N-cadherin promotes recruitment and migration of neural progenitor cells from the SVZ neural stem...

Development/Plasticity/Repair

N-Cadherin Promotes Recruitment and Migration of NeuralProgenitor Cells from the SVZ Neural Stem Cell Niche intoDemyelinated Lesions

Michael Klingener,1 Manideep Chavali,1,2 Jagdeep Singh,1 Nadia McMillan,1 Alexandra Coomes,1,4 Peter J. Dempsey,3

Emily I. Chen,1,4 and Adan Aguirre1

State University of New York at Stony Brook University, Departments of 1Pharmacological Science and 2Materials Science and Engineering, Stony Brook,New York 11794, 3Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan 48109, and 4Stony Brook UniversityProteomics Center, School of Medicine, Stony Brook, New York 11794

Discrete cellular microenvironments regulate stem cell pools and their development, as well as function in maintaining tissue homeo-stasis. Although the signaling elements modulating neural progenitor cells (NPCs) of the adult subventricular zone (SVZ) niche are fairlywell understood, the pathways activated following injury and the resulting outcomes, are less clear. In the present study, we used mousemodels of demyelination and proteomics analysis to identify molecular cues present in the adult SVZ niche during injury, and analyzedtheir role on NPCs in the context of promoting myelin repair. Proteomic analysis of SVZ tissue from mice with experimental demyelina-tion identified several proteins that are known to play roles in NPC proliferation, adhesion, and migration. Among the proteins found tobe upregulated were members of the N-cadherin signaling pathway. During the onset of demyelination in the subcortical white matter(SCWM), activation of epidermal growth factor receptor (EGFR) signaling in SVZ NPCs stimulates the interaction between N-cadherinand ADAM10. Upon cleavage and activation of N-cadherin signaling by ADAM10, NPCs undergo cytoskeletal rearrangement and polar-ization, leading to enhanced migration out of the SVZ into demyelinated lesions of the SCWM. Genetically disrupting either EGFRsignaling or ADAM10 inhibits this pathway, preventing N-cadherin regulated NPC polarization and migration. Additionally, in vivoexperiments using N-cadherin gain- and loss-of-function approaches demonstrated that N-cadherin enhances the recruitment of SVZNPCs into demyelinated lesions. Our data revealed that EGFR-dependent N-cadherin signaling physically initiated by ADAM10 cleavageis the response of the SVZ niche to promote repair of the injured brain.

Key words: brain repair; cell migration; demyelination; neural progenitor cells; SVZ

IntroductionThe adult brain, specifically the subventricular zone (SVZ) niche,contains multipotent neural progenitor cells (NPCs; Doetsch etal., 1999; Gage, 2000). Microenvironments, or niches, regulate

NPC development and population size to maintain tissue ho-meostasis in the adult CNS (Kokovay et al., 2010; Kazanis andFrench-Constant, 2011). NPCs born in the SVZ migrate longdistances along the rostral migratory stream, reaching the olfac-tory bulb and terminally differentiating into neurons (Menezes etal., 1995). Furthermore, NPCs retain the ability to migrate intothe subcortical white matter and differentiate into myelinatingoligodendrocytes, both in normal and pathological conditions(Aguirre et al., 2007; Etxeberria et al., 2010).

Progress in stem cell biology has expanded expectations fortherapeutic approaches to neurological disorders (Lim et al.,2007; De Filippis and Binda, 2012). It has been suggested thatmanipulation of endogenous NPCs might be an ideal strategy topromote cellular repair, because of their relative abundance, highproliferation rate, and migratory capacity (Taupin, 2007). Cur-rent theories suggest that NPCs respond to brain injury in a man-ner that is believed to be an effort to stop ongoing damage andpromote repair (Sellers and Horner, 2005; Felling et al., 2006). Insupport of this hypothesis, increased cell proliferation and migra-tion have been reported in the acute phases of brain injury in bothhypoxic/ischemic and demyelination conditions (Decker et al.,2002; Felling et al., 2006; Menn et al., 2006; Nait-Oumesmar et al.,

Received Aug. 29, 2013; revised June 4, 2014; accepted June 6, 2014.Author contributions: M.K., E.I.C., and A.A. designed research; M.K., M.C., J.S., N.M., A.C., and A.A. performed

research; P.J.D. contributed unpublished reagents/analytic tools; M.K., M.C., E.I.C., and A.A. analyzed data; M.K. andA.A. wrote the paper.

This work was supported by NIH Grant 4R00NS057944-03 (A.A.). We thank Dr Gallo (CNMC, Washington, DC), DrRatner (CHMC, Cincinnati, OH), Dr Crawford (Department of Cancer Biology, Mayo Clinic, Jacksonville, FL), and DrPeter J. Dempsey (University of Michigan) for the CNP-EGFP, CNP-hEGFR, and ADAM10 fl/fl mice (Dr Crawford andDempsey), respectively; Dr T. Suda (Department of Cell Differentiation, Keio University, Japan) for the generous giftof the N-cadherin constructs; H. Colognato and M. Frohman for critically reading this manuscript; our colleagues atthe Pharmacology Department at SUNY, Stony Brook, and in the Aguirre Laboratory for discussion and support; theDLAR, particularly Laurie Levine, at SUNY Stony Brook for their support in the maintenance of the animal colony; andthe University of Iowa for the monoclonal antibody MNCD2 developed by M. Takeichi and H. Matsunami.

The authors declare no competing financial interests.Correspondence should be addressed to Dr Adan Aguirre, 442 Centers for Molecular Medicine, Department

of Pharmacological Science, SUNY at Stony Brook University, Stony Brook, New York 11794. E-mail:[email protected].

E.I. Chen’s present address: Herbert Irving Comprehensive Cancer Center, Proteomics Shared Resource and De-partment of Pharmacology, Columbia University Medical Center, New York, NY. 10032.

DOI:10.1523/JNEUROSCI.3699-13.2014Copyright © 2014 the authors 0270-6474/14/349590-17$15.00/0

9590 • The Journal of Neuroscience, July 16, 2014 • 34(29):9590 –9606

2008). Indeed, several studies have demonstrated mobilization ofendogenous NPCs from the SVZ to areas of damage, achievingsome degree of repair in several pathological brain settings(Barkho and Zhao, 2011; Winner et al., 2011).

Gradients of factors such as heparin-binding epidermalgrowth factor, vascular endothelial growth factor, and stromalcell-derived factor 1 can emanate from distant brain lesions andmay act as attractants for NPCs (Decker et al., 2002; Schmidt etal., 2005; Cantarella et al., 2008; Kokovay et al., 2010; Plane et al.,2010). Such signals have been shown to alter the migratory pathsof progenitors, redirecting them toward areas of brain damage(Cayre et al., 2009). In particular, EGFR signaling has proven tobe crucial in promoting NPC proliferation, migration, and re-cruitment during development and in pathological conditions(Aguirre and Gallo, 2007; Aguirre et al., 2007; Gonzalez-Perez etal., 2009). Although the signaling pathways acting on NPCs of theadult SVZ niche are fairly well understood, the pathways acti-vated following injury, and the resulting outcomes, are less clear.Identifying signaling cues that promote mobilization and recruit-ment of NPCs in the injured brain is of great importance to thedesign of cellular strategies for brain repair.

In the present study, we used mouse models of demyelinationalong with a proteomics approach to identify signaling cues pres-ent in SVZ NPCs following injury. Given the importance of cell–cell and cell–ECM complexes in the maintenance of the SVZ andfor cell migration, we focused on cues present during the peak ofdemyelination, when the SVZ is actively adapting to the injury,which could alter either of these cellular functions.

Materials and MethodsTransgenic mice. Details regarding the generation and characterization ofCNP-EGFP (Yuan et al., 2002), GFAP-GFP (Nolte et al., 2001), CNP-hEGFR (Ling et al., 2005), and NG2-dsRED mice (Zhu et al., 2008) havebeen previously reported. Tamoxifen inducible Nestin-Cre (stock no.012906), and Wa2 mice (waved-2 mutation; Egfrwa2; Wa2/Wa2) wereobtained from Jackson Laboratories. The ADAM10 fl/fl mouse was kindlydonated by Dr Dempsey (Department of Pediatrics and CommunicableDiseases, University of Michigan, Ann Arbor, MI) and Dr Howard Craw-ford (Department of Cancer Biology, Mayo Clinic Florida, Jacksonville,FL). Genotyping of these transgenic mice was performed by PCR (addi-tional information regarding genetic background, primers, and PCRprograms can be obtained on request). For induction of recombinationand deletion of ADAM10 in nestin � cells, adult mice (fl/fl and controlmice) received three tamoxifen injections (24 h apart; 45 �g/g bodyweight from a 10 mg/ml tamoxifen stock in sesame oil). Littermate micewild-type for ADAM10 or negative for nestin-Cre in the ADAM10 linewere used as controls and were also treated with tamoxifen concurrentwith fl/fl mice. In experiments using Nestin::ADAM10 f/fl mice, SVZ sam-ples were obtained at 48 h after the last tamoxifen injection, and pro-cessed for Western blot or cell culture, as indicated. In this study, we usedequal numbers of male and female mice for analysis. All animal proce-dures were performed according to the Institutional Animal Care andUse Committee of SUNY Stony Brook and the National Institutes ofHealth “Guide for the Care and Use of Laboratory Animals.”

