Direct-Current Electrical Field Guides Neuronal ... - Wiley Online Library

TRANSLATIONAL AND CLINICAL RESEARCH Direct-Current Electrical Field Guides Neuronal Stem/Progenitor Cell Migration LEI LI,a,b YOUSSEF H. EL-HAYEK,a BAOSONG LIU,a YONGHONG CHEN,a EVERLYNE GOMEZ,a XIAOHUA WU,a,b KE NING,a LIJUN LI,a NING CHANG,a LIANG ZHANG,a ZHENGGUO WANG,b XIANG HU,c QI WANa,d a

Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Ontario, Canada; bResearch Institute of Surgery and Daping Hospital, State Key Laboratory of Trauma, Burns and Combined Injury of China, Chongqing, People’s Republic of China, cBeike Biotech Co., Ltd., Shenzhen, People’s Republic of China; dDepartment of Physiology and Cell Biology, School of Medicine, University of Nevada, Reno, Nevada, USA Key Words. Direct-current electrical fields • Neuronal stem/progenitor cells • Migration • N-Methyl-D-aspartate receptor • GTPase

ABSTRACT Direct-current electrical fields (EFs) promote nerve growth and axon regeneration. We report here that at physiological strengths, EFs guide the migration of neuronal stem/progenitor cells (NSPCs) toward the cathode. EF-directed NSPC migration requires activation of Nmethyl-D-aspartate receptors (NMDARs), which leads to an increased physical association of Rho GTPase Rac1associated signals to the membrane NMDARs and the intracellular actin cytoskeleton. Thus, this study iden-

tifies the EF as a directional guidance cue in controlling NSPC migration and reveals a role of the NMDAR/Rac1/ actin signal transduction pathway in mediating EF-induced NSPC migration. These results suggest that as a safe physical approach in clinical application, EFs may be developed as a practical therapeutic strategy for brain repair by directing NSPC migration to the injured brain regions to replace cell loss. STEM CELLS 2008;26: 2193–2200

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

INTRODUCTION Although neuronal stem/progenitor cell (NSPC) migration is essential for early neuronal development, it is also a crucial process for the functioning of the mature central nervous system (CNS) in both physiological and pathological conditions [1–10]. Pathological insults such as cerebral ischemia not only stimulate increased generation of endogenous NSPCs in the subventricular zone but also induce migration of the NSPCs to the damaged brain regions [9 –13]. However, only a portion of the newly generated NSPCs is found to migrate to the damaged brain areas [10, 13, 14]. Understanding the cellular and molecular mechanisms that are responsible for NSPC migration in both biological and pathological conditions is the first step toward the development of efficient self-repair strategies for clinical application. The N-methyl-D-aspartate receptors (NMDARs) are ligand-gated, Ca2⫹-permeable ion channels that mediate a wide variety of physiological and pathological processes in the CNS, including synapse formation, synapse plasticity, neuronal development, and excitotoxicity [15, 16]. NMDARs are tetramers comprising NR1, NR2 (NR2A–NR2D), and NR3 (NR3A–NR3B) subunits [15, 17]. Recent studies reveal

that NMDARs are involved in neuronal migration in the developing brain [18, 19]. Activation of NMDARs increases the rate of neuronal migration, and blockage of NMDAR activity reduces cell movement [18, 19]. Although evidence suggests that calcium influx through NMDAR channels may play an important role in mediating NMDAR regulation of neuronal migration [18, 19], the cellular and molecular mechanisms underlying the effects of NMDARs in the migrating cells remain unclear. Neuronal migration requires rearrangement of the cytoskeletal network [20], and this dynamic reorganization is controlled by intracellular signal transduction cascades [6]. The Rho GTPase Rac1, RhoA, and Cdc42 play crucial roles in the regulation of actin cytoskeletal remodeling and have been implicated in axon guidance, neurite outgrowth, and neuronal migration [21, 22]. Rho GTPase cycles between an active, GTP-bound state and an inactive, GDP-bound state. The activation state of Rho GTPases is controlled by guanine nucleotide exchange factors (GEFs), which stimulate Rho GTPases by catalyzing the exchange of GDP for GTP [22]. The Rac1-specific GEF Tiam1 (the invasion-inducing Tlymphoma and metastasis 1) was originally isolated as an invasion-inducing gene product [21]. A recent study showed that Tiam1/Rac1 signals were involved in upregulation of

