that this effect depends critically on PI3K activity.

PSA Modulates the Sensitivity of ESGPs Toward Soluble Cues Within Different Brain Regions
In the normal and pathological CNS, neural cell migration can be modulated by a large number of environmental factors, including extracellular matrix components, cell adhesion molecules, chemoattractants, and chemorepellents [56, 57]. The broadly enhanced chemotactic responses of PSA-overexpressing ESGPs in vitro (Fig. 4A) suggest that PSA-NCAM may also influence the readout of migratory cues in different CNS regions. To approach this question, explants from adult rat SVZ, olfactory bulb, hippocampus, and cerebellum were tested for their ability to stimulate migration of STX- or ECFP-transduced ESGPs in an in vitro transfilter migration assay. Interestingly, STX-transduced cells showed enhanced chemotaxis toward all four brain regions compared with control cells. This effect was particularly pronounced in response to SVZ and olfactory bulb explants (Fig. 4C). These observations suggest that an increase in NCAM polysialylation also enhances the response of ESGPs toward putative guidance cues within different CNS regions.

PSA Overexpression Modulates Migration Routes of Transplanted ESGPs In Vivo
To explore whether overexpression of PSA-NCAM can modulate the migration of transplanted ESGPs in vivo, STX- and ECFP-transduced ESGPFGF2·EGF·PROL were injected into the anterior part of the adult rat striatum. Fourteen days after transplantation, the recipient animals were sacrificed, and the transplanted cells were visualized using DNA in situ hybridization with a mouse-specific probe. The location of labeled cells of the two experimental groups in coronal brain sections is represented in Figure 5A. The ECFP-expressing control cells were predominantly found in a dense core at or near the implantation site (Fig. 5B,5F). Occasionally, a slight spread toward the corpus callosum could be observed, possibly reflecting dispersion alongside the injection tract. In contrast, the STX/PSA-overexpressing glial precursors were more widely dispersed and showed a pronounced migration toward the lateral ventricle (Fig. 5C,5D,5G,5H), where some of them accumulated in small clusters in a subependymal location (Fig. 5C,5D,5H).


Figure 5
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  		Figure 5. PSA overexpression modifies the migration pattern of ESGPs in vivo. STX- and ECFP-transduced ESGPFGF2·EGF·PROL were transplanted into the striatum of adult rats. Fourteen days after transplantation, donor cells were identified by ISH with a mouse-specific DNA probe, and the distribution of transplanted cells was analyzed in coronal (A–D) and sagittal (E–H) sections. STX-transduced cells migrated away from the injection site and toward the ventricle (C, D, G, H), where they formed occasional clusters in the subependyma (C, D, H). In contrast, ECFP-transduced control cells remained largely restricted to the striatal injection site (B, F). (F–H): Superimpositions of dark-field images and fluorescence ISH; in (G), the inset shows the hybridized section double-labeled with an antibody to PSA. The schematic drawings ([A], coronal plane; [E], sagittal plane) illustrate the implant site (asterisks) and the distribution of STX- or ECFP-transduced ESGPs. For quantification the host tissue in sagittal sections was divided into territories anterior (area I) and posterior (area II) to the anterior commissure (E). Numbers show the percentages of the donor cells found in each territory. Scale bar = 500 µm. Abbreviations: CC, corpus callosum; ECFP, enhanced cyan fluorescent protein; ESGP, ES cell-derived glial precursor; ISH, in situ hybridization; PSA, polysialic acid.