Mouse models of demyelination and remyelination: cuprizone and lyso-lecithin. For the cuprizone model, adult mice (45- to 90-d-old; P45–P90)were fed ad libitum with 0.2% cuprizone (cyclohexylidenehydrazide;Sigma-Aldrich) mixed into a pelleted Teklad chow diet (TD.8604; TekladLaboratories) for 12 weeks to induce chronic demyelination of the sub-cortical white matter (SCWM; Hibbits et al., 2009). Untreated controlmice were fed pelleted Teklad chow concurrently, whereas experimentalmice were being treated with cuprizone. Following 12 weeks of treat-ment, some of the mice receiving cuprizone were returned to a standardpelleted chow diet for a period of 4 weeks to promote remyelination (theremaining mice were processed for analysis at the 12 week time point).For all experiments, 12 weeks of cuprizone treatment served as the de-

myelination time point, whereas 12 weeks of cuprizone plus 4 weeks ofregular chow served as the recovery time point (Fig. 1A). To induce acutedemyelination, adult mice (P45–P90) were injected with 1.0% lysoleci-thin (Sigma-Aldrich) into the corpus callosum (CC; 1.5 mm anterior/posterior; 1 mm medio/lateral and 2.2 mm dorso/ventral from bregma).The time of the injection was noted, and this served as the starting timepoint for all measurements of days postlesion (dpl). For this model, 5–7dpl served as the demyelination time point, whereas 21 dpl served as therecovery time point (Fig. 1B).

SVZ microdissection. SVZ tissue was microdissected from 200-�m-thick coronal brain sections using a tissue chopper (McIlwian) and fineforceps to avoid tissue contamination from neighboring brain regions, aspreviously described (Aguirre et al., 2010). Dissected SVZ tissue was usedfor Western blots, immunohistochemistry, and cell cultures as indicated.For all experiments, dorsolateral SVZ tissue was used (see Fig. 5B, boxedarea; Ortega et al., 2013).

Western blots and immunoprecipitation. SVZ tissue was microdissectedas described above, and tissue was used for whole protein extractionusing RIPA lysis buffer (Santa Cruz Biotechnology) with inhibitors;PMSF in DMSO, protease inhibitors, and sodium orthovanadate, as in-structed by the manufacturer. Protein samples (10 –15 �g) were clearedby centrifugation (10,000 � g � 5 min), separated on acrylamide gels,and then transferred to polyvinylidene difluoride membranes (Milli-pore) at 25 V for 12–16 h (overnight) at 4°C. For the detection ofN-cadherin C-terminal fragments (CTFs), 30 �g of protein was loaded.Antibodies were used for detection of the indicated proteins (Table 1) incombination with secondary horseradish peroxidase-conjugated anti-bodies using an enhanced chemiluminescence substrate mixture (ECLPlus, GE Healthcare; Santa Cruz Biotechnology; 1:5000). Where indi-cated, protein levels are quantified in arbitrary units (A.U.) followingnormalization to �-actin levels (used as the loading control for all West-ern blot experiments, where indicated).

For immunoprecipitation experiments, SVZ tissue from wild-type,transgenic, cuprizone, and lysolecithin adult mice was treated with RIPAbuffer to obtain protein extracts (Aguirre et al., 2007). Aliquots of 100 �g(for in vitro studies) or 200 �g (for in vivo studies) of protein extract wereincubated with antibodies against N-cadherin (Santa Cruz Biotechnol-ogy; 1 �g) and 10 �l of Protein-A-conjugated agarose beads (Santa CruzBiotechnology) for 12–16 h at 4°C. Proteins associated with N-cadherinwere concentrated by centrifugation at 10,000 � g for 3 min at 4°C andwashed twice with cold RIPA buffer to remove nonspecific binding part-ners. N-cadherin complexes were resolved on acrylamide gels and de-tected as described above.

Immunohistochemistry. Brain sections were processed for immunohis-tochemistry analysis as previously described with minor modifications(Yuan et al., 2002). Briefly, tissue sections were blocked for 1 h usingblocking solution (10% goat serum, 1% BSA, and 0.3% Triton X-100diluted in PBS) and incubated with primary antibodies overnight at 4°Cin carrier solution (4% goat serum and 0.3% Triton X-100 diluted inPBS; dilutions and companies for antibodies are displayed in Table 1).The following day, sections were washed three times in carrier solutionfollowed by incubation with the appropriate highly cross-absorbed sec-ondary antibodies. Following four additional washes, nuclei were stainedwith DAPI and sections were mounted using MOWIOL mounting me-dia. Histological preparations were imaged using confocal imaging. Pro-liferation in vivo was assessed by injecting BrdU intraperitoneally at 100mg/kg 2–3 h before the end of the experiment as previously described(Aguirre et al., 2007).

Immunocytochemistry. For immunocytochemistry analysis, cells wereplated onto poly-L-lysine (50 �g/ml) and fibronectin (10 �g/ml) coatedcoverslips to test cell differentiation potential and for characterization. Atthe conclusion of the respective experiments, cells were fixed with 4%PFA and then blocked using 20% goat serum/0.05% Triton X-100 in PBSfor 10 min at room temperature. The coverslips were then processed withthe indicated primary antibodies followed by secondary antibody incu-bation (Table 1 shows a list of antibodies used in this study).

Proteomic analysis. Proteomic analysis was performed as previouslydescribed (Sinnamon et al., 2012). Briefly, SVZ tissue was dissected fromadult brains and homogenized in lysis buffer (50 mM ammonium bicar-

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Figure 1. Acute and chronic demyelination of the subcortical white matter expands the pool of neural progenitor cells in the SVZ niche. A, Timeline illustrating the course of cuprizone-inducedchronic demyelination of the SCWM. Adult mice were fed a 0.2% cuprizone diet (Cupz.) for 12 weeks (12 w; demyelination phase), leading to global demyelination of the SCWM. At the conclusionof this period, a diet consisting of regular chow was resumed for 4 weeks (12w/4w; remyelination phase), to allow for remyelination of demyelinated lesions. Control mice were supplied with aregular diet (Reg.), concurrently, for the full 16-week period; 12w � peak of global SCWM demyelination in the CNS and the time point chosen for proteomic analysis of SVZ tissue (caret symbol).B, Timeline illustrating the course of lysolecithin-induced acute focal demyelination of the SCWM. Adult mice received a single injection of lysolecithin (2 �l; 1.0%) directly into the CC (ipsilateralside) leading to acute focal demyelination at 5–7 d after injection. The lesion resulting from lysolecithin injection promoted recruitment of local resident NPCs in the SCWM as well as migratory NPCsfrom the SVZ (10 –14 d after lysolecithin injection), and further differentiation of NPCs (21 d after lysolecithin injection), leading to the eventual repair of demyelinated lesions. The same mice wereinjected with 0.9% NaCl into the contralateral hemisphere and used as controls. C, Immunohistochemistry analysis at the peak of chronic demyelination with anti-BrdU, anti-Olig2, anti-EGFR, andanti-Mash1 antibodies was used to characterize SVZ cells that respond to injury. Quantifications are presented as the number of cells displaying immunoreactivity to the indicated antibodies, relativeto the total number of cells counted. Cell quantifications were determined by analyzing images of the SVZ stem cell niche obtained by confocal microscopy. D, Representative confocal images in theSVZ niche with anti-EGFR and anti-Olig2 antibodies at the peak of demyelination of adult control and cuprizone fed mice. A higher number of Olig2 �EGFR � cells were present in the SVZ niche ofdemyelinated mice. The dotted lines indicate the boundaries of the SVZ niche and represent the area taken into consideration for analysis. E, Representative confocal images in the SVZ niche withanti-NG2 and anti-Ki67 antibodies of control and lysolecithin-injected hemispheres. NG2-dsRED transgenic mice were used to detect the response of NG2 � cells within the SVZ niche duringdemyelination. Using this approach, cells displaying NG2 immunoreactivity were expanded at the peak of acute demyelination. NG2 � cell expansion in the SVZ was a result of both active cell divisionand lineage commitment from pre-existing NPCs (right panels). The dotted lines indicate the boundaries of the SVZ and area that was taken into consideration for analysis. F, SVZ tissue was dissectedfrom control or cuprizone mice at 12 weeks of treatment, and single cell suspensions were prepared for floating cell culture (neurosphere assay) to assay proliferation dynamics during this condition.Cells were plated at clonal density and maintained in stem-cell medium under proliferative conditions. At 7 DIV the numbers of neurospheres were quantified. (Figure legend continues.)

9592 • J. Neurosci., July 16, 2014 • 34(29):9590 –9606 Klingener et al. • Recruitment of NPCs from the SVZ to Brain Lesions

bonate, 8M Urea, 1 � invitrosol, protease and phosphatase inhibitors)using a tissue homogenizer (Precellys 24, Precellys). Tissue lysates werecleared by centrifugation following homogenization and the proteinconcentration in individual samples was determined using an EZQprotein assay (Invitrogen). We generated protein lysate of SVZ tissuefrom metabolically labeled mice as internal protein standards. Lysatesfrom wild-type mice were metabolically labeled using stable isotope-labeled ( 15N) amino acids (SILAM) according to the feeding regimenestablished in the Chen laboratory (Koller et al., 2013). 15N incorpo-ration into peptides was verified by 2D LC-MS/MS as being �97%.Age and sex-matched 15N-labeled SVZ tissues were dissected fromthree labeled brains and homogenized using the produces describedabove. Approximately 30 �g of unlabeled SVZ lysate (from regulardiet or cuprizone-treated samples) was mixed with an equal amountof 15N-labeled SVZ lysate and diluted in 50 mM Ammonium Bicar-bonate (1:1) for trypsin digestion. For detailed methods on the detec-tion and identification of peptide fragments, see Sinnamonet al. (2012). Protein expression changes were calculated as the ratiosof signals from cuprizone-treated SVZ and untreated SVZ(treated/control).