Author contributions: Lei Li and Y.H.E.-H.: collection and assembly of data, data analysis and interpretation, manuscript writing; B.L.: collection and assembly of data; Y.C., E.G., X.W., K.N., Lijun Li, and N.C.: technical support; L.Z., Z.W., and X.H.: technical support, final approval of manuscript; Q.W.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. Correspondence: Qi Wan, M.D., Ph.D., Department of Physiology and Cell Biology, School of Medicine, University of Nevada, Reno, Nevada 89557-0271, USA. Telephone: 775-784-4352; Fax: 775-784-6903; e-mail: [email protected] Received December 5, 2007; accepted for publication May 30, 2008; first published online in STEM CELLS EXPRESS June 12, 2008; available online without subscription through the open access option. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-1022

STEM CELLS 2008;26:2193–2200 www.StemCells.com

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neuronal migration in the developing cerebral cortex [21]. However, it is not known whether this regulatory migration process is dependent on NMDAR activation. It has been well established that direct-current electrical fields (EFs) play crucial roles in the nervous system [23, 24]. Early in development, nervous system construction requires the presence of EFs [23, 24]. Naturally occurring EFs (10 –1,800 mV/mm) occur in frog and chick embryos during neurogenesis, and the disruption of these endogenous EFs causes failure of the nervous system to form [23]. Application of EFs in vitro has profound effects on nerve growth, guidance, and branching during neural construction. An EF as low as 10 mV/mm causes growth cones to turn, usually toward the cathode [23]. The endogenous EFs also play a critical role in pathological conditions. EFs are induced in damaged axons, and these injuryinduced EFs are believed to contribute to axonal regeneration [23]. In a corneal wound, injury-induced EFs direct and enhance nerve sprouting, implicating EF as a key factor controlling the orientation of nerve sprouts toward a wound [23]. On the basis of the effects of EFs on nerve growth, small EFs have been applied in animal models of spinal cord injury (SCI) [25]. The EF applications in these CNS injury models result in functional improvements [25]. Recently, weak EF stimulation has been applied in human SCI in a phase I trial [26]. The results indicate considerable clinical benefit. Given the involvement of EFs in neurogenesis, axon guidance, and nerve growth [23], we reasoned that physiological EFs might act as a guidance cue to regulate NSPC migration in the mammalian CNS. As a safe physical approach in clinical application, the EFs could have the potential to be developed as a practical therapeutic strategy for the repair of brain damage. We set up first to examine whether EFs could exert effects on NSPC migration and then to determine how membrane and intracellular signals were involved in mediating EF-induced NSPC migration. We have demonstrated that physiological EFs direct and facilitate NSPC migration toward the cathode and that the NMDAR-activated NMDAR/Rac1/actin signal transduction pathway may be responsible for the EF-induced NSPC migration.

MATERIALS

AND

METHODS

Explant Cultures of Rat Embryonic Lateral Ganglionic Eminence The explant cultures of rat (Wistar) lateral ganglionic eminence (LGE) were prepared from embryonic day (E) 17–18 rats [27] and placed on L-lysine/laminin-coated coverslips in a microchamber built for EF application as described elsewhere [28]. To allow the cultures to recover from the trauma of the dissection process, the cultures were placed in a 5% CO2 incubator for at least 2 hours before use. The 2-hour incubation was also required for the cultures to completely adhere to the poly-L-lysine/laminin substratum. The incubation medium was composed of minimum essential medium supplemented with 10% fetal bovine serum and 24 mM NaHCO3.

EF Application and the Imaging of NSPC Migration Explants (100 –300 ␮m in diameter) were selected for observation. For the EF application, agar-salt bridges were used to connect silver/silver chloride electrodes in beakers of Steinberg’s solution, to pools of excess culture medium at either side of the chamber. Field strengths were measured directly at the beginning and end of the observation period. For time-lapse observation, HEPES acid (25 mM) was added to the culture medium, with pH adjusted to 7.4. Field strengths of 50, 100, and 250 mV/mm were used for these experiments, with an exposure time of 3 hours. Time-lapse imaging was performed with an inverted microscope (Zeiss Axiovert 200M;

