Neural stem cells (NSCs) and neural progenitors (NPs) in the mammalian neocortex give rise to the main cell types of the nervous system. The biological behavior of these NSCs and NPs is regulated by extracellular niche derived autocrine-paracrine signaling factors on a developmental timeline. Our previous reports [Plos One 2010;5:e15341; J Neurochem 2011;117:565–578] have shown that chondroitin sulfate proteoglycan and ApolipoproteinE are autocrine-paracrine survival factors for NSCs. NogoA, a myelin related protein, is expressed in the cortical ventricular zones where NSCs reside. However, the functional role of Nogo signaling proteins in NSC behavior is not completely understood. In this study, we show that NogoA receptors, NogoR1 and PirB, are expressed in the ventricular zone where NSCs reside between E10.5 and 14.5 but not at E15.5. Nogo ligands stimulate NSC survival and proliferation in a dosage-dependent manner in vitro. NogoR1 and PirB are low and high affinity Nogo receptors, respectively and are responsible for the effects of Nogo ligands on NSC behavior. Inhibition of autocrine-paracrine Nogo signaling blocks NSC survival and proliferation. In NSCs, NogoR1 functions through Rho whereas PirB uses Shp1/2 signaling pathways to control NSC behavior. Taken together, this work suggests that Nogo signaling is an important pathway for survival of NSCs. Stem Cells2014;32:1636–1648
Neuroepithelial cells, the first neural stem cells (NSCs), are a single layer of cells that line the ventricles as early as E9.5 during mouse brain development . NSCs are cells that give rise to astrocytes, neurons, and oligodendrocytes in vitro and in vivo. The NSCs self-renew for a number of generations, while differentiating into the lineages of the neural tissue through the intermediate of neural progenitors (NPs). In the embryonic mouse brain, NSCs reside in the ventricular zone (VZ) and subventricular zone (SVZ; for review see ). NSCs and NPs are generated all along the dorsal and ventral VZs in the cortex (CX) and ganglionic eminences (GE). There is an increase in NSC number and neurogenesis at E14.5 with the expansion of the VZs, after which decline in neurogenesis is observed at E17.5 postnatal birth in mammalian brain [2-5].
The behavior of NSCs is regulated by extrinsic factors present in their niche during development . The NSC niche includes factors such as cystatin C, Wnts, Sonic Hedgehog (Shh), and bone morphogenetic protein [7-10]. The exact function of these niche factors in controlling NSC behavior is not understood [11-14]. The behavior and fate of NSCs can be followed in vitro using the neurosphere (nsph) assay . We have developed a clonal nsph formation assay that allows quantitative determination of NSC numbers. Using this clonal assay we have shown that chondroitin sulfate proteoglycan (CSPG) and ApolipoproteinE (ApoE) are autocrine-paracrine factors that stimulate NSC survival and proliferation [16, 17].
NogoA and OmGP are present in the extracellular niche of the embryonic brain VZ where NSCs reside in vivo [18-22]. Nogo isoforms belong to the family of reticulon (RTN) domain proteins. NogoA is primarily expressed in the central nervous system (CNS), NogoB is found in various cell types, whereas NogoC is expressed mainly in muscle. NogoA was first identified as a protein inhibiting neurite outgrowth and signals through at least two receptors, Nogo66 receptor 1 (NogoR1) and leukocyte immunoglobulinlike receptor, also known as PirB, to mediate axonal outgrowth inhibition [23-27]. Nogo RTN homology domain proteins contain two transmembrane regions separated by a 66 amino acid loop, Nogo66, which is able to inhibit neurite outgrowth in vitro. To increase solubility and ease of purification, Nogo66 can be reduced to Nogo54, which still inhibits neurite outgrowth. Like NogoA, Nogo66 and 54 function through NogoR1 and PirB [24, 28-32]. NogoA is also important for maturation of oligodendrocytes and neuronal fasciculation and guidance during early brain development [33-36]. Soluble ectodomains generated from the receptors NogoR1 and PirB have been shown to block Nogo66 and improve neuronal sprouting in spinal cord injury [26, 27, 37]. Recent studies have shown that NogoR1 and NogoR3 receptors enable CSPG-dependent inhibition of neuronal outgrowth, providing evidence of a shared mechanism . NogoA and NogoR1 have been shown to play distinct roles in adult NSC homeostasis . Nogo has also been shown to regulate Nanog expression in embryonic stem cells. Different regions of Nogo and Nogo 66 have been shown to play a role in differentiation of neural progenitors [40-42].