Neurosphere cultures. Adult SVZ tissue was dissected out and trans-ferred to chilled HBSS containing 26 mM HEPES, 0.3% glucose, and0.75% sucrose. Cells were dissociated into single cell suspensions using0.1% trypsin and 100 U/ml DNase I (Sigma-Aldrich) in HBSS for 25 min

at room temperature. Single-cell suspensions were plated in floating cul-tures at a clonal dilution in stem-cell medium (SCM; 1:1 DMEM:F12medium, 1x B27 supplement, 1% penicillin/streptomycin with daily ad-dition of EGF, Millipore Bioscience Research Reagents,10 ng/ml; andbFGF2, Millipore Bioscience Research Reagents,10 ng/ml) as previouslydescribed (Aguirre et al., 2010). Counting of spheres was performed 7 dafter plating. For differentiation experiments, neurospheres were disso-ciated after 7 d in vitro (DIV) using TrypLE (Invitrogen) and single cells(10,000/well) were plated onto poly-L-lysine (50 �g/ml) and fibronectin(10 �g/ml) coated coverslips. These cultures were maintained for 5 DIVin SCM without growth factors, and then processed for immunocyto-chemistry analysis with neural lineage-specific antibodies. All neuro-sphere, cell, and tissue explant cultures were performed in a humidifiedincubator maintained at 37°C and 5% CO2.

EGF time-course stimulation. SVZ explants were extracted from adultmice (P45–P90), dissociated into single cells as above, and cells wereplated (20,000 –50,000 cells/well in 24-well plates with coverslips or200,000 –250,000 cells/well in 6-well plates) in wells coated with poly-L-lysine (50 �g/ml) and fibronectin (10 �g/ml). Cells were maintained for24 – 48 h under proliferating conditions in SCM using bFGF2 (2.5 ng/ml)and EGF (2.5 ng/ml) before the time course experiment. Following thisperiod, cell culture media was replaced with fresh SCM 4 – 6 h before theEGF time course experiment. EGF (10 ng/ml) was used in all time courseexperiments for stimulation. At the end of the respective experiments,cells were processed for Western blot or immunocytochemistry analysis.Additionally, the supernatant was collected for each time point for theexperiments in Figure 5C. For all experiments involving detection ofN-cadherin fragments in the supernatant, we used the same protocol asfor immunoprecipitation with minor modifications. Briefly, 250 �l ofsupernatant (from each respective time point) was mixed with an equalvolume of RIPA, 10 �l of Protein-A beads (Santa Cruz Biotechnology),and 1 �g of antibody against N-cadherin (Santa Cruz Biotechnology)and incubated overnight at 4°C. The 95 kDa N-cadherin fragment wasdetected using MNCD2 primary antibody against the N-terminus ofN-cadherin (Developmental Studies, Hybridoma Bank).

Biotinylation of cell-surface proteins. SVZ cell cultures were prepared asabove and cells were plated onto poly-L-lysine (50 �g/ml) and fibronec-tin (10 �g/ml)-coated plates (250,000 cells/well in 6-well plates) andmaintained in SCM for 48 h with EGF and bFGF2 at 37°C before per-forming the assay. Following 0 –24 h of EGF stimulation (as indicated inFig. 5A), cell-surface proteins were biotinylated using the EZ-Link Sulfo-NHS-SS-Biotinylation Kit (Thermo) following the manufacturer’s pro-tocol with minor modifications. In brief, cell cultures were washed twicewith ice-cold PBS and incubated in 1.5 ml biotinylation solution (Sulfo-NHS-SS-biotin dissolved in aCSF) for 30 min at 4C with gentle shaking.The solution was aspirated, and un-reacted biotin was quenched by in-cubating cells with aCSF containing 100 mM glycine for 25 min at 4C withgentle agitation, followed by two washes with PBS. Following biotin la-beling, cells were lysed in RIPA buffer containing inhibitors. Lysates werecleared by centrifugation at 10,000 � g for 5 min at 4C, and biotinylatedproteins (membrane proteins) were selectively isolated using streptavi-din beads. Membrane proteins and intracellular material was analyzed bySDS-PAGE and Western blotting as described above.

SVZ niche explant cultures. Adult brains (P45–P90) were dissected,washed twice in HBSS, then transferred to fresh ice-cold HBSS mediumbefore SVZ dissection. The brains were cut in 200-�m-thick sections,and only the sections containing the SVZ were selected for additionalprocessing. Explant migrations were performed essentially as previouslydescribed (Aguirre et al., 2005). At 24 h after culturing, explants wereretrovirally transduced with the N-cadherin constructs as indicated inFigure 6 (a gift from Dr Suda; Department of Cell Differentiation, Tokyo,Japan; Hosokawa et al., 2010) at a titer of 2– 4 � 10 6 plaque-formingunits ml �1 for 4 consecutive days. At the conclusion of this period,explants were cocultured with heparin beads (Sigma-Aldrich) loadedwith EGF (2 �g/ml) or BSA (0.1% BSA). Explants were positioned 300 –400 �m from each set of beads (Pozas et al., 2001). Morphological anal-ysis and cell migration quantifications were obtained at 24 and 48 h,respectively, after EGF-bead treatment, as previously described (Aguirreet al., 2005).

4

(Figure legend continued.) The colony-forming ability of NPCs derived from demyelinatedsamples was significantly higher until the second passage, and subsequently returned to non-significant levels (data not shown). G, Neurospheres from control and cuprizone-treated micewere plated onto poly-L-lysine and fibronectin-coated coverslips and maintained for 5 DIVunder differentiating conditions to assay cell differentiation potential. A higher number of Tuj �

neurons (green) and CNP � oligodendrocytes (red) were detected after demyelination, com-pared with control cultures. LV, Lateral ventricle. Data are shown as mean � SEM (n � 4);*p � 0.01 for in vivo, *p � 0.05 for in vitro data using the Student’s t test. Scale bars: D, E, G, 40�m; F, 300 �m.

Table 1. Antibodies used in this study

Primary antibody Supplier Dilution Technique*

N-cadherin (MNCD2) Iowa Developmental StudiesHybridoma Bank

1:1000 WB

N-cadherin Santa Cruz Biotechnology 1 �g/sample IPN-cadherin BD Biosciences 1:5000 IP/IHC/ICC/WBNG2 Chemicon 1:500 IHCGFAP Abcam 1:2000 IHCTuj1 Sigma-Aldrich 1:500 IHCCC1 Calbiochem 1:500 IHCEGFR Cell Signaling Technology 1:4000 IHC/ICC/WBMash1 BD Biosciences 1:3000 IHCOlig2 Millipore 1:500 IHCCD31 Abcam 1:200 IHCKi67 Novocastra 1:500 IHCGFP Invitrogen 1:500 IHCCNP Covance 1:500 ICCADAM10 Novus 1:3000 ICC/IHC/WBADAM10 Abcam 1:4000 ICC/IHC/WBp-ERK1/2 Cell Signaling Technology 1:1000 WBp-EGFR Cell Signaling Technology 1:2000 WBp120 BD Biosciences 1:5000 WBpp120 BD Biosciences 1:5000 WB�-Actin Abcam 1:8000 WB�-catenin BD Bioscience 1:6000 WBBrdU Accurate Chemical & Science 1:700 IHCMBP Covance 1:600 IHCNestin Millipore 1:200 ICC

*Some primary antibodies were used for both immunohistochemistry (IHC) and Western blotting (WB). If nototherwise described, the dilution for IHC/immunocytochemistry (ICC) is 10 times less than the WB dilution. IP,Immunoprecipitation.


In vivo expression of N-cadherin constructs by retroviral infection. SVZNPCs were infected using retroviral particles containing N-cadherinconstructs (Hosokawa et al., 2010). Retroviral production and titer de-termination was done as previously described (Aguirre et al., 2007). Con-trol and transgenic mice were injected with the retroviral stock (2 �l, titer2– 4 � 10 6 plaque-forming units ml �1). Injections were performed ste-reotaxically using the following coordinates (relative to bregma): 0.5,0.85, and 2.2 mm, anterior/posterior, medio/lateral, and dorso/ventral,respectively. Brains were processed at 15 and 30 d postinfection (dpi)using immunofluorescence to analyze cell migration and differentiationof SVZ NPCs, respectively.

Microscopy and cell counting. A Leica DMI6000B confocal laser-scanning microscope TCS-SP5 was used for image localization of FITC(488 nm laser line excitation; 522/35 emission filter), Cy3 (570 nm exci-tation; 605/32 emission filter), and Cy5 (647 excitation; 680/32 emissionfilter). Optical sections (z � 0.5 �m) of confocal epifluorescence imageswere sequentially acquired using LAS AF software (Leica). Images weremerged using NIH ImageJ software and merged images were processedin Photoshop Cs4 software (Adobe) with minimal manipulation of con-trast. At least 3– 4 different brains for each strain and each experimentalcondition were analyzed and counted, as indicated in each respectiveexperiment. Cell counting was performed blindly, analyzing the dorso-lateral region of the SVZ, and tissue sections were matched across sam-ples. For characterization of cells in the SVZ at different time points afteracute and chronic demyelination, a minimum of six correlative slicesfrom a 1-in-10 series located between �0.2 and �1.2 mm (anterior toposterior) from bregma were analyzed. On average, 15–20 sections werequantified using unbiased stereological morphometric analysis of theSVZ to obtain an estimate of the total number of positive cells. Forthe quantifications in Figure 7, GFP � cells in volume matched regions,the center of which contained the injection site, were taken into consid-eration. All cell quantification data were obtained by cell counting usingImageJ software (NIH). Statistical analysis was performed by the Stu-dent’s t test. All data are presented as the mean � SEM, as indicated.