Carl Zeiss, Jena, Germany, http://www.zeiss.com) that was used to digitally record NSPC migration for 3 hours. The inverted microscope was equipped with an ORCA-ER camera (Hamamatsu, Bridgewater, NJ, http://www.hamamatsu.com) and Uniblitz brightfield shutter (Zeiss), allowing acquisition of transmitted phasecontrast or differential interference contrast images. Hardware was controlled by Axiovision software (Zeiss). Images were acquired every 10 minutes. For long-term observations, the cultures and the EF stimulation device were kept in the CO2 incubator. A field strength of 30 mV/mm was used for these experiments, with an exposure time of 10 hours. Exposure was initiated at 2–3 hours postplating and was terminated at 12–13 hours postplating. At the end of exposure period, explants were fixed in 4% paraformaldehyde and then digitally photographed for quantification.

Quantification of Cell Motion The explant was divided into four diagonal quadrants for the analysis of cell motion (Fig. 2B) [27]. Two of the quadrants were designated as cathode-facing and anode-facing (Fig. 2B). To quantify velocity and directedness of cell motion in time-lapse experiments, the cell centroids for eight outlying cells per quadrant (32 cells per explant) were calculated with ImageJ software (NIH). This yielded (x, y) coordinates in micrometers. The starting and final positions of cell centroids were exported to Minitab software (Minitab Inc., State College, PA, http://www.minitab.com). Absolute motion in the x- and y-planes was used to calculate displacement for each cell according to the Pythagorean theorem. Displacement was divided by 3 (hours) to yield velocity in ␮m/hour. Directedness of motion was expressed as a function of cosine [28]. It was calculated by defining movements in the x-plane toward the cathode (or mock-cathode) as negative. This was divided by displacement to yield directedness for each cell. Therefore, a cell moving directly toward the cathode would have a value of ⫺1, whereas a cell moving directly toward the anode would have a value of ⫹1. For quantification of long-term EF-exposed cells, the symmetry was expressed as a function of the distribution of cells around the explant. Total cell numbers in the cathode- and anode-facing quadrants were calculated. The data were expressed as a ratio of cell counts in the cathode-facing quadrant divided by the sum of cell counts in both the cathode-facing and anode-facing quadrants. This ratio would be equal to ⫹1 if all cells were located in the cathode quadrant, 0 if all cells were located in the anode quadrant, and 0.5 if cells were evenly distributed.

Immunocytochemistry and Coimmunoprecipitation The methods for immunocytochemistry and coimmunoprecipitation have been described in detail in our recent studies [29, 30]. For immunocytochemical labeling, the guinea-pig anti-doublecortin (anti-DCX; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http:// www.scbt.com), mouse anti-nestin (Chemicon, Temecula, CA, http://www.chemicon.com), rabbit anti-NR1 (Chemicon), and rabbit anti-NR2B (Novus Biologicals, Inc., Littleton, CO, http://www. novusbio.com) primary antibodies were used. Secondary antibodies, consisting of Alexa Fluor 488 and 596, were purchased from Molecular Probes (Eugene, OR, http://probes.invitrogen.com). Imaging was performed with a Zeiss LSM 510 META confocal microscope, and image processing was performed with ImageJ software (NIH). For the coimmunoprecipitation assay, rabbit anti-Tiam1 (Santa Cruz Biotechnology), rabbit anti-NR2B (Novus Biologicals), rabbit antiphosphorylated Pak1 at serine 423 (Santa Cruz Biotechnology), and mouse anti-actin (Chemicon) were used.

Data Analysis All data are presented as mean ⫾ SEM. Statistical significance was placed at p ⬍ .05. Significance was assessed with the two-tailed t test.

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Figure 1. Neuronal stem/progenitor cell (NSPC) migration in the explant cultures of rat embryonic lateral ganglionic eminence. (A): At 1 hr postplating, explants exhibited a generally circular appearance, with essentially no cell migration out of the periphery (left). By 10 hr a symmetrical cell migration out of the explant was observed (right). (B): The majority of the cells migrating away from the explant were immature NSPCs, as verified by nestin (green) and DCX (red) double staining. Higher-magnification images (bottom row) correspond to the boxed areas in the lower-magnification images (top row). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DCX, doublecortin; hr, hour(s).