In this study, we sought to understand the function of NogoA, NogoR1, and PirB in NSC behavior. We found NogoR1 and PirB to be expressed in the VZ/SVZ regions and GE at E14.5. However, the expression of these receptors is absent in the VZ/SVZ regions at E15.5. Interestingly, picomolar (pM) and nanomolar (nM) doses of Nogo66/54 stimulated NSC survival and proliferation via PirB and NogoR1, respectively. Blocking of endogenous NogoA signaling by using a combination of NogoR1 and PirB receptor knockout (KO) mice-derived NSCs/NPs and ectodomains led to decrease in nsph size and NSC numbers. Lastly, Nogo stimulation of NSC survival via NogoR1 required Rho proteins, while PirB required Shp1/2. Thus NogoA, acting through NogoR1/Rho and PirB-Shp1/2 signaling pathways, is an autocrine-paracrine factor that stimulates NSC survival and proliferation.
Animals and Tissue Collection
The animal procedures were performed in accordance with the IACUC and NACLAR guidelines and approved by the animal department (http://www.brc.astar.edu.sg/index.php? sectionID=11). The morning of plug detection was considered as embryonic day 0.5 (E0.5), and fetuses were removed from pregnant females at respective stages of development, euthanized in CO2 chambers, and individual brains were dissected before mounting in boats with cryo-mounting media and isopentane-dryice freezing the specimen. Sections of 12–14 µM thicknesses were obtained from the brains after mounting in chucks fitted in Leica cryostat. The sections were embedded in poly-l-lysine (PLL)-coated super frost slides and dried in room temperature for a few hours before proceeding for immunohistochemistry procedures.
Nogo54 Protein Purification, Constructs and Inhibitors
Constructs pGEX-GST, pGEX-GST Nogo54 were used for protein overexpression and purification as described previously [31, 43, 44]. Plasmids that have alkaline phosphatase (AP) fusions with “His” tag (pAP, pAP-Nogo66, pAP-NogoR1 ectodomain (NogoR1ecto), pAP-PirB ectodomain (PirBecto), Phospho Tyrosine phosphatase (PTP) σ ectodomain (PTP σ ecto) constructs (PTP δ Lys ecto control) were transfected in HEK 293T cells for overexpression, and purification was performed as described previously [26, 45, 46]. Receptor associated protein inhibitor (RAP) was used to inhibit ApoE receptors in NSCs/NPs as described previously .
Neuroblastoma Cultures and Neurite Outgrowth Assays
Neuroblastoma (NB) cultures for N1E-115 and ND7 cell lines were maintained and used in neurite outgrowth assays as described previously [43, 47].
Isolation and Culturing of nsphs
NSCs/NPs were isolated from forebrain embryonic cortex at various developmental stages (E10.5–E15.5) from wild type C57BL/6 (+/+) mice. NSCs/NPs were also isolated from NogoR1−/−, PirB−/− (both from MMRC) and PirB−/− (Tohoku University) at E14.5. The characterization of the homozygote and heterozygote mice genotypes for NogoR1 and PirB was according to the protocols from (http://www.mmrrc.org/catalog/sds.php?mmrrc_id=30668) and [48, 49]. Dissociated cortical tissue per cells were triturated and seeded at 1 × 105 cells per mL (bulk density), 2 x 103 cells per mL (low density), 50 cells per mL (clonal density) in NSC growth medium [Dulbecco's modified Eagle's medium (DMEM)/F-12, B27 supplement, 20 ng/mL epidermal growth factor (EGF), 10 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, http://www.invitrogen.com)] [16, 17]. Cells were grown at 37°C in a 5% CO2 atmosphere in a humidified incubator. Nsphs were passaged every 4–7 days. We estimate that under low-density culture conditions approximately 98% of nsphs are clonal . Clonal cultures were cultured at 50–100 cells per well where aggregation is not observed . In some experiments, dissociated NSCs/NPs were plated at 2.5 × 103 cells per mL in a 1.2% w/v hydrogel solution that was made by dissolving the arginine polymer (a gift from Dr. Kurisawa, IBN, Singapore) in NSC growth medium at 37°C as previously described . In adherent cultures, dissociated cells were plated at 1 × 104 cells per mL on 0.1% poly-l-ornithine (PLO) (Sigma)-coated plates. Nsph numbers were assessed wherein NSCs/NPs were cultured for 5–7 days with recombinant Nogo54 after cleavage from GST (Nogo54), alkaline phosphatase (AP), AP-Nogo66 (Nogo66), NogoR1ecto, PirB ecto, Human RAP (Innovative Research), PTP σ receptor ecto, PTP δ lys ecto control. For secondary nsph formation in self-renewal assays, nsphs were collected, triturated, reseeded at respective clonal densities and grown for another 7 days. Nsph numbers are expressed as nsph formation unit (NFU) which refers to the number of nsphs formed for every 100 cells plated.