 
To quantify the migration behavior, we divided the target area in sagittal sections into two territories (Fig. 5E). Area I comprised the more anterior part of the striatum and the overlaying corpus callosum; area II included the posterior aspect of the striatum and the adjacent SVZ. Whereas ECFP-expressing control cells were exclusively located in area I, 43% ± 5% of STX-transduced glial precursors had populated area II. STX-transduced cells were frequently arranged in chains of tightly associated cells (Fig. 5G, inset; supplemental online Fig. 6A), reminiscent of the "chain migration" of endogenous PSA-NCAM-positive progenitors within the RMS. However, we found no donor cells in the RMS or the olfactory bulb. Immunohistochemical characterization of incorporated cells detected by in situ hybridization revealed that the STX-transduced ESGPs can generate GFAP-positive astrocytes and CNP-positive oligodendrocytes (supplemental online Fig. 6B, 6C), whereas double-labeled β-III tubulin-positive neuronal cells were not detected. Taken together, these data indicate that PSA-NCAM not only enhances cell migration but also modifies the migration routes of ESGPs in vivo.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The results of this study demonstrate that an increased polysialylation of NCAM in ESGPs enhances their response to chemotactic factors and guidance cues within different brain regions, thereby promoting directional migration. Specifically, we show that cells transduced with the polysialyltransferase STX and exposed to defined chemoattractants or brain explants show enhanced chemotaxis and that this effect is (a) distinct from a general increase in cell motility or chemokinesis and (b) dependent on PI3K activity. In vivo, PSA-overexpressing but not control cells transplanted into the striatum display a targeted migration toward the ventricle, suggesting that polysialylation may also be instrumental in the readout of migration guidance cues in vivo. Together, these data indicate that PSA plays an important role in modulating the sensitivity of neural precursor cells to chemoattractive cues in vitro and in vivo.

Sustained Overexpression of PSA Does Not Interfere with Lineage Specification
During normal development, glial precursors such as oligodendrocyte progenitors express PSA-NCAM, whereas mature oligodendrocytes are PSA-negative [23, 36, 58]. This decrease in PSA-NCAM expression with increased differentiation has been shown to be caused by a downregulation of polysialyltransferases [59]. The low level of PSA observed in ESGPs might indicate that these in vitro-derived glial precursors are at an earlier lineage stage or have not yet acquired all the characteristics of postnatal glial precursors [60]. When these cells were engineered to overexpress PSA, differentiation into astrocytes and oligodendrocytes was not perturbed. Similarly, overexpression of PSA had no effect on the lineage diversification of ESNPs into neurons and glia (supplemental online Fig. 4). The potential for astrocytic and oligodendroglial differentiation of ESGPs was also preserved after in vivo transplantation into the adult rat striatum. Moreover, STX-transduced cells cocultured with cerebellar slice cultures derived from myelin-deficient rats incorporated into the host tissue and synthesized PLP-positive myelin sheaths around axons. This is in keeping with the ability of CNS neural precursors overexpressing PSA to generate myelin-forming oligodendrocytes after transplantation into shiverer mice and of Schwann cells overexpressing PSA to myelinate cerebellar axons in slices [24, 26]. From these observations, we conclude that overexpression of PSA in ESGPs and precursors of myelin-forming cells causes no major interference with their ability to generate myelinating oligodendrocytes and astrocytes.

PSA Overexpression Enhances Chemotaxis In Vitro
Negatively charged PSA chains are thought to reduce adhesion forces between cells, allowing dynamic changes in membrane contacts that favor migratory processes [6, 61]. We show here that PSA-overexpressing ESGPs more rapidly bridge a gap created in monolayers than EGFP-transduced controls. STX-transduced ESGPs also migrated faster and deeper into acutely prepared brain slices. These observations are in agreement with previous reports showing that PSA-NCAM is involved in migration of oligodendrocyte precursor cells in response to injury of glial monolayers [62] and that overexpression of PSA in Schwann cells enhances their migratory potential in a scratch wound assay and following transplantation into slice cultures of the neonatal brain [26]. Although the mechanisms underlying PSA-mediated migration enhancement have remained largely elusive, our transfilter assays demonstrate that PSA can modulate the ability of cells to sense and respond to concentration gradients of the chemoattractants FGF2, PDGF, and BDNF, that is, candidate factors that are known to stimulate the directed migration of brain-derived neural precursors (PDGF and BDNF) [40, 43–45], as well as oligodendrocyte progenitor cells (PDGF and FGF2) [41, 42]. Such a role of PSA in modulating growth factor-mediated signaling is consistent with previous studies showing that PSA-NCAM modulates the effects of BDNF and ciliary neurotrophic factor on neuronal survival, differentiation, and function [63–65], as well as the migratory response of cultured oligodendrocyte progenitors to PDGF [25].

The signaling pathways and molecules that have been implicated in directional sensing and chemotaxis include the activation of G protein receptor systems, PI3K, the lipid phosphatase PTEN, and the Rho family of small GTPases [51, 52, 66]. Our in vitro migration data obtained in the presence of the general PI3K inhibitors wortmannin and LY294002 support the notion that PI3K is required for the chemotactic responses of PSA-overexpressing ESGPs. The MAPK inhibitor PD98059 had little effect, suggesting that MAPKs are not directly involved in this process.