RESULTS EFs at Physiological Strengths Guide and Speed NSPC Migration Toward the Cathode Explant cultures of the LGE from E 17–18 rats, a well-established in vitro model for the study of neuronal migration, were used to study the effect of EFs on NSPC migration [27]. In the control cultures, cells moved radially out of the explants and were symmetrically distributed around the circumference of each explant (Fig. 1A). To characterize the phenotypes of cells that migrate out of the explants, immunocytochemical staining with an antibody against nestin (an intermediate filament protein that is typical for undifferentiated NSPCs) and an antibody against DCX (a protein specifically expressed in immature, migrating neurons) was performed. To determine how many cells in the explant express each marker, cells were also labeled with 4,6-diamidino-2-phenylindole (nuclear labeling). For control experiments, primary antibodies were omitted (data not shown). We found that 73% of the cells migrating out of the explants were nestin-positive cells and that 75% of nestinpositive cells were positive for DCX labeling (Fig. 1B). These data indicate that the majority of cells migrating out of the LGE explants are immature, migrating neurons that are derived from NSPCs. The explant cultures were exposed to EFs at the range of physiologically relevant strengths (30 –250 mV/mm). Our results showed that EFs directed the migration of cells on the cathode side of explants toward the cathode but prevented the migration of cells on the anode side of explants toward anode www.StemCells.com

(Fig. 2A–2C; time-lapse video movie is given in the supplemental online data). EFs also increased the speed of cells on the cathode side of the explants migrating toward the cathode (Fig. 2C). Immunocytochemical labeling showed that 68% of the cells migrating toward the cathode were nestin- and DCX-positive cells (Fig. 2D). Together, these results led us to conclude that EFs may act as a directional guidance cue to control and expedite NSPC migration toward the cathode.

EF-Directed NSPC Migration Requires NMDAR Activation To understand how EF-directed NSPC migration is triggered at the cellular level, we next examined the molecular signals that might be responsible for EF-directed NSPC migration. As important membrane channel proteins, NMDARs have been shown to play a key role in regulating neuronal migration by affecting Ca2⫹ transient frequency [18]. However, the molecular mechanisms mediating the function of NMDARs in neuronal migration remain largely unknown. We therefore set up to determine whether NMDARs were involved in EF-directed NSPC migration, and the downstream signals mediating the effect of NMDARs were dissected. We first examined whether NMDARs were expressed in NSPCs. Immunocytochemical staining showed that the NR1 and NR2B, but not NR2A (data not shown), subunits of NMDARs were expressed in the majority (87%) of the cells migrating toward the cathode (Fig. 3A). Since majority of the cells migrating toward the cathode were found to be nestin ⫹ DCX-positive (Fig. 2D), these data suggest that NR2B-containing NMDARs may play a role in mediating NMDAR

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Figure 2. Physiological EFs direct neuronal stem/progenitor cell (NSPC) migration toward the cathode. (A): Radial migration of cells in lateral ganglionic eminence (LGE) explant culture at 10 hr postplating (left). Exposure of an LGE explant culture to an EF (30 mV/mm) for 10 hr resulted in an asymmetrical distribution with a higher number of cells in the cathodal side of the explant than in the anodal side (right). (B): A diagram illustrating the division of quadrants around an LGE explant (left). For long-term exposures, the effect of the EF was expressed as a ratio of cell counts in the cathode-facing quadrant divided by the sum of cell counts in both the cathode and anode-facing quadrants (right; control, n ⫽ 226; EF, n ⫽ 205; ⴱ, p ⬍ .05). (C): In time-lapse experiments, directedness (expressed as a function of cosine) of cell motion toward the cathode was EF strength-dependent (left; 0 mV, n ⫽ 256; 50 mV, n ⫽ 96; 100 mV, n ⫽ 96; 250 mV, n ⫽ 96; ⴱ, p ⬍ .05 compared with 0 mV). EFs also increased the speed of NSPC migration in a strength-dependent manner (right; 0 mV, n ⫽ 256; 50 mV, n ⫽ 96; 100 mV, n ⫽ 96; 250 mV, n ⫽ 96; ⴱ, p ⬍ .05 compared with 0 mV). (D): Double labeling of nestin (green) with DCX (red) shows that the majority of cells migrating toward the cathode were immature, migrating neurons that were derived from NSPCs. Higher-magnification images (bottom row) correspond to the boxed areas in the lower-magnification images (top row). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DCX, doublecortin; EF, electric field; hr, hour(s).