Differentiation and Immunocytochemistry
Single nsphs from low or clonal density cultures were transferred to each well of a 50 well cover glass (Sigma) coated with PLO (0.01%) and laminin (10 µg/mL; Invitrogen). Nsphs were differentiated for 5 days in differentiation medium [DMEM/F-12, B27 supplement, 1% penicillin/streptomycin, and 0.5% foetal bovine serum (FBS) (Invitrogen)]. After plating cells overnight or differentiating the nsphs, the coverslips were fixed with 4% paraformaldehyde (PFA) for 20 minutes and washed several times with and without permeabilization (0.1% Triton X-100) before blocking with 3% bovine serum albumin (BSA). NSCs/NPs were stained with mouse IgM anti-O4 (Chemicon, Temecula, CA, http://www.chemicon.com), mouse IgG2a anti-Tuj1 (βIII-tubulin) (Covance, Princeton, NJ, http://www.covance.com), and rabbit IgG anti-glial fibrillary acidic protein (GFAP) (1:1,000; Dako, Glostrup, Denmark, http://www.dako.com). The secondary antibodies were Alexa Fluor antibodies 488 anti-mouse IgM, 594 anti-mouse IgG2a, and 647 anti-rabbit IgG (Invitrogen). NSCs/NPs were also stained with rabbit anti-NogoR1 and goat anti-LLIRB (PirB) or rat anti-LLIRB. The cells were subsequently stained with 488 anti-rabbit IgG and 488 anti-rat IgG. The cells or the flattened nsphs were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and differentiated neurons, astrocytes, and oligodendrocytes were scored based on staining and morphology.
Total RNA was isolated from nsphs at relevant days (E10.5–E15.5) according to manufacturer's instruction (Trizol) and RNA columns (Qiagen, Hilden, Germany, http://www1.qiagen.com) were used to purify total RNA. Total RNA was used to reverse transcribe and obtain cDNA using first super script RTIII (Life Technologies, Rockville, MD, http://www.lifetech.com). The cDNA was diluted and used for PCR reaction using Taq polymerase (NEB) with respective primers as mentioned in Supporting Information Table 1.
NogoR1 and PirB were detected by Western blotting. NSCs/NPs were lysed with RIPA lysis buffer [150 mM NaCl, 50 mM Tris HCl pH 7.8, 0.25 mM EDTA pH 8.0, 1% sodium deoxycholate, 1% Triton X-100] and cleared lysate was used for Western blotting receptor NogoR1 or immunoprecipitation of PirB. 100–200 µg of protein was loaded on SDS-PAGE gel and probed with the following antibodies: anti-NogoR1 (1:100), anti-LLIRB3 (1:100), anti-Sox2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), anti-Nestin (1:500; Millipore, Billerica, MA, http://www.millipore.com), anti-β-actin (1:1,000).
For immunohistochemistry, primary brain slices were embedded on PLL-coated slides and dried for 4–6 hours at room temperature. The sections were washed with PBS before fixation with 50% cold methanol-acetone solvent (−20°C). The sections were further washed with PBS several times with and without permeabilization (PBS containing 0.1% Triton X-100) and blocked with 5% normal goat serum (NGS) and 2% BSA mixture. The sections were then incubated with primary and secondary antibodies overnight and at room temperature, respectively, before counterstaining with DAPI. Immunoreagents were diluted in PBS containing NGS and BSA. The following primary and secondary antibodies were used in the immunohistochemistry procedure: NogoA (Santa Cruz), NogoR1 (Millipore), PirB (Santa Cruz; BD), Nestin (BD), Tuj1 (Covance) (1:100–500), Alexa Fluor 568 anti-rabbit, Alexa Fluor 488 anti-mouse, Alexa Fluor 568 anti-goat, Alexa Fluor 647 anti-rabbit, and Alexa Fluor 568 anti-mouse (1:500).