Different signaling pathways may be involved in PI3K activation during NCAM-dependent chemotaxis in response to growth factor and neurotophin receptor activation (Fig. 6). The candidate factors used in this study (PDGF, FGF2, and BDNF) activate specific receptor tyrosine kinases (i.e., FGF and PDGF receptors and TrkB), and PI3K is known to be activated by interaction with phosphotyrosine residues of activated receptors either directly or via adaptor proteins [67]. Direct binding of PI3K to activated Ras further stimulates PI3K activity. In addition, homophilic and heterophilic NCAM interactions have been shown to activate intracellular signal transduction pathways [68, 69]. Upon NCAM ligation, the nonreceptor tyrosine kinases Fyn and FAK become activated, resulting in Ras-MAPK signaling [70, 71]. Recently, PI3K has been demonstrated to be a downstream transducer of NCAM-mediated signaling [72]. Moreover, heterophilic cis-interactions between NCAM and growth factor/neurotrophin receptors have been described (e.g., between NCAM and FGF receptor or glial cell line-derived neurotrophic factor family receptor {alpha}), which result in different modes of signaling cooperation [73, 74]. Thus, both signals triggered by the individual receptors (i.e., FGF and PDGF receptors and TrkB) and those induced by NCAM engagement could be responsible for the enhanced PI3K-dependent chemosensitivity to PDGF, FGF2, and BDNF observed in this study. Furthermore, an additive stimulation of both signaling cascades, possibly mediated by physical interaction between NCAM and the receptors, could converge on PI3K activation (Fig. 6).


Figure 6
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  		Figure 6. Potential signaling pathways involved in PI3K activation during PSA-NCAM-dependent chemotaxis. Details given in text. Abbreviations: BDNF, brain-derived neurotrophic factor; FGFR, fibroblast growth factor receptor; NCAM, neural cell adhesion molecule; P, phosphorylated (activated) tyrosine kinase receptor; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3'-kinase; PSA, polysialic acid.

 
There are several possibilities as to how PSA-NCAM may sterically enhance the impact of chemoattractants on these signaling pathways. Long PSA chains, possibly associated in filament bundles [64], could serve as tentacles, which trap chemoattractive ligands and guide their movement toward their receptors. The PSA moiety could also delay ligand diffusion away from the cell membrane, thereby maintaining a high local concentration for receptor activation.

PSA Modulates Sensitivity to Chemotactic Cues in CNS Tissue
Remarkably, explants from different brain regions, also, stimulated a much stronger, gradient-dependent chemotactic response of PSA-overexpressing cells than of ECFP-transduced cells. This effect was particularly pronounced for explants derived from the olfactory bulb and the subventricular zone. To explore whether this phenomenon also holds true in vivo, we transplanted PSA-overexpressing ESGPs and control cells into the striatum of adult rats. Whereas ECFP-transduced control cells remained mostly confined to the injection site, STX-transduced ESGPs showed a directed movement toward the lateral ventricle and often formed streams of closely aligned cells, resembling the chain migration of PSA-NCAM-expressing SVZ precursors in the RMS [75–77]. In the current study, no STX-transduced glial precursors were found within the RMS, probably because glial precursors tend to diverge/avoid the RMS milieu favorable to neuronal migration [78]. Previous studies have shown that PSA-NCAM overexpressing neural precursors transplanted into the early trunk neural crest migration pathway of chick embryos change their migration from a dorsal to a ventral route [24]. Our findings extend these observations in that they provide the first evidence that PSA modulates and promotes the directional migration of transplanted glial precursors in the adult brain. Moreover, our in vitro studies suggest that these changes in directional migration may be due to a central role of PSA-NCAM in sensitizing migrating glial precursors toward chemotactic cues. Additional studies will have to address whether PSA has similar effects on the sensitization of neural precursors in general. In addition, long-term studies are required to thoroughly assess the potential tumorigenicity and immunogenicity of PSA-overexpressing transplants. Elucidation of the molecular mechanisms mediating PSA-dependent responses to migratory cues might enable new strategies for the guided migration of endogenous and transplanted precursors in neural repair.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by European Union Project QLG3-CT-2000-00911, the Hertie Foundation, BMBF (Bundesministerium für Bildung und Forschung) Grant 01GN0502, and the Institute for Multiple Sclerosis Research in Göttingen. We thank Geneviève Rougon for providing the PSA antibody and endoneuraminidase, as well as for helpful comments. We acknowledge I. Griffith for providing the PLP antibody, M. Fukuda for providing human STX cDNA, and Ian Duncan for providing the myelin-deficient rats. M.D.-D. is currently affiliated with the National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center; Bethesda, MD; I. F. is currently affiliated with INRA, Unité de Physiologie de la Reproduction et des Comportements, Nouzilly, France.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Zhang SC. Neural subtype specification from embryonic stem cells. Brain Pathol 2006;16:132–142.[CrossRef][Medline]