ResultsProteins that play pivotal roles in neural stem and progenitorcell biology are upregulated in the SVZ niche duringdemyelinationTo identify candidate signaling pathways that could enhance cellrecruitment to areas of brain damage we used the followingmouse models of demyelination and remyelination: (1) dietarycuprizone-fed mice as a model of chronic demyelination of theSCWM (Fig. 1A; Hiremath et al., 1998) and (2) lysolecithin-treated mice as a model of induced acute focal demyelination inthe CC (Fig. 1B; Woodruff and Franklin, 1999). Using the cupri-zone model we applied an unbiased proteomics-based approachto elucidate signaling cues present in the SVZ that could be in-volved in promoting repair. SVZ tissue was collected from micetreated with a cuprizone diet or a regular diet for 12 weeks. SVZtissues collected from control mice fed with 15N-SILAM-Mousediet (Silantes, Germany) were used as internal standards forquantification (Kruger et al., 2008; Sinnamon et al., 2012). Usingthis approach, we aimed to identify signaling elements present inthe SVZ niche during the peak of demyelination when the SVZniche was actively adapting to the injury, which could be manip-ulated to promote repair. Protein expression changes were calcu-lated as the ratios of signals from cuprizone-treated SVZ anduntreated SVZ (treated/15N labeled control; Sinnamon et al.,2012 ). Using this novel approach we identified several proteinsthat are known to play roles in neural stem and progenitor cellbiology (Table 2). Of note, proteins including Cadherin super-family members (cadherin-2/N-cadherin and cadherin-13/T-cadherin), Ig superfamily CAMs (neural cell adhesion molecule1/2, CHL1, L1 cell adhesion molecule, Neurofascin, and CADM1;Katidou et al., 2008), Catenin family members (�-catenin,

�-catenin, and delta-catenin; Zhao et al., 2011) and proteins in-volved in focal adhesion formation and cytoskeletal protrusion(Talin 1, vinculin, and actin-related protein 2/3 complex subunit4; DeMali et al., 2002) were found to be differentially regulatedduring the peak of ongoing demyelination.

EGFR� neural progenitor cells expand within the SVZ nichein response to demyelinating injuryUpregulation of molecular signatures in specific cells populationsof the SVZ during demyelination could be an adaptation of theCNS to promote repair. Consistent with this hypothesis, therewas a significant expansion of cells in the dorsolateral SVZ at thepeak of SCWM demyelination (Fig. 1C–E). Immunohistochem-istry (IH) analysis was used to characterize the expanded cellpopulations. A higher number of cells coexpressing EGFR andOlig2 or Mash1 were observed in in the SVZ during the peak ofdemyelination compared with control conditions (Fig. 1C,D anddata not shown). We denoted EGFR� cells as SVZ NPCs in thisstudy (type C cells; also called transit-amplifying cells; Doetsch etal., 1999, 2002), and used the EGFR as a cellular marker for NPCcharacterization. Hence, our data revealed that the expanded cellpopulations corresponded to NPCs. Progenitor populations inthe SVZ at the peak of demyelination were actively dividing(EGFR�BrdU�, Olig2�BrdU�, and Mash1�BrdU� cells) dem-onstrating that their expansion was as a result of the higher pro-liferation rate observed following injury (Fig. 1C–E). Moreover,in mice subjected to acute demyelination, along with an enrich-

Table 2. Shotgun proteomics analysis of SVZ niche tissue during chronicdemyelination of the SCWM

Accession no. Protein descriptionSpectracounts

Treated/control SD

Cell adhesiontr Q6GU11 Q6GU11 Cadherin 2 (N-cadherin) 9 1.6 0.19tr A2AFG8 A2AFG8 L1 cell adhesion molecule 37 1.5 0.07sp Q9WTR5 CAD13 Cadherin-13 5 1.5 0.01sp Q81OU3 NFASC Neurofascin 31 1.5 0.17sp P13595 NCAM1 Neural cell adhesion molecule 1 59 1.4 0.1sp 035136 NCAM2 Neural cell adhesion molecule 2 10 1.4 0.09tr A2AIM8 A2AIM8 Talin 1 9 1.4 0.09sp Q64727 VINC Vinculin 7 1.4 0.3tr A2RRK1 A2RRK1 Cell adhesion molecule with

homology to L1CAM (CHL1)3 1.3 0.05

sp Q8R5M8 CADM1 Cell adhesion molecule l (CADM1) 4 1.3 0.23Migration

sp Q8K3E5 AHI1 Jouberin 2 2.3 0.13sp Q9QWY8 ASAP1 Arf-GAP with SH3 domain, ANK repeat

and PH domain-containingprotein I

2 1.8 0.03

sp Q02248 CNTB1 Catenin beta-1 20 1.8 0.16sp Q9R1V6 ADA22 Disintegrin and metalloproteinase

domain-containing protein 2213 1.7 0.25

tr A2AS97 A2AS97 NCK-associated protein 1 31 1.6 0.33tr Q5SRA0 Q5SRA0 A disintegrin and metalloprotease

domain 239 1.4 0.02

Proliferationsp P63216 GBG3 Guanine nucleotide-binding protein

G(I)/G(S)/G(O) subunit gamma-38 1.7 0.01

tr Q3UPA1 Q3UPA1 Guanine nucleotide binding protein,alpha 11

23 1.3 0.09

sp Q61301 CTNA2 Catenin alpha-2 11 1.3 0.17sp O35927 CTND2 Catenin delta-2 25 1.2 0.48sp p59999 ARPC4 Actin-related protein 2/3 complex

subunit 412 1.2 0.01


ment of SVZ NPCs, we also detected a higher number of SVZNG2� cells and NPCs undergoing OL linage commitment (NG2-dsRed�Ki67�;Fig. 1E, and data not shown). Of interest, the pro-liferation rate was higher in conditions of chronic demyelinationas compared with acute demyelination (650 � 36 cells/10 5mm 3

BrdU� cells and 352 � 16 cells/10 6mm 3 BrdU� cells, respec-tively; p � 0.03). Our analysis suggests that stimuli present duringboth chronic and acute injury keep NPCs in an active cell cyclestate, as cellular expansion during the recovery phase was nolonger different between control and demyelination conditions(data not shown).

To further confirm that the expansion of NPCs in the SVZniche resulted from cell division, we measured proliferation andthe self-renewal capacity of SVZ NPCs after demyelination. Asignificantly higher number of neurospheres were generatedfrom tissue derived from the SVZ of cuprizone-treated mice (Fig.1F). The enhanced colony-forming ability was present exclu-sively during the first and second passages (Fig. 1F), as nonsig-nificant differences were observed after the third passage (datanot shown). Enhanced neurosphere colony-forming capacity wasalso found during acute focal demyelination of the SCWM, as asignificantly increased number of neurospheres were formedfrom NPCs isolated from lysolecithin-injected mice (28 � 3 neu-rospheres/1000 cells) compared with NaCl-injected mice (16 � 4neurospheres/1000 cells, p � 0.05). We also analyzed the differ-entiation potential of these NPCs in vitro. In both treatments,NPCs were multipotent, generating neurons (Tuj� cells), oligo-dendrocytes (CNP� cells), and astrocytes (GFAP� cells); how-ever, higher differentiation capacity was observed during chronicdemyelinating conditions (Fig. 1G).

N-cadherin signaling pathway components are upregulated inthe SVZ in response to demyelinationAmong the proteins identified by the proteomics analysis werecomponents of the N-cadherin signaling pathway, which havebeen implicated in many cellular functions in the nervous systemincluding cell adhesion, proliferation, and migration (Chalasaniand Brewster, 2011; Shikanai et al., 2011; Table 2). In an initialcharacterization, NPCs were found to express N-cadherin, asanalysis of SVZ cells dissociated from adult mice demonstratedthat the majority of EGFR� cells display N-cadherin immunore-activity on the cell surface (Fig. 2A). In an extension of our in vitrocharacterization, we used immunohistochemistry analysis to de-termine which cell populations in the SVZ express N-cadherin invivo. Using this approach, N-cadherin expression was detected inquiescent neural stem cells (type B cells; GFAP� EGFR�), acti-vated neural stem cells (GFAP�EGFR�), and type C cells ortransit-amplifying NPCs (GFAP�EGFR�) (Fig. 2B, and data notshown; see Doetsch et al., 1999 for SVZ cell classification). Inter-estingly, N-cadherin was found to be upregulated during demy-elination, and this newly detected N-cadherin expression waslocalized to NPCs (EGFR� cells) responding within the SVZ tothe white matter injury (Fig. 2B). In agreement with our pro-teomics analysis, N-cadherin and its interaction partners�-catenin and pp120 were found to be upregulated during thepeak of both acute and chronic demyelination (Fig. 2C–F). Thisupregulation was evident only during the peak of demyelination,as expression levels of N-cadherin pathway members returned toor toward control levels during the recovery phase. Together, ourdata indicate that higher expression levels of N-cadherin pathwaycomponents are present during the peak of demyelination in SVZNPCs, suggesting a potential role for this signaling pathway inmyelin repair.