TUNEL and BrdU Assays
NSCs/NPs were dissociated and plated in PLO/laminin-coated coverslips. The reagents/purified protein samples were added to growth medium. Cells were monitored and fixed with 4% PFA at relevant time points. The coverslips were washed with PBS extensively to remove excess fixative before permeabilization of cell membrane with Triton X-100 (0.2%–0.5%). Detergent-treated cell samples were washed, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction was performed at 37°C as per the assay to label apoptotic nuclei (Promega, Madison, WI, http://www.promega.com). 5-bromo-2-deoxyuridine (BrdU) assays were performed by adding BrdU to cultures in growth medium after treatment with proteins/inhibitors for 2 hours as per the manufacturer (Neomarkers). The cells were later fixed using 4% PFA, washed, and permeabilized. Cells were then blocked with (NGS/BSA) reagent and incubated with anti-BrdU antibody overnight and signal was detected using secondary Alexa Fluor 488 anti-mouse (Invitrogen). The nuclei were counterstained with DAPI. The TUNEL positive or BrdU positive NSCs/NPs were scored as percentage cells positive in comparison to total DAPI nuclei using Metamorph (cell scoring) imaging tool.
Results are represented as mean ± SEM where n means number of experiments or samples unless otherwise stated. Statistical significance was performed using One-way ANOVA with post hoc comparisons using Bonferroni test, p values denoted in the figures are *, p < .05; **, p < .01; ***, p < .001. The phase images of NB cultures were taken with the Nikon Eclipse TS100 microscope and confocal images with Olympus point scanning FV-1000 confocal microscope by sequential scanning in order to avoid crosstalk of fluorophores.
NogoR1 and PirB are Expressed in NSCs In Vitro and in the VZ and GE of Developing Forebrain
NogoA is expressed in the VZ and SVZ of developing forebrain . Using reverse transcriptase polymerase chain reaction, we show that NogoA, NogoR1, and PirB transcripts are expressed in developmental stages (E10.5–E14.5) using nsphs derived from embryonic mouse brain (Fig. 1A). Interestingly, NogoR1 and PirB receptor expression is not seen at E15.5. There is a distinct localization pattern of endogenous NogoR1 and PirB receptors in vitro in embryo (E14.5) derived NSCs/NPs (Fig. 1C, 1E). NogoR1 seems to be present in both plasma membrane and endoplasmic reticulum-like compartments whereas PirB is asymmetrically localized in the membrane in 10%–15% of NSCs/NPs as shown (Fig. 1C, 1E). The antibodies against NogoR1 and PirB receptors are specific because there was only background staining from NSCs, NSC lysates, and cortical coronal brain slices derived from respective NogoR1 (−/−) and PirB (−/−) KO mice (Supporting Information Fig. S1).
To determine whether NogoR1 and PirB are expressed in NSCs and NPs in vivo we turned to in situ analysis of brain slices. E14.5 brain slices were probed with DAPI and nestin antibodies and the VZ and SVZ (VZ's) were localized (Fig. 2). The specificity of the antibodies was shown by using tissues from NogoR1−/− and PirB−/− knockout mice (see in situ in Supporting Information Fig. S1A, S1B and western in Fig. S1C). Nestin is an early marker for NSCs/NPs and is turned off as cells differentiate into neurons, astrocytes, and oligodendrocytes. We were able to colocalize NogoR1 (Fig. 2A) and PirB (Fig. 2B) with nestin in the VZ and SVZ. We looked in detail at two regions of the E14.5 brain—the CX (panels e–h) and the GE (i–l). The CX and GE are adjacent regions of the brain where NSCs/NPs differentiate and neurons and axons begin to migrate. Cellular colocalization of NogoR1/PirB with nestin can be seen in the inserts to panels f, g, and h (CX) and j, k, and l (GE). The data show that both NogoR1 and PirB are expressed in NSCs/NPs but are not restricted to these cells. Interestingly, NogoR1 and PirB receptors are not expressed in the apical VZ/SVZ zones at E15.5. Nestin labeling is specific to the apical VZ/SVZs of the dorsal cortex at E15.5 (Fig. 3A, 3B) while Tuj1 is mostly present at the basal part of the cortex and does not colocalize with nestin. NogoR1 and PirB receptors follow Tuj1 expression and not Nestin at E15.5 (Fig. 3). These results show a developmental change in expression and localization pattern of NogoR1 and PirB receptors during the transition from E14.5 to E15.5, which might signify a switch in functional roles of Nogo signaling. These results clearly show that NogoR1 and PirB are expressed in areas of embryonic brain where NSCs reside and are therefore able to play a role in NSC survival and proliferation.