  2. Bruses JL, Rutishauser U. Roles, regulation, and mechanism of polysialic acid function during neural development. Biochimie 2001;83:635–643.[Medline]

  3. Kiss JZ, Muller D. Contribution of the neural cell adhesion molecule to neuronal and synaptic plasticity. Rev Neurosci 2001;12:297–310.[Medline]

  4. El Maarouf A, Petridis AK, Rutishauser U. Use of polysialic acid in repair of the central nervous system. Proc Natl Acad Sci U S A 2006;103:16989–16994.[Abstract/Free Full Text]

  5. Rutishauser U, Acheson A, Hall AK et al. The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions. Science 1988;240:53–57.[Abstract/Free Full Text]

  6. Rutishauser U, Landmesser L. Polysialic acid in the vertebrate nervous system: A promoter of plasticity in cell-cell interactions. Trends Neurosci 1996;19:422–427.[Medline]

  7. Seki T, Arai Y. Distribution and possible roles of the highly polysialylated neural cell adhesion molecule (NCAM-H) in the developing and adult central nervous system. Neurosci Res 1993;17:265–290.[CrossRef][Medline]

  8. Figarella-Branger DF, Durbec PL, Rougon GN. Differential spectrum of expression of neural cell adhesion molecule isoforms and L1 adhesion molecules on human neuroectodermal tumors. Cancer Res 1990;50:6364–6370.[Abstract/Free Full Text]

  9. Fukuda M. Possible roles of tumor-associated carbohydrate antigens. Cancer Res 1996;56:2237–2244.[Abstract/Free Full Text]

  10. Gluer S, Schelp C, von Schweinitz D et al. Polysialylated neural cell adhesion molecule in childhood rhabdomyosarcoma. Pediatr Res 1998;43:145–147.[Medline]

  11. Daniel L, Durbec P, Gautherot E et al. A nude mice model of human rhabdomyosarcoma lung metastases for evaluating the role of polysialic acids in the metastatic process. Oncogene 2001;20:997–1004.[CrossRef][Medline]

  12. Daniel L, Trouillas J, Renaud W et al. Polysialylated-neural cell adhesion molecule expression in rat pituitary transplantable tumors (spontaneous mammotropic transplantable tumor in Wistar-Furth rats) is related to growth rate and malignancy. Cancer Res 2000;60:80–85.[Abstract/Free Full Text]

  13. Gluer S, Schelp C, Madry N et al. Serum polysialylated neural cell adhesion molecule in childhood neuroblastoma. Br J Cancer 1998;78:106–110.[Medline]

  14. Tanaka F, Otake Y, Nakagawa T et al. Prognostic significance of polysialic acid expression in resected non-small cell lung cancer. Cancer Res 2001;61:1666–1670.[Abstract/Free Full Text]

  15. Angata K, Fukuda M. Polysialyltransferases: Major players in polysialic acid synthesis on the neural cell adhesion molecule. Biochimie 2003;85:195–206.[Medline]

  16. Ong E, Nakayama J, Angata K et al. Developmental regulation of polysialic acid synthesis in mouse directed by two polysialyltransferases, PST and STX. Glycobiology 1998;8:415–424.[Abstract/Free Full Text]

  17. Weinhold B, Seidenfaden R, Rockle I et al. Genetic ablation of polysialic acid causes severe neurodevelopmental defects rescued by deletion of the neural cell adhesion molecule. J Biol Chem 2005;280:42971–42977.[Abstract/Free Full Text]