N-cadherin signaling in SVZ neural progenitor cells isinitiated by enhanced proteolytic shedding duringdemyelinationFull-length (135 kDa) N-cadherin undergoes proteolytic cleav-age at the extracellular domain by metalloproteinase activity,generating an intracellular 40 kDa C-terminal fragment (CTF1),which can be processed further by a �-secretase complex into asoluble 35 kDa fragment (CTF2; Nakazora et al., 2010). The ect-odomain cleavage of N-cadherin, which disrupts cell– cell con-tact, is required before PS1/�-secretase cleavage (Nakazora et al.,2010). During demyelination, N-cadherin cleavage was signifi-cantly higher in the SVZ niche of mice with experimentally in-duced demyelination, as indicated by the accumulation of CTF1(Fig. 3A,B, arrowhead) and CTF2 (Fig. 3A,B, line). Interestingly,N-cadherin cleavage returned to control levels after remyelina-tion, indicating that cleavage is temporally regulated (Fig. 3A,B).The cleavage of N-cadherin by a disintegrin and metalloprotei-nase domain-containing protein 10 (ADAM10) leads to changesin cellular behavior, resulting from active N-cadherin signalingupon CTF generation (Kohutek et al., 2009). To determinewhether ADAM10 cleavage of N-cadherin in SVZ NPCs activatesN-cadherin signaling during demyelination, we first character-ized ADAM10 expression in resident cells of the SVZ by immu-nohistochemistry analysis. Our data showed that GFAP� NSCsand EGFR� NPCs, but not CD31� endothelial cells expressADAM10 in the SVZ (Fig. 3C). We next questioned whetherADAM10 in NPCs is directly involved in N-cadherin cleavageduring demyelination. To this end, we first assayed the total ex-pression levels of ADAM10 in the SVZ during injury. ADAM10was upregulated during the peak of demyelination in the SVZ,when N-cadherin cleavage was also at its highest level, indicatinga possible link between these two proteins (Fig. 3A,B,D,E). Wethen performed a series of immunoprecipitation (IP) experi-ments to further elucidate whether ADAM10 regulatesN-cadherin cleavage and signaling activation during demyelina-tion. Using this approach, enriched ADAM10/N-cadherin com-plexes were detected under demyelinating conditions, whichresolved to control levels during recovery (Fig. 3F,G). The tran-sient increase in the association between these signaling elementsprovides evidence they function in the adaptation of the SVZniche to injury.

ADAM10 processes N-cadherin in the SVZ by an EGFR-mediated mechanism during demyelinationEGF is a potent mitogen for NPCs in the SVZ niche, and alsopromotes recruitment of NPCs to acute demyelinating lesions,acting as a positive signal to accelerate the process of remyelina-tion (Aguirre et al., 2007; Cantarella et al., 2008; Gonzalez-Perezet al., 2009). Taking this into consideration, we asked whetherEGFR signaling regulates N-cadherin processing. We first inves-tigated whether the EGFR signaling pathway is activated duringacute and chronic demyelination in the SVZ niche. By Westernblot analysis, higher levels of EGFR phosphorylation and thepresence of the downstream target phospho-ERK1/2 were ob-served in SVZ niche tissue isolated from lysolecithin- andcuprizone-treated mice, confirming EGFR pathway activation(Fig. 4A,B).

In prostate epithelial cells, cleavage of E-cadherin byADAM10 is mediated by EGFR signaling (Grabowska et al.,2012). To address the possibility that a similar mechanism leadsto N-cadherin cleavage in NPCs, we performed cell culture ex-periments from NPCs isolated from the SVZ and then evaluatedN-cadherin expression and colocalization with ADAM10 in the


Figure 2. Elements of the N-cadherin signaling pathway are upregulated in the SVZ niche during the peak of demyelination. A, Characterization of N-cadherin in cellular residents of the adult SVZniche. EGFR � cells (NPCs) of the SVZ express N-cadherin. Analysis by immunocytochemistry revealed that EGFR � NPCs of the adult SVZ display immunoreactivity to N-cadherin on the cellmembrane. The arrowhead indicates the cell selected from a low-magnification view (Low Mag., left) depicted in the right panels at a higher-magnification. B, Cells residing within the adult SVZniche express N-cadherin in vivo. Immunohistochemistry analysis with anti-GFAP and anti-EGFR antibodies was used to characterize N-cadherin expression in neural stem cells (NSCs) and NPCs inthe SVZ. Using this approach, N-cadherin expression was detected in quiescent NSCs (GFAP �EGFR �; data not shown), activated stem cells (GFAP �EGFR �), and NPCs (GFAP �EGFR �). Further,at 7 dpl, an increase in the total number of EGFR � progenitors, and EGFR �N-cadherin � cells was observed in the SVZ. White boxes depict higher-magnification views of the indicated region. C,D, N-cadherin signaling elements are upregulated in response to demyelination. SVZ tissue was dissected to detect total protein levels of N-cadherin signaling elements during acute (C) and chronic(D) demyelination. Western blot analysis demonstrated a transient upregulation of N-cadherin pathway members during the peak of acute (5 dpl) and chronic (12w) demyelination which returnedto or toward control levels after recovery (21 dpl and 12w/4w), respectively. Note that at the demyelination time points (5 dpl and 12w), a significant difference in the levels of phospho-p120(pp120), but not total p120, was detected. E, F, Quantification of data from (C) and (D), respectively. Histograms express results in A.U. after actin normalization. NaCl, Control; contralateral; Lyso,lysolecithin; ipsilateral; Cupz., cuprizone diet; Reg., regular chow diet; LV, lateral ventricle. Data are shown as mean � SEM (n � 3); *p � 0.05, Student’s t test. Scale bars: A, 10 �m; B(low-magnification), 60 �m; B (high-magnification), 15 �m.


presence of an EGF stimulus. Untreated NPCs from the adult SVZshowed only minimal N-cadherin/ADAM10 colocalization on thecell membrane surface (Fig. 4C, top). However, with 2 h of EGFstimulation, N-cadherin and ADAM10 displayed a strong mem-brane colocalization, an indication of N-cadherin cleavage and sig-naling activation (Fig. 4C, middle). This colocalization resolved tocontrol levels after prolonged EGF treatment (24 h; Fig. 4C, bottom).Our results indicate that EGFR signaling mediates N-cadherin cleav-age and pathway activation, as EGF treatment of NPC culturesdecreased levels of full-length N-cadherin and induced phosphory-lation of p120 after prolonged stimulation (Fig. 4D, top). To furthervalidate this finding, we performed IP experiments using proteinextracts of SVZ cells treated with EGF. Increased N-cadherin/ADAM10 interaction occurred during the first hours of EGF treat-ment and returned to normal levels after 24 h of treatment (Fig. 4D,bottom). Consistent with our hypothesis, an EGFR blocker (AG;AG-1478) prevented N-cadherin cleavage (Fig. 4E; CTF panel) andN-cadherin/ADAM10 interaction (Fig. 4E) in primary cell culturesderived from SVZ tissue, demonstrating the direct participation ofEGFR signaling in this process.

To substantiate the role of EGFR signaling in regulatingN-cadherin in the context of demyelination in vivo, we analyzedN-cadherin cleavage in the SVZ of adult Wa2 (Wa2/Wa2; hypo-morphic EGFR signaling; Aguirre and Gallo, 2007; Aguirre et al.,2007) and CNP-hEGFR (overexpressing EGFR in CNP-lineageprogenitors; Ling et al., 2005) mice. Higher CTF levels were ob-served in the CNP-hEGFR samples both under normal condi-tions and after demyelination as compared with wild-type andWa2/Wa2 mice (Fig. 4F). However, in Wa2/Wa2 mice, the accu-mulation of CTFs was not observed under normal conditions orafter demyelination (Fig. 4F). The lack of CTF accumulation inWa2/Wa2 mice could be explained, in part, by a defect in theEGFR-mediated ADAM10/N-cadherin interaction as demon-strated by IP analysis. After demyelination, N-cadherin/ADAM10 complexes were lower in Wa2/Wa2 samples comparedwith control samples, as Wa2/Wa2 mice did not activateADAM10 in response to injury (Fig. 4G).

We performed a biotin-labeling assay to measure cell surfaceexpression levels of full-length N-cadherin to gain further insightinto EGFR-mediated N-cadherin cleavage. SVZ tissue from adult

Figure 3. Increased N-cadherin shedding and ADAM10 expression levels in the SVZ niche during acute and chronic demyelination of the subcortical white matter. A, B, N-cadherin shedding andsignaling activation is enhanced in the SVZ in response to injury. SVZ tissue was dissected from lysolecithin- and cuprizone-treated mice at the indicated time points to perform Western blot (WB)analysis. Note that N-cadherin shedding is most robust in the SVZ niche during the peak of acute and chronic demyelination (7 dpl and 12w, respectively), before returning to control levels duringthe recovery phase (21 dpl and 12w/4w, respectively). Processing of N-cadherin was determined by the presence of the C-terminal fragments CTF1 (arrowhead) and CTF2 (line). C, Characterizationof ADAM10 in resident cells of the adult SVZ niche. Adult brains of wild-type and reporter GFAP-GFP mice were processed for immunohistochemistry analysis with antibodies against ADAM10, EGFR,and CD31 to characterize ADAM10 expression in neural stem cells (NSCs), NPCs, and endothelial cells. ADAM10 colocalized to the cell membrane of GFAP �GFP � NSCs and EGFR � NPCs, but not CD31 �

endothelial cells. D, E, Increased expression levels of ADAM10 were observed during the peak of acute and chronic demyelination in the SVZ. WB analysis using anti-ADAM10 against SVZ protein extracts fromlysolecithin (D) and cuprizone (E) treated mice revealed increased ADAM10 protein levels at the peak of demyelination. F, G, Equal amounts of SVZ lysate from (D) and (E) were incubated with protein-A beadscoupled to N-cadherin antibodies, and the precipitated material was analyzed by WB to assay N-cadherin and ADAM10 interaction during demyelination. Anti-N-cadherin immunoprecipitation followed byanti-ADAM10 immunoblotting demonstrated the enhanced interaction of ADAM10 and N-cadherin during the peak of demyelination. During the recovery period, this difference was no longer evident,suggesting a temporal regulation of this pathway only during the peak of demyelination. NaCl, Control: contralateral; Lyso, lysolecithin; ipsilateral; Cupz., cuprizone diet; Reg., regular chow diet; LV, lateralventricle. Data are shown as mean � SEM (n � 4); *p � 0.05, Student’s t test. Arrow, Full-length N-cadherin; arrowhead, CTF1; line, CTF2. Scale bar, 25 �m.