Nogo66/54 Increases nsph Number and Size
Since NogoA, NogoR1, and PirB are expressed in the VZ and SVZ at E14.5, where NSCs reside, we wanted to know whether NogoA regulates NSC behavior. For these experiments, we used Nogo66 and 54 polypeptides that mimic the NogoA ligand. Previous reports have shown that Nogo66 or Nogo54 can inhibit neurite outgrowth [27, 31, 52]. To confirm whether our Nogo polypeptides were active, we added them to differentiated NB cultures where we could score for neurite length. Both Nogo54 and Nogo66 significantly reduced neurite length (Supporting Information Figs. S2, S3). Specific ectodomains for NogoR1 and PirB receptors have been shown to reverse Nogo66-mediated neurite outgrowth inhibition [26, 27, 37, 53]. We confirmed that NogoR1 and PirB ectodomains were able to block the effect of Nogo54 and Nogo66 on neurite length (Supporting Information Figs. S2, S3). Next, we investigated the effect of Nogo ligands on nsph formation. We found that Nogo66 stimulated NFU by 1.7–2-fold over a wide range of concentration (100 pM to 50 nM; Fig. 4A) and this was correlated with a decrease in TUNEL positive cells and an increase in BrdU positive cells (Fig. 4B, 4C).
In order to assess the role of NogoR1 and PirB receptors in Nogo66 stimulation, we repeated the nsph formation experiment with NSCs/NPs derived from NogoR1−/− or PirB−/− mice. Nogo ligand between 10 and 50 nM did not affect NFU in NogoR1−/− NSCs/NPs, but at pM doses was still stimulatory (Fig. 4A). In contrast, Nogo ligand at 100–250 pM did not affect NFU from PirB−/− NSCs/NPs, but higher doses in the nM range were still stimulatory (Fig. 4A). Nogo66 concentrations that gave 50% stimulation (SC50) of NFUs were 35 pM for PirB and 13 nM for NogoR1 (Supporting Information Fig. S4). These results suggest that (a) Nogo66 stimulation of NFU requires both NogoR1 and PirB and (b) that NogoR1 and PirB are low and high affinity receptors for Nogo66, respectively. The Nogo54 C-terminal mutation (Lys50, Glu51, Arg53, and Arg54 changed to Ala) did not have any stimulatory effect on NSCs/NPs (data not shown; [31, 52]). The stimulation of NFU by Nogo66 and Nogo54 was found at both clonal (100 cells per mL) and low densities (2,000 cells per well). Furthermore, similar results with Nogo66 and Nogo54 were obtained when the nsph formation assay was carried out in hydrogel (Supporting Information Fig. S5A). The full-length NogoA (mouse and human) positive lysates increases nsph number in comparison to empty vector suggesting that the effect of Nogo on NSCs/NPs is not restricted to Nogo54/66 (Supporting Information Fig. S5C). It is not an artificial effect produced by Nogo-66 since NogoA lysates have the same effect. The sphere size was analyzed as a measure of proliferation in clonal hydrogel experiments. There was 1.61 (50–100 µm) and 3.33 (>100 µm) folds increase in size compared to that of GST/AP controls (Supporting Information Fig. S5B). Taken together, these results suggest that NogoA is important for NSC/NPs survival and proliferation.
Nogo54/66 Increases NSC Numbers
To determine whether Nogo ligand could affect NSC numbers, we quantified the number of multipotent nsphs generated (in clonal assays) in the presence and absence of different concentrations of Nogo66 (Fig. 5). The NSC numbers are increased approximately fourfold to fivefold by the addition of Nogo66 at pM or nM concentrations (Fig. 5H). Next we carried out nsph formation assays to see if Nogo66 influences self-renewal. In the absence of Nogo66 the stimulation of NFU was lost demonstrating the importance of Nogo signaling in self-renewal (Fig. 5G).
Autocrine-Paracrine Nature of Nogo Signaling on NSC Survival
In order to investigate the autocrine-paracrine nature of Nogo signaling, we blocked NogoR1 and PirB receptors and measured nsph size and NSC frequency in the absence of added ligand. Ectodomains for NogoR1 or PirB have been shown to functionally block these receptors in neurite outgrowth experiments (Supporting Information Figs. S2, S3). The ectodomains were added to NogoR1−/− and PirB−/− NSC/NP cultures and nsph size and NSC number measured. NogoR1 and PirB ecto are able to reduce nsph formation substantially in comparison to AP control (Fig. 6A, 6B). The decrease in nsph number was associated with significant decrease in nsph size (Fig. 6A–6D). NSC numbers are also reduced by approximately 3.5–4-fold, when both PirB and NogoR1 receptors are blocked (Fig. 6E, 6F). The decrease in nsph number and size in ectodomain assays was correlated to an increase in TUNEL-positive nuclei and decrease in BrdU-positive cells (Supporting Information Fig. S6A, S6B). Thus Nogo is an autocrine-paracrine survival factor for NSCs.