  18. Cremer H, Lange R, Christoph A et al. Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning. Nature 1994;367:455–459.[CrossRef][Medline]

  19. Ono K, Tomasiewicz H, Magnuson T et al. N-CAM mutation inhibits tangential neuronal migration and is phenocopied by enzymatic removal of polysialic acid. Neuron 1994;13:595–609.[CrossRef][Medline]

  20. Chazal G, Durbec P, Jankovski A et al. Consequences of neural cell adhesion molecule deficiency on cell migration in the rostral migratory stream of the mouse. J Neurosci 2000;20:1446–1457.[Abstract/Free Full Text]

  21. Nait-Oumesmar B, Decker L, Lachapelle F et al. Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur J Neurosci 1999;11:4357–4366.[CrossRef][Medline]

  22. Picard-Riera N, Decker L, Delarasse C et al. Experimental autoimmune encephalomyelitis mobilizes neural progenitors from the subventricular zone to undergo oligodendrogenesis in adult mice. Proc Natl Acad Sci U S A 2002;99:13211–13216.[Abstract/Free Full Text]

  23. Decker L, Picard-Riera N, Lachapelle F et al. Growth factor treatment promotes mobilization of young but not aged adult subventricular zone precursors in response to demyelination. J Neurosci Res 2002;69:763–771.[CrossRef][Medline]

  24. Franceschini I, Vitry S, Padilla F et al. Migrating and myelinating potential of neural precursors engineered to overexpress PSA-NCAM. Mol Cell Neurosci 2004;27:151–162.[CrossRef][Medline]

  25. Zhang H, Vutskits L, Calaora V et al. A role for the polysialic acid-neural cell adhesion molecule in PDGF-induced chemotaxis of oligodendrocyte precursor cells. J Cell Sci 2004;117:93–103.[Abstract/Free Full Text]

  26. Lavdas AA, Franceschini I, Dubois-Dalcq M et al. Schwann cells genetically engineered to express PSA show enhanced migratory potential without impairment of their myelinating ability in vitro. Glia 2006;53:868–878.[CrossRef][Medline]

  27. Swiatek PJ, Gridley T. Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev 1993;7:2071–2084.[Abstract/Free Full Text]

  28. Brustle O, Jones KN, Learish RD et al. Embryonic stem cell-derived glial precursors: A source of myelinating transplants. Science 1999;285:754–756.[Abstract/Free Full Text]

  29. Okabe S, Forsberg-Nilsson K, Spiro AC et al. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech Dev 1996;59:89–102.[CrossRef][Medline]

  30. Lopez-Lastra M, Gabus C, Darlix JL. Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors. Hum Gene Ther 1997;8:1855–1865.[Medline]

  31. Franceschini IA, Feigenbaum-Lacombe V, Casanova P et al. Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector. J Neurosci Res 2001;65:208–219.[CrossRef][Medline]

  32. Perez-Bouza A, Glaser T, Brustle O. ES cell-derived glial precursors contribute to remyelination in acutely demyelinated spinal cord lesions. Brain Pathol 2005;15:208–216.[Medline]

  33. Ullrich O, Diestel A, Eyupoglu IY et al. Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1. Nat Cell Biol 2001;3:1035–1042.[CrossRef][Medline]

  34. Scheffler B, Schmandt T, Schroder W et al. Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures. Development 2003;130:5533–5541.[Abstract/Free Full Text]

  35. Schmandt T, Glaser T, Brüstle O. Lineage selection and transplantation of ES cell-derived neural precursors. In: Notarianni E, Evans M, eds. Embryonic Stem Cells: A Practical Approach.Oxford, U.K.: Oxford University Press,2006;189–217.

  36. Decker L, Avellana-Adalid V, Nait-Oumesmar B et al. Oligodendrocyte precursor migration and differentiation: Combined effects of PSA residues, growth factors, and substrates. Mol Cell Neurosci 2000;16:422–439.[CrossRef][Medline]

  37. Seidenfaden R, Krauter A, Schertzinger F et al. Polysialic acid directs tumor cell growth by controlling heterophilic neural cell adhesion molecule interactions. Mol Cell Biol 2003;23:5908–5918.[Abstract/Free Full Text]

  38. Petridis AK, El-Maarouf A, Rutishauser U. Polysialic acid regulates cell contact-dependent neuronal differentiation of progenitor cells from the subventricular zone. Dev Dyn 2004;230:675–684.[CrossRef][Medline]