Figure 4. EGFR signaling regulates N-cadherin pathway activation via ADAM10 in SVZ neural progenitor cells. A, B, The EGFR signaling pathway is activated in the SVZ niche during the peak ofdemyelination. SVZ tissue was collected during the peak of demyelination and during the recovery phase, and processed for Western blot (WB) analysis. EGFR signaling is activated in acute (A) andchronic (B) demyelination as detected by immunoreactivity to phospho-EGFR (Tyr1068) and phospho-ERK1/2 (Thr202/Tyr204) antibodies. Histograms express results in A.U. after actin normaliza-tion and quantify the data at the 5 dpl and 12w time point, respectively. C, EGFR signaling activation promotes N-cadherin/ADAM10 interaction. Adult SVZ tissue was used to prepare NPC culturesto detect N-cadherin and ADAM10 colocalization in NPCs. In vitro EGF stimulation of cultured NPCs promotes ADAM10/N-cadherin interaction in a time-dependent fashion on the cell membrane, asindicated by immunocytochemistry analysis with anti-ADAM10 and anti-N-cadherin antibodies. Confocal images were processed to observe colocalization between ADAM10 and N-cadherin.Protein-protein interaction was most evident from 2 to 4 h of EGF stimulation. Arrow depicts areas of colocalization between ADAM10 and N-cadherin on the cell membrane and also indicates theregion cropped to show high-magnification images (right). D, Adult SVZ tissue was used to prepare NPC cell cultures as above. Cell cultures were treated with EGF at different time points to detectprocessing of full-length N-cadherin and N-cadherin pathway activation (top) and ADAM10/N-cadherin interaction (bottom). Note that processing and (Figure legend continues.)


mice was used to prepare cell cultures to assay N-cadherin bioti-nylation following EGF treatment. Beginning at 1 h after theaddition of EGF to the culture medium, there was a continualdecrease in the levels of biotinylated full-length N-cadherin (Fig.

5A). We next attempted to detect the 95 kDa fragment, generatedexclusively by proteolytic cleavage, in the culture media followingEGF stimulation of NPC cultures (Fig. 5B). Our Western blotdata showed that the 95 kDa N-terminal fragment of N-cadherinaccumulated in the supernatant (culture media) after prolongedEGF stimulation (4 –24 h; Fig. 5C).

To directly link EGFR signaling and ADAM10 activity in NPCs,we generated a Nestin::ADAM10fl/fl mouse line (fl/fl mice), enablingtamoxifen-inducible genetic deletion of ADAM10 in nestin� pro-genitors. Expression of the Nestin intermediate filament pro-tein has been detected in SVZ NPCs in the mature brain(Walker et al., 2010; Fig. 6B), validating nestin-cre as an appro-priate driver for the knockdown of ADAM10 in NPCs and theirprogeny, and not in other neural cell lineages that express ADAM10.In this ADAM10fl/fl mouse, processing of N-cadherin was noticeablydecreased, as CTFs were nearly undetectable (Fig. 5D). Furthermore,the expression levels of the downstream targets of N-cadherin,�-catenin and pp120, were also reduced, indicative of decreasedpathway activity (Fig. 5D). Of note, overall levels of full-lengthN-cadherin were also reduced in ADAM10fl/fl mice, indicating theregulation of N-cadherin in the SVZ depends, in part, on ADAM10expression. After treating NPCs with EGF, accumulation of the 95kDa fragment in the culture medium was absent in NPCs fromADAM10 fl/fl mice (Fig. 5C). Similar results were obtainedwhen Wa2/Wa2 cells were treated with EGF (Fig. 5C).

4

(Figure legend continued.) ADAM10/N-cadherin interaction were most robust at 2– 4 h of EGFRstimulation. E, Adult SVZ NPCs were cultured for 24 h and then pretreated with an EGFR blocker(AG 1478) for 30 min before EGF treatment. Following the indicated treatment, cells werecollected and processed for WB and immunoprecipitation analysis. Note that blocking EGFRphosphorylation using AG 1478 reduced EGFR-mediated N-cadherin shedding and N-cadherin/ADAM10 protein/protein interaction in NPCs of the SVZ. EGFR signaling activation in NPC cul-tures of the SVZ was determined by detecting phosphorylation of the EGFR and ERK by WBanalysis. F, Wild-type, CNP-hEGFR, and Wa2/Wa2 mice were injected with lysolecithin in the CCand at the peak of acute demyelination SVZ tissue was collected for WB analysis. Processing ofN-cadherin in wild-type mice was heightened in response to demyelination (left). In a transgenicmouse where EGFR signaling is enhanced in CNP lineage progenitors (CNP-hEGFR) a constitutiveN-cadherin processing was detected, in both the presence and absence of demyelination (middle). Incontrast, in a mouse in which the EGFR signal is hypoactive (Wa2/Wa2 mice), a reduced N-cadherinprocessing was observed both in control and demyelination conditions. N-cadherin processing wasdetermined by the accumulation of CTF1 (arrowhead) and CTF2 (line) fragments by WB. G, Totalprotein extracts isolated from the SVZ at the peak of acute demyelination from Wa2/� (control) andWa2/Wa2 (impaired EGFR signaling) mice demonstrated that ADAM10 protein expression (top) andADAM10/N-cadherin interaction (bottom) were less abundant in the Wa2/Wa2 samples, comparedwithWa2/�samples.NaCl,Control:contralateral;Lyso, lysolecithin: ipsilateral;Cupz.,cuprizonediet;Reg., regular chow diet. Data are shown as mean � SEM (n � 3); *p � 0.05, Student’s t test.Arrowhead, CTF1; line, CTF2; AG 1478, AG. Scale bar, 15 �m.

Figure 5. EGFR signaling and ADAM10 are required for N-cadherin processing. A, EGFR signaling induces cell surface shedding of N-cadherin in SVZ NPC cultures. Following EGF simulation for theindicated time points, Sulfo-NHS-biotin was used to label cell surface proteins with exposed lysine residues (i.e., N-cadherin), and the labeled material (membrane fraction) was precipitated withstreptavidin beads. Anti-N-cadherin was used with the lysate collected from the membrane fraction to determine the processing state of N-cadherin (unprocessed/full-length or processed).Following EGF stimulation, immunoreactivity against full-length N-cadherin markedly decreases, indicating increased processing in response to EGFR activity. Equal aliquots of intracellular lysatewere analyzed by WB with anti-�-Actin, to demonstrate equal amounts of material were collected, and subsequently used as input for the above analysis. B, Diagram indicating the workflow foranalysis of N-cadherin shedding used in (C). Equal numbers of SVZ NPCs were plated and maintained in vitro. Following EGF stimulation for the indicated time points, the culture media was collectedfor immunoprecipitation and Western blot (WB) analysis to detect the 95 kDa fragment of N-cadherin released upon shedding. C, NPCs from the SVZ of the wild-type, ADAM10 fl/fl, and Wa2/Wa2mice were treated with EGF in vitro for 0, 4, or 24 h. Wild-type mice showed an accumulation of the 95 kDa fragment, the product of N-cadherin cleavage, in the supernatant. Note that accumulationof the 95 kDa fragment was not detected in NPCs isolated from ADAM10 fl/fl or Wa2/Wa2 mice after EGF stimulation, indicating cleavage does not occur at detectable levels in response to treatment.D, Adult ADAM10 fl/fl mice received three tamoxifen injections 24 h apart over the course of 3 d to ablate the expression of ADAM10 in nestin � cells of the SVZ. Following tamoxifen treatment, SVZtissue was collected and processed for WB analysis. N-cadherin shedding and downstream signaling components of the N-cadherin pathway (�-catenin and pp120) were reduced in ADAM10 fl/fl

mice compared with control samples; n � 3. Arrow, Full-length N-cadherin; line, CTF.


Figure 6. EGFR signaling activates the N-cadherin pathway and leads to cell polarization and directional migration of adult SVZ neural progenitor cells. A, EGF treatment promotes cellularpolarization of NPCs. Wild-type SVZ NPCs were cultured on coverslips and maintained under proliferating conditions for 24 h. Cell cultures were maintained in basal media for 4 h before EGFstimulation at the indicated time points. After EGF stimulation, cell cultures were fixed and processed for immunocytochemistry analysis with anti-EGFR and anti-Nestin antibodies. Note that SVZNPCs undergo cytoskeletal rearrangement in a time-dependent fashion upon EGF stimulation. B, Graphical representation of the N-cadherin constructs used in this study (adapted from Hosokawaet al., 2010). The wild-type N-cadherin construct (WT-N-cadherin) retains all the features of endogenous N-cadherin; the extra-cellular domain (ECD), �-catenin binding domain, and p120-bindingdomain. In addition, a GFP tag is present on the C terminus of the protein. dn-N-cadherin retains the features of the WT construct; however, the ECD is truncated resulting in a dominant-negativeeffect. CBR-N-cadherin and JM-N-cadherin also retrain the features of the wild-type construct, with the exception of the deletion of the �-catenin and p120 binding sites, respectively. A GFPsequence coupled to an IRES was used as a control construct (MX-GFP). C, Primary NPC cultures were infected with retrovirus encoding the WT-N-cadherin (WT) and dn-N-cadherin constructs (dn)to analyze expression levels of N-cadherin signaling pathway components. At 3 d after viral transduction, NPCs were collected, lysed, and subjected to WB analysis to assay for N-cadherin pathwayactivity. NPCs transduced with dn-N-cadherin display decreased expression of N-cadherin binding partners �-catenin and pp120, as well as a decreased generation of CTF1 compared withWT-N-cadherin. A similar characterization using CBR-N-cadherin and JM-N-cadherin encoding retrovirus demonstrated decreased expression of �-catenin and p120, respectively (data not shown).D–L, Disrupting N-cadherin signaling prevents the directional migration of NPCs toward an EGF source. Adult SVZ tissue was dissected and prepared for tissue explant culture. Explants weretransduced with several N-cadherin retroviral constructs as indicated. Representative low- (D–G) and high- (H–L) magnification images obtained from the cultures for each condition are displayed.Following viral transduction explants were cultured with EGF-soaked heparin beads on Matrigel. Note that WT-N-cadherin/GFP � transduced cells displayed a migratory morphology (E, I) comparedwith N-cadherin mutant forms (F, G, J, K, L) and control conditions (D, H). M, A higher number of migratory GFP � cells en route to the EGF source were observed in the WT-N-cadherin/GFP conditionscompared with N-cadherin mutant forms and control conditions. Dotted lines indicate the edges of the SVZ explants. Data are shown as mean � SEM (n � 3); *p � 0.03, Student’s t test. Scale bars:A, 10 �m; D–G, 150 �m; H–L, 20 �m.