Nsphs obtained from experiments where Nogo66 showed effect on NSC/NP survival showed an increase in NSC and astro-glial NP frequency when differentiated. There was a 2.5–3-fold increase in the astro-glial lineage at both pM and nM doses of Nogo66 in comparison to AP control (Fig. 5E, 5F). Moreover, autocrine/paracrine inhibition of Nogo signaling by ectodomains showed threefold to fourfold reduction in number of astro-glial positive NPs (Fig. 6E). These results suggest a role for Nogo signaling in promoting the generation of astro-glial NPs. Further work needs to be done to understand the precise role of Nogo signaling in the glial lineage. Lastly, Nogo did not affect or protect against general cell toxicity induced by lipopolysaccharide, acrylamide, or dicoflenac (Supporting Information Table 2) suggesting that the Nogo stimulation of NSC survival was specific.
Nogo, CSPG, and ApoE and NSC survival
CSPG and ApoE found in E14.5 NSC/NP conditioned medium (CM) stimulate NSC proliferation and survival [16, 17]. In order to study the role of Nogo, CSPG, and ApoE in NSC/NP survival, we used antagonists to block signaling and measured nsph formation. To PirB−/− NSCs/NPs, we added NogoR1ecto to block NogoR1, PTP σ receptor ecto to block CSPG, and RAP inhibitor to block ApoE [17, 27, 45]. Inhibition of NogoR1 and CSPG in the absence of PirB receptor reduced nsph formation significantly in comparison to control (Supporting Information Fig. S7). However, addition of RAP inhibitor in these experiments did not further reduce nsph number. These results suggest that basal signaling maintains nsph formation even when Nogo, CSPG, and ApoE signaling are simultaneously inhibited.
NogoR1 and PirB Signal NSC Survival Through Distinct Pathways
NogoR1 receptor functions with cytoplasmic mediator Rho toward axonal plasticity and regeneration. We used signaling inhibitors to investigate the proteins involved downstream of NogoR1 and PirB receptors. The IC50 values for inhibitors obtained with nsph formation assays in the absence of Nogo66 ligand were used as a reference. The rationale for these signaling pathway experiments is as follows; we compare the dose response curves and IC50 values under three conditions in the presence of inhibitor. (a) with AP control, (b) with pM Nogo66 (PirB), and (c) with nM Nogo66 (NogoR1). If the Nogo66 curves/IC50 values follow AP controls, it suggests that the inhibitors have blocked the stimulation caused by Nogo66. In contrast if the dose response curves are shifted to the right and IC50 values higher, it suggests that the inhibitors are not blocking and the pathway is not involved. We used a similar strategy to investigate signaling pathways of NSC survival factors CSPG and ApoE [16, 17]. Inhibitor IC50 values were then measured in the presence of Nogo66 at pM or nM concentration; a decrease in IC50 suggested the protein was involved in Nogo signaling while an increase in IC50 suggested it was not.
Protein kinase C (PKC) has also been shown to be important for Nogo signaling, and inhibition of PKC signaling using Go 6796 reduced IC50 at both nM and pM concentrations of Nogo66 (Fig. 7A). The reference IC50 for Go 6796 was 3.83 ± 0.07, and this value was reduced to 1.58 ± 0.04 at nM Nogo66 and 1.49 ± 0.11 at pM Nogo66 treatments. Rho and Rho kinase inhibitors, Y27632 and C3, reduced IC50 at nM but not at pM (Fig. 7B, 7C). The changes in IC50 from reference for Y27632 and C3 were 5.87 ± 1.7 µM and 0.7 ± 0.09 µg/mL, and reduced to 1.34 ± 1.2 µM and 0.29 ± 0.05 µg/mL with nM Nogo66, respectively.
Shp1/2 inhibitors, NSC878777 and PHPS1, reduced IC50 at pM Nogo66 but not at nM. The NSC 87877 reference IC50 value was 15 ± 0.09 µM and decreased to 10 ± 0.06 µM with pM Nogo66. With PHPS1, the reference IC50 value was 20 ± 0.9 µM and decreased to 11 ± 0.3 µM with pM Nogo66. Taken together, these data suggest NogoR1 signals through Rho and Rho kinase (Fig. 7B, 7C) while PirB signals through Shp1/2 protein (Fig. 7D, 7E).