function in NSPCs. We then examined the effects of DAPV, a selective NMDAR antagonist, on NSPC migration in the explant cultures. We found that DAPV (10 ␮M) significantly inhibited NSPC migration toward the cathode on the cathode

side of explants in a small EF (30 mV/mm treatment for 10 hours; Fig. 3B, 3C). These data indicate that activation of NMDARs by EF stimulation mediates EF-induced NSPC migration, suggesting that altered activity of membrane chan-

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Figure 3. Activation of N-methyl-D-aspartate receptors (NMDARs) by EF stimulation mediates EF-guided NSPC migration. (A): NR1 and NR2B subunits of NMDARs were expressed on NSPCs migrating cathodally. Higher-magnification images (right) correspond to the boxed areas in the lower-magnification images (left). (B): NMDAR antagonist DAPV (10 ␮M) significantly attenuated EF-directed NSPC migration toward the cathode in lateral ganglionic eminence explants exposed to 30 mV/mm of EF at 10 hours after plating. (C): Summarized data showing effects of NMDAR inhibition on EF-directed NSPC migration (control, n ⫽ 192; DAPV, n ⫽ 175; EF, n ⫽ 187; DAPV⫹EF, n ⫽ 212; ⴱ, p ⬍ .05, EF vs. control or DAPV; #, p ⬍ .05, DAPV⫹EF vs. EF). Abbreviation: EF, electric field.

nel proteins may be a critical first step for a migrating cell to respond to EF stimulation.

EF Stimulation Enhances a Physical Association of NMDARs with the Activator of Rac1 Rearrangement of the actin cytoskeletal network is an essential process in neuronal migration [20, 31]. Recent evidence indicates that the Rho GTPase Rac1 plays an important role in mediating neuronal migration through regulating actin cytoskeletal remodeling [32]. If NMDARs are required for EF-induced NSPC migration, an intracellular signal pathway would link NMDARs to actin cytoskeletal remodeling. We therefore hypothesized that the activated NMDARs by EF stimulation might mediate NSPC migration through interacting with the Rac1dependent signal transduction pathway. To address this possibility, we performed coimmunoprecipitation assays to examine whether EFs could increase a coupling of NMDARs to the GEF Tiam1, a specific Rac1 activator that causes Rac1 activation and subsequent actin polymerization [21, 33]. Immunoprecipitation with an anti-Tiam1 antibody resulted in coprecipitation of NMDARs in control explants, suggesting a physical interaction between NMDARs and Tiam1 in biological conditions. Interestingly, our data showed that treatment of explants with physiological EFs at a strength of 250 mV/mm for 60 minutes significantly increased the association of NMDARs with Tiam1 (Fig. 4A). Importantly, we demonstrated that the NMDAR antagonist DAPV (10 ␮M) inhibited the EF-induced increase in NMDAR association with Tiam1 (Fig. 4A). These data indicate www.StemCells.com

that EF stimulation can activate NMDARs on the cell membrane, which leads to an increased association of NMDARs with the Rac1 activator Tiam1. Thus, through forming a complex with Rac1-associated signals, NMDARs may transmit extracellular EF stimulation to the intracellular Rac1 signaling transduction pathway and thereby mediate EF-directed NSPC migration. To obtain further evidence to support the interaction of Rac1 signals with NMDARs in the involvement of EF-induced NSPC migration, we examined whether p21-activated kinase 1 (Pak1), a downstream target of Rac1 for actin polymerization [34 –36], was involved in EF-induced interaction between NMDARs and Tiam1. By performing coimmunoprecipitation assays, we showed that there was an increased association of the phosphorylated Pak1 (p-Pak1; i.e., the activated form of Pak1) with Tiam1 in EF-exposed explants and that inhibition of NMDARs by DAPV abolished the enhanced association of p-Pak1 with Tiam1 (Fig. 4B). These data suggest that EF stimulation may lead to the recruitment of activated Pak1 to the NMDAR-Tiam1 complex, and this protein-protein interaction process requires NMDAR activation.