In several cell types, cadherins have been found to mediate thecell migration cycle by regulating the placement of focal adhe-sions to the leading edge of migratory cells (Nelson et al., 2004;Camand et al., 2012). Both talin 1 and vinculin, intricately in-volved in the formation of focal adhesions, were found to beupregulated at 12 weeks of cuprizone treatment, during whichhigh levels of pEGFR were detected (Table 2; Fig. 4A). To furtherdefine the functional consequences of N-cadherin activation byEGFR signaling in NPCs, we analyzed morphological changes inNPCs stimulated with EGF in vitro. Our immunocytochemicalanalysis demonstrated that 4 h of EGF stimulation of SVZ NPCspromotes cell polarization, as treated cells underwent cytoskeletalrearrangement; by comparison, untreated cells displayed a largercell body and multiple protrusions instead of a single leading edge(Fig. 6A). Altogether, our analysis indicates that N-cadherincleavage and ADAM10 activity are functionally linked by the ac-tivation of EGFR signaling, and that N-cadherin cleavage byADAM10 promotes cell polarization.

N-cadherin promotes cell migration and recruitment ofneural progenitor cells into demyelinating lesionsTo directly determine the functional role of N-cadherin signalingin the adaptation of the SVZ niche during injury, we used an invitro gain-/loss-of-function approach. Recombinant retrovirusesencoding different versions of N-cadherin were produced to sta-bly express N-cadherin variants in vitro and in vivo (Hosokawa etal., 2010; Fig. 6B,C). We sought to address whether N-cadherinpromotes SVZ NPC cell migration by using an in vitro migrationassay with SVZ explants. To this end, adult SVZ explants werecultured in three-dimensional Matrigel and, 24 h later, weretransduced with recombinant retroviruses encoding differentversions of N-cadherin. At 24 h after the completion of transduc-tion, migratory GFP� cells began to detach, acquired a migratorymorphology, and also started to migrate out of the explant to-wards the source of EGF, an indication of chemotaxis and direc-tional migration (Fig. 6D–L). Interestingly, a significantly highernumber of detached GFP� cells were observed in the WT-N-cadherin/GFP condition (Fig. 6D–G). In explants transducedwith mutant forms of N-cadherin (dn, containing a deletion ofthe extra cellular domain; JM, lacking the intracellular p120 bind-ing site; or CBR, lacking the intracellular �-catenin binding site;Hosokawa et al., 2010), neither detachment or notable direc-tional migration of GFP� cells was observed, indicating that thenative form of N-cadherin is necessary to promote chemotaxisand directional migration of NPCs toward an EGF source (Fig.6D–L). Only GFP� cells that had migrated out of the SVZ ex-plants displayed a typical migratory morphology characteristic ofdirectional migration (i.e., a small cell body and a thin leadingedge process; Fig. 6H–L). These morphological changes werenever observed with the N-cadherin mutant forms, suggestingthat N-cadherin-mediated cell polarization is a prerequisite fordirectional migration (Fig. 6H–L). At 48 h after the completion oftransduction, NPC migration distance (distance between theedge of the explant and the migrating cells farthest from the ex-plant) was also significantly enhanced in the WT-N-cadherincondition (Fig. 6M). These findings strongly indicate thatN-cadherin signaling promotes polarity, enabling directional mi-gration of adult SVZ NPCs.

Remyelination of demyelinated lesions is not only ensured bylocal SCWM progenitors, but also by migratory NPCs that aregenerated de novo in the SVZ (Aguirre et al., 2007; Nait-Oumesmar et al., 2008). To further dissect the role of N-cadherinsignaling during brain injury, we performed gain- and loss-of

function experiments in vivo. After removal from 12 weeks ofcuprizone diet, MX-GFP and N-cadherin-encoding retroviruseswere stereotaxically injected into the SVZ of treated mice. At 48 hpostinfection, MX-GFP� cells were found in the SVZ and mi-grating into the rostral migratory stream (RMS) and, between 5and 15 dpi, a higher number of GFP� cells were observed in theolfactory bulb (OB; Fig. 7A). These results confirmed that NPCsoriginally targeted were transduced within the SVZ. This migra-tory pathway was used as a model to elucidate the role ofN-cadherin in promoting recruitment of NPCs from the SVZinto demyelinating lesions.

At 15 dpi, we also observed MX-GFP� cells migrating enroute to the SCWM, with some already integrated into the demy-elinated lesions at this time point (Fig. 7B–D). Interestingly, thenumber of GFP� cells found within the SCWM was significantlyhigher in the WT-N-cadherin/GFP condition compared with thedn-N-cadherin/GFP or MX-GFP conditions (Fig. 7B–D,F). Inall circumstances, the GFP� cells that migrated to the SWCMwere oligodendrocytes (GFP�CC1�) or oligodendrocyte progeni-tors (GFP� NG2�), as determined by immunohistochemistry anal-ysis at 30 dpi (Fig. 7B–D, right, E). N-cadherin overexpressionpromotes enhanced NPC cell recruitment not only as evidencedby the higher number of GFP� cells within demyelinated lesions,but also by the fact that NPCs migrated farther and were moredistributed throughout the entire SCWM compared with thecontrol and dn-N-cadherin conditions (Fig. 7B–D). Of note, al-though the migration of NPCs following viral transduction wasthe focus of our analysis, we cannot exclude the possibility thataltering N-cadherin expression (either by overexpression of WT-N-cadherin or dn-N-cadherin) can also impact the course ofNPC differentiation.

To directly elucidate whether our in vitro finding that EGFRsignaling promotes N-cadherin migration through ADAM10 ac-tivity also acts in vivo, we performed the same analysis using viraltransduction in the Nestin::ADAM10 fl/fl mouse line. First, wedetermined whether there was a predisposition to demyelinationbetween the ADAM10 fl/fl and control mice using the lysolecithinand curpizone paradigm. Our analysis demonstrated that there isno predisposition to demyelination between control andADAM10fl/fl mice, as immunohistochemistry analysis with anti-MPB antibodies at 7 dpl displayed similar levels of demyelinationin the SCWM (Fig. 7G). Similar results were observed using thecuprizone paradigm (data not shown). Adult Nestin::ADAM10fl/fl

and Nestin::ADAM10control mice were fed cuprizone for 12 weeks,and upon cuprizone removal, received tamoxifen injections (3times, 24 h apart). Finally, these mice were transduced withN-cadherin retroviral constructs (Fig. 7H) at 48 h after the lasttamoxifen injection. At 15 dpi, the number of GFP� cells thatmigrated into the demyelinated lesions of the ADAM10 fl/fl micewas significantly reduced compared with the number inADAM10 control mice (Fig. 7H, I), confirming the critical role forADAM10 in EGFR-mediated N-cadherin signaling activation.Together, our data provides evidence that ADAM10 activity isrequired for EGFR-mediated N-cadherin processing, allowingfor NPC cell migration from the SVZ niche into demyelinatedlesions to enhance repair following injury.

DiscussionA number of studies have highlighted the potential for mobilizingendogenous progenitor cells for repair purposes. These studieshave suggested that activation of SVZ NPCs during injury fluc-tuates from transient (Arvidsson et al., 2002; Parent et al., 2002),to persistent (Thored et al., 2006), suggesting that the type of


injury differentially impacts the cellular and molecular propertiesof responding NPCs. Our data indicate that during the onset ofdemyelination, regardless of the course of the injury (acute vschronic), signaling pathways involved in proliferation, cell adhe-

sion, and migration are transiently activated in the SVZ niche. Oftherapeutic interest, recent studies of subjects with pathologicalbrain conditions have revealed that cellular and molecularchanges are also present in cases of human brain injury (Nait-

Figure 7. N-cadherin signaling promotes migration and recruitment of SVZ NPCs into demyelinating lesions and requires ADAM10 activity. A, NPCs retrovirally transduced with MX-GFP withinthe SVZ migrate via the rostral migratory stream to reach the olfactory bulb and the SCWM. B–D, N-cadherin promotes the recruitment of SVZ NPCs into demyelinated lesions of the SCWM.Retroviral-based control (MX-GFP), wild-type N-cadherin (WT-N-cadh/GFP), and dn-N-cadh/GFP constructs were injected directly into the SVZ of adult WT mice after 12 weeks of cuprizonetreatment. Images were taken at 15 dpi (left). Overexpression of N-cadherin promoted the recruitment of NPCs into the demyelinated SCWM. Characterization of GFP � cells that have migrated intothedemyelinatinglesionsoftheSCWM(right)demonstratedthatthemajorityofGFP �cellswereOLlineagecellsasdeterminedbycellmorphologyandCC1immunoreactivity inGFP �cellsat30daftertheviraltransduction (right). E, After 30 dpi, GFP � cells in the SCWM were characterized by immunohistochemistry. GFP � cells at time point were identified as either mature oligodendrocytes (as determined by CC1immunoreactivity and analysis of cell morphology) or oligodendrocyte progenitor cells (NG2 � cells). See B–D (right) for further characterization of GFP � cells found in the SCWM after retroviral transduction inthe SVZ. F, Quantification of data from B–D. G, ADAM10 fl/fl mice display the same susceptibility to demyelination as control mice. Adult mice received a single injection of lysolecithin (2 �l; 1.0%) directly intotheCC.BothADAM10 fl/fl miceandcontrolsreceivedthreetamoxifeninjections(24hapart,3consecutivedays)at24hafter lysolecithininjection.Representativeconfocal imagesat7dplwithanti-MBPantibodiesin the CC of ADAM10 control and ADAM10 fl/fl mice. Note that the same extent of demyelination is observed between the two genotypes. H, Retroviral infection (MX-GFP construct) of NPCs in ADAM10 fl/fl micedemonstrated that a very small percentage of GFP �cells reached the SCWM, compared with control mice. I, Quantification of data from H. LV, Lateral ventricle; OB, olfactory bulb; RMS, rostral migratory stream.Data are shown as mean � SEM; n � 3; *p � 0.05, Student’s t test. Scale bars: A, 200 �m; B–D, 60 �m; E, 15 �m; G, 100 �m; H, 30 �m.