NogoA proteins were first identified as inhibitors of neurite regeneration . Subsequent studies have revealed a role for NogoA in neuronal and oligodendrocyte differentiation, neuronal pruning and plasticity [18, 19, 36, 54], and induction of neural cell types in the early developing brain during neural plate formation . NogoA is specifically expressed in the VZ/SVZ layers of mammalian embryonic brain . However, the function of Nogo signaling in embryonic NSC behavior is not known. Here we have investigated the functional role for Nogo signaling in NSC/NP behavior.
Expression of NogoA Receptors in NSCs
NogoA and NogoR1 transcripts are localized to human brain VZs and postmitotic cells of the cortical plate by in situ hybridization [56, 57]. We find that NogoR1 and PirB are expressed between E10.5 and E14.5 in NSCs/NPs in vitro but not at E15.5. In vivo, at E14.5, NogoR1 and PirB were found to be expressed in both VZ and SVZ layers where NSCs reside. The presence of NogoA, NogoR1, and PirB in the VZ/SVZ zone suggests potential roles for Nogo signaling in NSC behavior and neurogenesis in vivo . Moreover, Nogo receptors are also expressed in the GE. The expression of Nogo receptors in the ventral regions (GE) of the embryonic brain suggests roles in migrating neuronal and glial progenitor populations responsible for cortical expansion. By E15.5, both NogoR1 and PirB are found only in the non VZ/SVZ basal cortex, suggesting an interesting switch in Nogo function from E14.5. The regulation of Nogo receptors at E15.5 could be addressed by conditional gain of function studies in NSCs. Thus, expression pattern studies reveal important changes in expression and localization pattern of Nogo signaling receptors in the cortical neuroepithelium during early cortical expansion of the embryonic brain.
NogoR1 and PirB Receptors are Involved in NSC Survival and Proliferation
To investigate the role of NogoR1 and PirB in NSC function, we used Nogo ligands 66 and 54 and cell lysates expressing full-length NogoA. We found that over a wide range of concentrations, Nogo54/66 ligands stimulated NSC/NP survival, proliferation, potency, and frequency. Using NSCs/NPs derived from KO mice, we were able to demonstrate that NogoR1 and PirB receptors sense nM and pM quantities of Nogo66 ligand. Thus, NogoR1 and PirB are low and high affinity Nogo receptors, respectively, involved in NSC behavior. Further work will be required to characterize the exact ligand receptor binding characteristics (Kd) of NogoR1 and PirB in NSCs. The high affinity of Nogo66-PirB activity in NSCs/NPs in vitro does not have its counterpart in Nogo inhibition neurite outgrowth inhibition. The presence of both pM and nM range of affinities suggest a wide range of sensitivity in NogoR1 and PirB receptor biological function in vitro in NSCs/NPs. It would be interesting to know whether gradient expression of NogoA at early development of brain dictates the affinity characteristics of NogoR1 and PirB receptors function in vivo. The NSC/NP assays have been studied under clonal conditions as a stringent requirement to report NSC activity distinct from NP activity in vitro [16, 17].
The increase in bipotential astro-glial positive nsphs (NPs) after differentiation at both pM and nM stimulatory doses suggests potential roles of Nogo signaling in promoting differentiation into the glial lineage. Interestingly, selection of NSCs/NPs positive for NogoR1 and PirB receptors by fluorescence-activated cell sorting (FACS) analysis show a bias toward nsphs differentiating into oligodendrocytes (data not shown). Further studies are necessary to identify how Nogo signaling pathways may promote oligodendrocyte differentiation in vitro and in vivo.
NSC Niche and Autocrine-Paracrine Nogo Signaling in NSC Survival
The function of the niche and its associated autocrine-paracrine factors, extracellular matrix, and membrane bound molecules, is important for survival and regulation of both embryonic and adult NSCs [58-61]. Our studies identified a number of proteins, for example, DSD1-proteoglycan and ApoE, from CM of NSCs/NPs suggesting novel functions for myelin-related and lipid transporting proteins in NSC biology [16, 17, 62-64]. In an autocrine-paracrine context, the ectodomains of NogoR1 and PirB receptors have been used to antagonize Nogo ligand, in spinal cord injury [24, 26, 53, 65]. To test the role of autocrine-paracrine signaling for NogoA in NSC behavior, we used combinations of NogoR1 or PirB KO NSCs/NPs and ectodomains to inhibit the wild type receptor; NogoR1ecto with PirB KO cells and PirBecto with NogoR1 KO cells. When both NogoR1 and PirB receptors were blocked there was a significant reduction in NSC/NP survival, proliferation and NSC frequency. This is good evidence for NogoA as an autocrine paracrine factor and further supports the idea that NogoR1 and PirB are individually redundant in NSC survival.