EF-Induced NMDAR Activation Leads to Increased Activity of the Rac1 Signal Pathway The observed formation of NMDAR/Tiam1/p-Pak1 protein complex in an EF suggests that the activity of Pak1 may be enhanced because of the activation of the NMDAR/Tiam1/pPak1 signal cascade. Because increased activation of Pak1 rep-

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Figure 4. EF stimulation promotes a physical association of Nmethyl-D-aspartate receptors (NMDARs) with Rac1 signals. (A): EF exposure (250 mV/mm for 60 minutes) significantly enhanced an association of NMDAR NR2B subunit with the specific Rac1 activator Tiam1, and the increased association was suppressed by inhibition of NMDARs with DAPV (10 ␮M) in cultured lateral ganglionic eminence explants (left). Summarized data indicate that the EF-induced increase in NR2B-Tiam1 association is dependent on NMDAR activation (right; n ⫽ 3; ⴱ, p ⬍ .05, EF vs. control; #, p ⬍ .05, DAPV⫹EF vs. EF). (B): EF stimulation enhanced the association of p-Pak1 with Tiam1, and the increased association of p-Pak1 with Tiam1 was abolished by inhibition of NMDARs (left). Summarized data indicate that the EF-induced increase in p-Pak1 association with Tiam1 is dependent on NMDAR activation (right; n ⫽ 3; ⴱ, p ⬍ .05, EF vs. control; #, p ⬍ .05, DAPV⫹EF vs. EF). Abbreviations: Ab, antibody; EF, electric field; IP, immunoprecipitation; p-Pak1, phosphorylated p21-activated kinase 1.

Figure 5. EF stimulation increases levels of p-Pak1. EF treatment significantly increased the levels of p-Pak1, and this increase was blocked by the N-methyl-D-aspartate receptor (NMDAR) antagonist DAPV (left). Summarized data show that the EF-induced increase in Pak1 phosphorylation requires NMDAR activation (right; n ⫽ 3; ⴱ, p ⬍ .05, EF vs. control; #, p ⬍ .05, DAPV⫹EF vs. EF). Abbreviations: EF, electric field; p-Pak1, p21-activated kinase 1 phosphorylation.

resents an enhanced activity of the Rac1 signal pathway, we set up to determine whether the phosphorylation levels of Pak1 are altered by EF stimulation. Using an antibody against p-Pak1 at serine 423 in immunoblot assays, we found that EF treatment (250 mV/mm) for 60 minutes significantly enhanced Pak1 phosphorylation (Fig. 5), indicating an increased activity of Pak1 by EF stimulation [34, 37, 38]. Moreover, treatment with the NMDAR antagonist DAPV significantly attenuated the EFmediated increase in Pak1 phosphorylation (Fig. 5), suggesting that NMDAR activation contributes to the EF-induced increase in Pak1 activity. These data suggest that EF stimulation, via activation of NMDARs, may promote a physical association of NMDARs with Rac1-associated signals and thereby enhance the activity of the Rac1-dependent signal transduction pathway. Thus, these results support the possibility that the NMDAR/ Tiam1/Rac1/Pak1 pathway may play a crucial role in mediating EF-induced NSPC migration.


Figure 6. EF-induced N-methyl-D-aspartate receptor (NMDAR) activation leads to an enhanced association of Rac1 activator Tiam1 with actin cytoskeleton. EF exposure increased an association of actin with Tiam1, and the increased actin-Tiam1 association was inhibited by the NMDAR antagonist DAPV (left). Summarized data show that the EF-induced increase in actin-Tiam1 association is dependent on NMDAR activation (right; n ⫽ 3; ⴱ, p ⬍ .05, EF vs. control; #, p ⬍ .05, DAPV⫹EF vs. EF). Abbreviations: Ab, antibody; EF, electric field; IP, immunoprecipitation.

EF-Induced NMDAR Activation Promotes Association of Tiam1 with Actin Cytoskeleton If the activated NMDAR/Tiam1/Rac1/Pak1 signal pathway is responsible for the EF-induced NSPC migration, the actin cytoskeleton should respond to Rac1 signaling, which is known to couple to the actin cytoskeletal remodeling process to mediate cell migration [20, 31]. To determine whether there was an EF-induced interaction between Rac1 signals and the actin cytoskeleton, we performed coimmunoprecipitation assays using protein from control and EF-exposed explants. We found that anti-Tiam1 antibody led to the coprecipitation of actin in the control explants (Fig. 6) and that the coprecipitated amount of actin protein was significantly increased in EF-exposed explants (Fig. 6). We further showed that DAPV treatment suppressed the EF-induced increase in association between actin and Tiam1 (Fig. 6). These data provide evidence that EF-controlled NSPC migration may be mediated by a physical interaction of the NMDAR/Tiam1/Rac1/Pak1 signal complex with the actin cytoskeleton and that this interaction is dependent on NMDAR activation.