  39. Vutskits L, Gascon E, Zgraggen E et al. The polysialylated neural cell adhesion molecule promotes neurogenesis in vitro. Neurochem Res 2006;31:215–225.[CrossRef][Medline]

  40. Forsberg-Nilsson K, Behar TN, Afrakhte M et al. Platelet-derived growth factor induces chemotaxis of neuroepithelial stem cells. J Neurosci Res 1998;53:521–530.[CrossRef][Medline]

  41. Armstrong RC, Harvath L, Dubois-Dalcq ME. Type 1 astrocytes and oligodendrocyte-type 2 astrocyte glial progenitors migrate toward distinct molecules. J Neurosci Res 1990;27:400–407.[CrossRef][Medline]

  42. Simpson PB, Armstrong RC. Intracellular signals and cytoskeletal elements involved in oligodendrocyte progenitor migration. Glia 1999;26:22–35.[CrossRef][Medline]

  43. Behar TN, Dugich-Djordjevic MM, Li YX et al. Neurotrophins stimulate chemotaxis of embryonic cortical neurons. Eur J Neurosci 1997;9:2561–2570.[CrossRef][Medline]

  44. Lu Q, Sun EE, Klein RS et al. Ephrin-B reverse signaling is mediated by a novel PDZ-RGS protein and selectively inhibits G protein-coupled chemoattraction. Cell 2001;105:69–79.[CrossRef][Medline]

  45. Borghesani PR, Peyrin JM, Klein R et al. BDNF stimulates migration of cerebellar granule cells. Development 2002;129:1435–1442.[Medline]

  46. Van Haastert PJ, Devreotes PN. Chemotaxis: Signalling the way forward. Nat Rev Mol Cell Biol 2004;5:626–634.[CrossRef][Medline]

  47. Firtel RA, Chung CY. The molecular genetics of chemotaxis: Sensing and responding to chemoattractant gradients. Bioessays 2000;22:603–615.[CrossRef][Medline]

  48. Parent CA, Devreotes PN. A cell's sense of direction. Science 1999;284:765–770.[Abstract/Free Full Text]

  49. Sasaki AT, Firtel RA. Regulation of chemotaxis by the orchestrated activation of Ras, PI3K, and TOR. Eur J Cell Biol 2006;85:873–895.[CrossRef][Medline]

  50. Funamoto S, Meili R, Lee S et al. Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 2002;109:611–623.[CrossRef][Medline]

  51. Chung CY, Potikyan G, Firtel RA. Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa. Mol Cell 2001;7:937–947.[CrossRef][Medline]

  52. Chung CY, Funamoto S, Firtel RA. Signaling pathways controlling cell polarity and chemotaxis. Trends Biochem Sci 2001;26:557–566.[CrossRef][Medline]

  53. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–225.[CrossRef][Medline]

  54. Chen HC, Appeddu PA, Isoda H et al. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol Chem 1996;271:26329–26334.[Abstract/Free Full Text]

  55. Beggs HE, Baragona SC, Hemperly JJ et al. NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn). J Biol Chem 1997;272:8310–8319.[Abstract/Free Full Text]

  56. Jarjour AA, Kennedy TE. Oligodendrocyte precursors on the move: Mechanisms directing migration. Neuroscientist 2004;10:99–105.[Abstract/Free Full Text]

  57. Sobeih MM, Corfas G. Extracellular factors that regulate neuronal migration in the central nervous system. Int J Dev Neurosci 2002;20:349–357.[Medline]

  58. Trotter J, Bitter-Suermann D, Schachner M. Differentiation-regulated loss of the polysialylated embryonic form and expression of the different polypeptides of the neural cell adhesion molecule by cultured oligodendrocytes and myelin. J Neurosci Res 1989;22:369–383.[CrossRef][Medline]

  59. Stoykova LI, Beesley JS, Grinspan JB et al. ST8Sia IV mRNA corresponds with the biosynthesis of alpha2,8sialyl polymers but not oligomers in rat oligodendrocytes. J Neurosci Res 2001;66:497–505.[CrossRef][Medline]

  60. Ben-Hur T, Rogister B, Murray K et al. Growth and fate of PSA-NCAM+ precursors of the postnatal brain. J Neurosci 1998;18:5777–5788.[Abstract/Free Full Text]