Oumesmar et al., 2007; Tepavcevic et al., 2011), and adult sub-jects with ischemic injuries showed increased cell proliferation in theanatomical region corresponding to the SVZ (Macas et al., 2006). Inlight of the fact that the proliferation and the differentiation potentialof NPCs are improved under these pathological conditions, our dataare consistent with the hypothesis that activation of SVZ NPCs is anadaptation of the brain to promote regeneration.

Despite promising findings, many of the cues and molecularevents that promote activation and expansion of NPCs in theSVZ during pathological brain conditions are still largely un-known. Several studies have performed transcript and proteomicprofiling assays to determine the basic molecular changes thatunderlie neural stem-cell properties in vitro, and show a relation-ship to the changes in vivo (Gurok et al., 2004; Aiba et al., 2006).Whether those elements govern stem cell properties during path-ological conditions remains poorly understood. In the presentwork, we report the application of a mouse SILAC diet to analyzenewly synthesized proteins from brain tissue in vivo, and com-pare the protein profiles that are present during the adaptation ofSVZ niche in conditions of demyelination (Kruger et al., 2008,Sinnamon et al., 2012). To date, many studies attempting to ob-tain quantitative gene expression profiles in higher organisms

have used microarray-based approaches. Although this and othertranscriptome profiling techniques have a number of advantages,regulation above the level of transcription often complicates theinterpretation of such data (Kruger et al., 2008). Our analysis bySILAC to quantify changes of protein expression on a large scalein the SVZ after demyelination is a novel, reliable, and compre-hensive approach to identify proteins and ultimately test theirrole in brain repair. We focused on the N-cadherin pathway, inpart, based on findings that this pathway regulates the mainte-nance of neural precursors during embryonic development(Zhang et al., 2010, 2013). Although a role for N-cadherin inNPCs of the pathological SVZ has not been established before thisstudy, it has been suggested that N-cadherin could be involved inthe initiation of remyelination in the spinal cord in a mousemodel of multiple sclerosis (Hochmeister et al., 2012). In supportof this, our data demonstrated higher levels of N-cadherin pro-cessing and activation of its downstream elements (�-catenin andpp120) in both acute and chronic demyelination, suggesting arole for this signaling pathway in myelin repair.

In pathological processes, several mechanisms of migrationhave been identified for the guidance of neural progenitors fromthe SVZ towards brain lesions (Aguirre et al., 2007; Jablonska et

Figure 8. Model for N-cadherin regulated neural progenitor cell recruitment to areas of demyelinated lesions following induction of experimental acute or chronic demyelination. Upondemyelination of the SCWM, secretion of HB-EGF and TGF� ligands induce the activation of EGFR signaling in NPCs of the SVZ. Activation of ADAM10 activity by EGFR signaling at the peak ofdemyelination mediates N-cadherin shedding. ADAM10 is required for EGF-mediated N-cadherin activation, allowing for NPC cell polarization and subsequent directional migration from the SVZniche into demyelinating lesions to promote the recruitment of regenerative NPCs and enhance repair.


al., 2010), but the downstream targets have remained elusive. Ourdata indicate that N-cadherin signaling pathway activation dur-ing the peak of demyelination promotes migration of NPCs.N-cadherin is the most common cadherin in the vertebrate CNS,and has been shown to play a role in cell adhesion, migration, anddifferentiation, in this context (Bronner-Fraser et al., 1992; Corellet al., 2010). Inactivation of N-cadherin in mice and zebrafishresults in notable defects in neural tube formation (Radice et al.,1997; Lele et al., 2002), and it was shown that those defects arecorrelated with aberrant cell migration (Taniguchi et al., 2006).Classically, N-cadherin has been associated with the maintenanceof tissue integrity through its mediation of cell– cell adhesionbetween stationary cells (Kostetskii et al., 2001; Kim et al., 2005).This observation is in agreement with recent findings for epithe-lial cells, in which disruption of ADAM10-dependent N-cadherinshedding lead to increased cell– cell adhesion and inhibition ofcell motility (Linford et al., 2012). Further, E-cadherin has beenimplicated as being part of a regulatory circuit that includes bothADAM10 and the EGFR in hepatocellular carcinoma (Yue et al.,2013). A positive role for N-cadherin in NPC migration in theadult brain in pathological conditions was further reinforced byour N-cadherin gain-of-function experiments in which we over-expressed WT-N-cadherin in SVZ NPCs of mice that were sub-jected to 12 weeks of cuprizone treatment. A significantly greaternumber of WT-N-cadherin GFP� cells migrated into demyeli-nated lesions of the CC) and furthermore, there were WT-N-cadherin GFP� cells observed migrating long distances withinthe SCWM, including the external capsule. However, althoughNPCs infected with control virus and dn-N-cadherin constructsmigrate along similar routes, fewer dn-N-cadherin GFP� cellswere observed in the CC, with the overall migration distancebeing more limited, as well.

ADAM10 and ADAM17 were found to be upregulated in as-trocytes and macrophages in mouse models of multiple sclerosis(Kieseier et al., 2003; Plumb et al., 2005). In these studies it wasproposed that ADAM10 and ADAM17 contribute to the inflam-matory response by making the cytokine TNF� available, whichis intricately involved in the pathogenesis of multiple sclerosis(Hofman et al., 1989). Although current data demonstrate thatADAM10 and ADAM17 transactivate the EGFR by promotingthe cleavage of pro-heparin-binding EGF ligand (Prenzel et al.,1999; Yan et al., 2002), our data also suggest that during demy-elination EGFR signaling could mediate ADAM10 activation aspart of the cellular and molecular adaptation of the SVZ niche topromote repair. Consistent with this, higher expression levels ofHB-EGF are present in demyelinated tissues (Aguirre et al.,2007). Previously, we demonstrated that manipulation of EGFRsignaling promotes proliferation and migration of NPCs bothduring development and after acute demyelination (Aguirre etal., 2007, and this work). Here, we provide in vitro and in vivoevidence that EGFR signaling is upstream of ADAM10, as NPCsfrom Wa2/Wa2 mice did not activate ADAM10 or theN-cadherin pathway in response to EGF stimulation or demyeli-nating injury. Furthermore, NPCs from ADAM10 fl/fl mice weredefective in promoting N-cadherin cleavage in the presence ofEGF stimulation, supporting the idea that EGFR signaling is up-stream of ADAM10 in mediating N-cadherin activation. Alto-gether, our study supports the hypothesis that the EGFR pathwayand ADAM10 play significant roles in the maintenance of theSVZ niche, but also are key regulators of N-cadherin signalingunder conditions of demyelination (Fig. 8).

Interestingly, our data demonstrates that EGFR mediatedN-cadherin shedding induces the polarization of NPCs allowing

for migration toward demyelinated lesions. The cell migrationcycle is a complex event requiring coordination between matrixproteins, transmembrane receptors that sense the matrix, andintracellular signaling pathways that regulate cell polarity, pro-mote membrane protrusion/retraction, and ultimately drive theprocess of migration (Lauffenburger and Horwitz, 1996). Ourmigration assay using explants from tissue isolated from the SVZniche in cell culture demonstrates that EGFR pathway activationpromotes N-cadherin-mediated cell polarization in NPCs, a pre-requisite for cell migration. Importantly, N-cadherin-mediatedmigration can be regulated at several different levels. A recentstudy has demonstrated that Rab14 plays a role in cell migrationevents by regulating N-cadherin levels at the cell surface and thatthis was the result of altered transport of ADAM10, which pre-vented N-cadherin shedding (Linford et al., 2012). Further workis required to create a complete picture of the pathways regulatingmigration in the pathological SVZ and their role in promotingbrain repair. However, from the present study, it is evident thatNPC chemotaxis toward demyelinated lesions and cell polariza-tion, both mediated in part by EGFR, ADAM10, and N-cadherinsignaling, are critical in the process of myelin repair. Decipheringhow to enhance cell migration and recruitment at the molecularlevel is vital to developing cellular strategies for brain repair fromendogenous progenitor cells.

In summary, by using proteomics analysis in the SVZ duringdemyelination, we identified N-cadherin as a key element in pro-moting repair. We propose that the response of the SVZ niche todemyelination includes altered expression of molecules involvedin proliferation, cell adhesion, and migration during the peak ofdemyelination that are important for regeneration and func-tional recovery. Our study provides critical insights for the designof cell-based therapies to promote more efficient brain repair, byenhancing the recruitment of endogenous progenitor cells fromthe stem cell niche toward the lesion. A deeper understanding ofhow neural stem and progenitor cells and their progeny integratesignals from the microenvironment will lend insight into themechanisms that underlie the adaptation of these cells duringinjury, and ultimately allow targeted therapies to be developed.

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N-cadherin promotes recruitment and migration of neural progenitor cells from the SVZ neural stem...

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