DISCUSSION The prospect of stem cell-based neuroregeneration brings hope for patients suffering from brain diseases such as stroke and neurodegenerative disorders. To increase the pace of the stem cell therapeutic strategy toward clinical application, it is important to understand how neural stem cell proliferation, differentiation, and migration are controlled. We have investigated the role of EFs in directing the migration of mammalian NSPCs. We provide evidence that physiological EFs direct and speed NSPC migration toward the cathode. Because EF treatment, as a physical approach, has proved safe in neurorehabilitation and in the clinical trial of human spinal cord injury [26, 39], our results suggest a possibility that EFs may be developed as a practical cell-based therapy for brain repair by directing NSPC migration to the injured brain regions to replace cell loss. Our data show that NMDARs are activated by EF stimulation and that activation of NMDARs leads to an increase in physical association of these channels with the Rac1 activator Tiam1 and effector Pak1, and subsequently an enhancement of association with actin cytoskeleton. These results suggest that NMDAR may act as a membrane transducer to couple the extracellular EF stimulation to the intracellular Tiam1/Rac1/ Pak1/actin pathway and thus play a role in mediating NSPC migration. Thus, our study identifies the protein complex

Li, El-Hayek, Liu et al. NMDAR/Tiam1/Rac1/Pak1/actin as a novel signal pathway in the EF-exposed migrating NSPCs. Although the Rho-family GTPases and many of their upstream regulators and downstream effectors have been shown to be critically involved in the cell migration process [21, 22], several receptor kinases, G protein-coupled receptors, and a variety of intracellular signaling proteins and scaffolds are also shown to play important roles in the cell migration [40]. Cellular signals that link cell surface receptors and integrins to intracellular pathways promote cytoskeletal reorganization. The transmembrane subclass of guanylyl cyclases, which catalyze the conversion of GTP to the second messenger cyclic GMP (cGMP), has been implicated in chemotactic cell migration and axon guidance [41, 42]. The cGMP, along with a few cGMPdependent kinases and phosphodiesterases, has been implicated in cell motility [43]. Also, a signaling link between the Rac GTPase and the transmembrane guanylyl cyclases has been shown to mediate growth factor-induced migration of fibroblasts [44]. Recently, Rho, Rac, Cdc42, and their effectors have been shown to play coordinated roles in growth cone guidance by EFs [45]. The Rho GTPases have also been shown to interact with the cytoskeleton and growth cone dynamics in an EF [46]. NMDARs have been shown to play a crucial role in regulating neuronal migration [18, 19]. However, the cellular and molecular mechanisms by which the NMDAR exerts its role in neuronal migration remain largely unknown. Our study provides novel evidence suggesting that the protein-protein interactions of NMDARs with Rac1 signals and actin cytoskeleton may represent a general cellular and molecular mechanism underlying NMDAR-mediated neuronal migration in the CNS.

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CONCLUSION The present study provides the first evidence that at physiological strength EFs direct NSPC migration towards the cathode. EF-directed NSPC migration requires NMDAR activation, which leads to enhanced coupling of the Rho GTPase Racl to both the upstream membrane NMDARs and the downstream cytoskeletal protein actin. Thus, this study uncovered the EF as a directional guidance cue in controlling NSPC migration and revealed the NMDAR/Racl/actin protein complex as a novel signal transduction pathway in mediating EF-induced NSPC migration. Because EF application has proved safe in humans, EFs may be developed as a practical therapeutic strategy for brain damage repair by directing NSPC migration to the injured brain regions.

ACKNOWLEDGMENTS We thank Dr. Chiping Wu for technical assistance. This study was carried out and completed in the Toronto Western Research Institute and supported by the start-up fund of University Health Network (to Q.W.).

DISCLOSURE

OF POTENTIAL OF INTEREST

CONFLICTS

The authors indicate no potential conflicts of interest.

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