We also assessed the roles of blocking ApoE signaling (using RAP) and CSPG receptor ectodomain PTP σ ecto in embryonic NSCs/NPs derived from PirB KO mice. We found that RAP and the PTP σ ecto did not reduce NSC/NP survival further. These results indicate that EGF/FGF growth factors provide a basal survival of NSCs/NPs and that additional protein factors are stimulatory but not essential. However, we cannot rule out that additional as yet unidentified proteins support basal NSC/NP survival in addition to EGF/FGF. Mice that lack NogoA do not have any obvious NSC survival problems but have defective migration of BrdU positive neuronal precursor cells at E17.5 . This suggests that more ligands or extracellular proteins have the potential to be redundant in NSC survival function in vivo. It is essential to investigate further the NSC survival, and related phenotypes, in mice that lack a combination of the abovementioned ligands or their respective receptors.
Nogo Stimulates NSC Survival in a Dose-Dependent Manner Through NogoR1-Rho and PirB-Shp Signaling Pathways
Several survival signaling pathways including EGF/FGF, Notch, Wnt, and Shh have been known to affect either NSC or NP survival [7, 10, 66-71]. Mice that lack Notch receptor or Shh/Smoothened (Smo) have decreased NSC survival in vivo [69, 70, 72]. Addition of Notch ligand Delta or Wnt3a has been shown to promote NSC survival in clonal assays [73, 74]. Our studies show that addition of CSPG, ApoE, and Nogo increases NSC frequency by 3.5-, 2.2-, and 3.7-fold, respectively. Notch signaling has also been shown to regulate NSC survival through CBF-1 transcriptional regulation and Notch activation induces Hes3, Shh through rapid activation of serine/threonine kinase Akt, and STAT3 promotes the survival of NSCs [71, 73]. Our previous studies show the importance of epidermal growth factor receptor (EGFR) and extracellular-signal-regulated kinases (ERKs) survival signaling pathways for CSPG and ApoE in NSC survival [16, 17].
Nogo proteins have been shown to function through Rho-Rho kinase and PKC in axonal injury . NogoR1 forms a complex with p75 coreceptor and signals through Rho and Rho kinase proteins [23, 32, 76]. PirB has been shown to associate with PTPN (Shp1/2) adaptor proteins in B cells and neurons [46, 77-79].
For NSCs/NPs, we found that NogoR1 and PirB signal through independent cytoplasmic effector proteins RhoA and Shp, respectively. At pM doses of Nogo66, we found that receptor PirB activates Shp adaptor proteins and at nM doses, NogoR1 activates Rho kinase protein in a specific manner to regulate NSC survival.
Our studies on expression, characterization and in vitro activity of NogoR1 and PirB suggest novel roles for NogoA signaling in embryonic NSCs/NPs survival pathways through Rho and Shp1/2. Through these NSC studies an understanding of signaling pathways that control NSC/NP survival and proliferation is emerging. An interesting question for future studies is to evaluate the roles of NogoA and other signaling pathways in regulating NSCs in vivo and how they may be involved in disease states such as neurodevelopmental disorders and oligodendrogliomas [80-83].
We thank Dr. Motoichi Kurisawa from the Institute of Bioengineering and nanotechnology, Singapore for providing the hydrogel, and Dr. Alyson Fournier and Dr. Philip Baker for providing us with necessary Nogo and CSPG receptor (PTP) constructs for this study. We would like to acknowledge Dr. Carlos Ibanez and team for carefully editing the manuscript. We also thank the Biological Resource Centre for maintenance of mice. This work was funded by Agency for Science, Technology and Research, Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
S.R.: manuscript writing, conception and design, collection and/or assembly of data, and data analysis and interpretation; F.Y.: collection and/or assembly of data; Y.H.Y. and S.H.: provision of tools and collection and/or assembly of data; G.S.D.: data analysis and interpretation; S.A.: manuscript writing, conception and design, data analysis and interpretation, and final approval of manuscript.
Disclosure of Potential Conflicts of Interests
The authors indicate no potential conflicts of interest.