  61. Yang P, Major D, Rutishauser U. Role of charge and hydration in effects of polysialic acid on molecular interactions on and between cell membranes. J Biol Chem 1994;269:23039–23044.[Abstract/Free Full Text]

  62. Barral-Moran MJ, Calaora V, Vutskits L et al. Oligodendrocyte progenitor migration in response to injury of glial monolayers requires the polysialic neural cell-adhesion molecule. J Neurosci Res 2003;72:679–690.[CrossRef][Medline]

  63. Muller D, Djebbara-Hannas Z, Jourdain P et al. Brain-derived neurotrophic factor restores long-term potentiation in polysialic acid-neural cell adhesion molecule-deficient hippocampus. Proc Natl Acad Sci U S A 2000;97:4315–4320.[Abstract/Free Full Text]

  64. Vutskits L, Djebbara-Hannas Z, Zhang H et al. PSA-NCAM modulates BDNF-dependent survival and differentiation of cortical neurons. Eur J Neurosci 2001;13:1391–1402.[CrossRef][Medline]

  65. Vutskits L, Gascon E, Kiss JZ. Removal of PSA from NCAM affects the survival of magnocellular vasopressin- and oxytocin-producing neurons in organotypic cultures of the paraventricular nucleus. Eur J Neurosci 2003;17:2119–2126.[CrossRef][Medline]

  66. Merlot S, Firtel RA. Leading the way: Directional sensing through phosphatidylinositol 3-kinase and other signaling pathways. J Cell Sci 2003;116:3471–3478.[Abstract/Free Full Text]

  67. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:1655–1657.[Abstract/Free Full Text]

  68. Povlsen GK, Ditlevsen DK, Berezin V et al. Intracellular signaling by the neural cell adhesion molecule. Neurochem Res 2003;28:127–141.[CrossRef][Medline]

  69. Walmod PS, Kolkova K, Berezin V et al. Zippers make signals: NCAM-mediated molecular interactions and signal transduction. Neurochem Res 2004;29:2015–2035.[CrossRef][Medline]

  70. Schmid RS, Graff RD, Schaller MD et al. NCAM stimulates the Ras-MAPK pathway and CREB phosphorylation in neuronal cells. J Neurobiol 1999;38:542–558.[CrossRef][Medline]

  71. Kolkova K, Novitskaya V, Pedersen N et al. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway. J Neurosci 2000;20:2238–2246.[Abstract/Free Full Text]

  72. Ditlevsen DK, Kohler LB, Pedersen MV et al. The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival. J Neurochem 2003;84:546–556.[CrossRef][Medline]

  73. Doherty P, Walsh FS. CAM-FGF receptor interactions: A model for axonal growth. Mol Cell Neurosci 1996;8:99–111.[CrossRef][Medline]

  74. Paratcha G, Ledda F, Ibanez CF. The neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands. Cell 2003;113:867–879.[CrossRef][Medline]

  75. Rousselot P, Lois C, Alvarez-Buylla A. Embryonic (PSA) N-CAM reveals chains of migrating neuroblasts between the lateral ventricle and the olfactory bulb of adult mice. J Comp Neurol 1995;351:51–61.[CrossRef][Medline]

  76. Lois C, Garcia-Verdugo JM, Alvarez-Buylla A. Chain migration of neuronal precursors. Science 1996;271:978–981.[Abstract]

  77. Bonfanti L, Theodosis DT. Expression of polysialylated neural cell adhesion molecule by proliferating cells in the subependymal layer of the adult rat, in its rostral extension and in the olfactory bulb. Neuroscience 1994;62:291–305.[CrossRef][Medline]

  78. Hack MA, Saghatelyan A, de Chevigny A et al. Neuronal fate determinants of adult olfactory bulb neurogenesis. Nat Neurosci 2005;8:865–872.[Medline]




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S. E. Kendall, J. Najbauer, H. F. Johnston, M. Z. Metz, S. Li, M. Bowers, E. Garcia, S. U. Kim, M. E. Barish, K. S. Aboody, et al.
Neural Stem Cell Targeting of Glioma Is Dependent on Phosphoinositide 3-Kinase Signaling
Stem Cells, June 1, 2008; 26(6): 1575 - 1586.
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